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Methods in Cell
Biology
The Neuronal Cytoskeleton,
Motor Proteins, and Organelle
Trafficking in the Axon
Volume 131
Series Editors
Leslie Wilson
Department of Molecular, Cellular and Developmental Biology
University of California
Santa Barbara, California
Phong Tran
University of Pennsylvania
Philadelphia, USA &
Institut Curie, Paris, France
Methods in Cell
Biology
The Neuronal Cytoskeleton,
Motor Proteins, and Organelle
Trafficking in the Axon
Volume 131
Edited by
K. Kevin Pfister
Department of Cell Biology, Charlottesville, USA
AMSTERDAM • BOSTON • HEIDELBERG • LONDON

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ISBN: 978-0-12-803344-9
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Contributors
Stefanie Alber
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot, Israel
C.J. Alexander
Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National
Institutes of Health, MD, USA
Adam W. Avery
Department of Genetics, Cell Biology, and Development, University of Minnesota,
Minneapolis, MN, USA
Peter W. Baas
Department of Neurobiology and Anatomy, Drexel University College of Medicine,
Philadelphia, PA, USA
Alexandre D. Baffet
Department of Pathology and Cell Biology, Columbia University, New York,
NY, USA
Lisa Baker
Marine Biological Laboratory, Woods Hole, MA, USA
Gary Banker
Jungers Center for Neurosciences Research, Oregon Health and Science
University, Portland, OR, USA
Marvin Bentley
Mark M. Black
Department of Anatomy and Cell Biology, Temple University School of Medicine,
Kiev R. Blasier
Department of Cell Biology, University of Virginia, Charlottesville, VA, USA
Scott T. Brady
Marine Biological Laboratory, Woods Hole, MA, USA; Department of Anatomy
and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA
Anthony Brown
Department of Neuroscience, The Ohio State University, Columbus, OH, USA
xiii
xiv Contributors
Kristy J. Brown
Research Center for Genetic Medicine, Children’s National Health System,
Washington, DC, USA; Department of Integrative Systems Biology, Institute of
Biomedical Sciences, The George Washington University, Washington, DC, USA
Alma L. Burlingame
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF,
San Francisco, CA, USA
John C. Cain
Aurélie Carabalona
NY, USA
Anaël Chazeau
Cell Biology, Department of Biology, Faculty of Science, Utrecht University,
Utrecht, The Netherlands
Michael Chein
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
Tiago J. Dantas
NY, USA
David D. Doobin
NY, USA
Ella Doron-Mandel
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot,
Israel
Catherine M. Drerup
Department of Cell, Developmental and Cancer Biology, School of Medicine,
Oregon Health & Science University, Portland, OR, USA
Noelle D. Dwyer
Department of Cell Biology, University of Virginia School of Medicine,
Charlottesville, VA, USA
Mike Fainzilber
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot, Israel
Contributors xv
J. Daniel Fenn
Xiaoqin Fu
Center for Neuroscience Research, Children’s National Health System,
Washington, DC, USA
Kathlyn J. Gan
Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, BC, Canada
Archan Ganguly
Department of Pathology, University of California, San Diego, La Jolla, CA, USA
Shani Gluska
J.A. Hammer, III
Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National
Institutes of Health, MD, USA
Thomas S. Hays
Erika L.F. Holzbaur
Department of Physiology, University of Pennsylvania Perelman School of
Medicine, Philadelphia, PA, USA; Neuroscience Graduate Group, University of
Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Casper C. Hoogenraad
Daniel J. Hu
NY, USA
Chung-Fang Huang
University, Portland, OR, USA; National Laboratory Animal Center, NARLabs,
Taipei, Taiwan
Ariel Ionescu
xvi Contributors
Kerstin M. Janisch
Department of Cell Biology, University of Virginia School of Medicine,
Minsu Kang
Marine Biological Laboratory, Woods Hole, MA, USA; Department of Anatomy
and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA
Lukas C. Kapitein
Eugene A. Katrukha
Noopur V. Khobrekar
NY, USA
Eva Klinman
Kelsey Ladt
Department of Neurosciences, University of California, San Diego, La Jolla,
CA, USA
Zofia M. Lasiecka
Children’s National Medical Center, Washington, DC, USA
Seung Joon Lee
Department of Biological Sciences, University of South Carolina, Columbia,
SC, USA
Lanfranco Leo
Min-gang Li
Judy S. Liu
Center for Neuroscience Research, Children’s National Health System,
Washington, DC, USA
Contributors xvii
James B. Machamer
Department of Neurology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Katalin F. Medzihradszky
David J. Mitchell
Paula C. Monsma
Gerardo Morfini
Department of Anatomy and Cell Biology, University of Illinois at Chicago,
Chicago, IL, USA; Marine Biological Laboratory, Woods Hole, MA, USA
Kanneboyina Nagaraju
Alex V. Nechiporuk
Department of Cell, Developmental and Cancer Biology, School of Medicine,
Oregon Health & Science University, Portland, OR, USA
Amanda L. Neisch
Jeffrey J. Nirschl
Juan A. Oses
Eran Perlson
K. Kevin Pfister
xviii Contributors
Sree Rayavarapu
Mitchell W. Ross
Nimrod Rotem
Subhojit Roy
Department of Neurosciences, University of California, San Diego, La Jolla, CA,
USA; Department of Pathology, University of California, San Diego, La Jolla, CA,
USA
Philipp Schätzle
Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
Michael A. Silverman
Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, BC, Canada; Department of Biological Sciences, Simon Fraser
University, Burnaby, BC, Canada; Brain Research Centre, University of British
Columbia, Vancouver, BC, Canada
Yuyu Song
Marine Biological Laboratory, Woods Hole, MA, USA; Yale School of Medicine,
Department of Genetics and Howard Hughes Medical Institute, Boyer Center,
New Haven, CT, USA
Jeffery L. Twiss
Department of Biological Sciences, University of South Carolina, Columbia,
SC, USA
Atsuko Uchida
Richard B. Vallee
NY, USA
Bettina Winckler
Department of Neuroscience, University of Virginia Medical School,
Contributors xix
Rui Yang
Julie Yi
NY, USA
Wenqian Yu
Jie Zhou
NY, USA
Preface
Investigations into fundamental questions in cell biology have long benefited from
experiments that utilize neuronal systems. Neurons have proven particularly useful
model systems for enhancing our understanding of intracellular transport. Their long
thin axons, which can comprise 95% of the cellular volume, cannot be maintained by
diffusion alone, and thus they may be regard as specialized for transport. In addition,
their morphology makes axons ideal systems to image motor protein-based move-
ment with live cell microscopy. These properties render axonal transport an effective
model for investigating the cytoskeleton, motor proteins, and organelle transport.
The chapters in this volume describe methods that utilize live cell imagining,
genetic, molecular, biochemical, and proteomic approaches in neuronal systems to
characterize and explore fundamental questions related to intracellular motility,
especially axonal transport. The contributors employ a wide variety of culture
systems including sympathetic, cortical, hippocampal, dorsal root ganglion, and
Purkinje neurons as well as in vitro slice cultures and axoplasm from the squid giant
axon; as well as model organisms Drosophila, zebrafish, and mice. The first chapter
introduces the basic paradigm for the mechanism(s) of movement in the axon. It
reviews in vivo pulse labeling experiments which identified the movement of three
distinct sets of structural components from the cell body down the axon; membrane-
bounded organelles, microtubule, and neurofilaments, and actin with the over 200
remaining axonal proteins. The chapter continues by discussing recent live cell
imaging data, utilizing the excellent optical properties of long thin axons, to define
the mechanisms for the moment of the structures. The volume is then organized into
three overlapping areas with methods chapters that focus on (1) cytoskeletal protein
dynamics and filament transport, (2) the motor proteins responsible for transport,
and (3) the transport of membrane-bounded organelle cargos.
Procedures are given for the live imaging of neurofilament transport and actin
dynamics and transport in cultured neurons. In addition, methods are described to
image tubulin dynamics in cultured hippocampal slices and single molecule resolu-
tion of tubulin and microtubule plus-end-tracking proteins in cultured neurons.
Techniques for live imaging of the movement of cytoplasmic dynein and the initia-
tion of retrograde organelle transport in axons of cultured neurons are also
presented. Assays to probe kinesin motor domain function and the role of a kinesin
family member in cytokinesis in neuroprogenitors are reviewed. Genetic and imag-
ing approaches to analyze motor protein function and organelle motility and neuro-
progenitor migration are provided using zebrafish, Drosophila, and mouse models.
A variety of approaches to image and analyze membrane-bounded organelle and
other cargo motility (including endosomes, lysosomes, autophagosomes, mitochon-
dria, signaling endosomes, viruses, and ribonucleoprotein particles) in axons, den-
drites, and squid axoplasm are discussed. These include utilizing microfluidics
chambers for culturing neurons; labeling the membrane-bounded organelle cargos
with dyes or fluorescent-tagged proteins; tracking internalized transmembrane
xxi
xxii Preface
proteins with quantum dot- or fluorochrome-labeled ligands or antibodies; and

investigating effect of the Alzheimer’s disease peptide b-amyloid on organelle
transport.
Several chapters take advantage of molecular and biochemical methods to
analyze cytoskeletal and motor protein activity. The squid axoplasm system is
utilized to investigate kinase pathways of phosphorylation of filament subunits
and motor proteins. A proteomics method is presented to probe the effects of mouse
mutations on the cytoskeleton and motor proteins; and affinity chromatography is
used to investigate motor proteins association with ribonucleoprotein particle trans-
port in axons. Two contributions discuss methods for knocking down the expression
of neuronal proteins using RNAi, one focuses on using siRNA in sympathetic and
hippocampal neurons; the second describes a plasmid-based approach to reduce
myosin Va levels in cultured Purkinje cells.
CHAPTER
Axonal transport: The

orderly motion of axonal
structures 1
Mark M. Black
Department of Anatomy and Cell Biology,
Temple University School of Medicine, Philadelphia, PA, USA
E-mail: mark.black@temple.edu
CHAPTER OUTLINE
1. Pulse-Labeling Studies of Axonal Transport ............................................................. 2
2. Live-Cell Imaging of Axonal Transport...................................................................... 7
2.1 FC and the Movement of Vesicular Cargoes................................................ 7
2.2 Slow Axonal Transport and the Movement of Cytoskeletal Polymers ............. 8
2.3 Neurofilaments are Transported in Axons................................................... 8
2.4 Microtubules and Slow Axonal Transport ................................................. 10
2.5 SCb and the Movement of Soluble Proteins of Axoplasm........................... 12
3. Summary ............................................................................................................. 15
References ............................................................................................................... 15
Abstract
Axonal transport is a constitutive process that supplies the axon and axon terminal with
materials required to maintain their structure and function. Most materials are supplied
via three rate components termed the fast component, slow component a, and slow
component b. Each of these delivers a distinct set of materials with distinct transport
kinetics. Understanding the basis for how materials sort among these rate components
and the mechanisms that generate their distinctive transport kinetics have been long-
standing goals in the field. An early view emphasized the relationships between axonally
transported cargoes and cytological structures of the axon. In this article, I discuss key
observations that led to this view and contemporary studies that have demonstrated its
validity and thereby advanced the current understanding of the dynamics of axonal
structure.
Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.001 1

© 2016 Elsevier Inc. All rights reserved.
2 CHAPTER 1 Axonal transport: The orderly motion of axonal structures
Axonal transport is the process by which proteins and other materials synthe-
sized in the neuronal cell body are delivered to the axon and axon terminal.
This is a constitutive process that occurs throughout the life of neurons, supply-
ing axons with materials needed to maintain their structure and function. The
notion that the axon depends on the cell body dates back to the nineteenth cen-
tury, based on the observation that axons disconnected from their cell bodies
degenerate (Ramon y Cajal, 1928). However, it was not until 1948 that movement
of materials in axons was first revealed by Weiss and Hiscoe, who partially con-
stricted axons and observed that axoplasm accumulated immediately proximal to
the constriction, suggesting a proximal-to-distal movement of axonal materials.
Upon release of the constriction, the accumulated axoplasm moved anterogradely
at z1 mm/day, thus identifying what later came to be known as slow axonal
transport.
Since this pioneering work, axonal transport has been studied extensively with
two experimental approaches providing most of the current understanding. One
uses radioactive precursors to pulse-label axonally transported materials and the
other uses imaging techniques to directly observe transport in living axons. These
two approaches provide distinct but complementary information (Brown, 2009).
Pulse-chase approaches provide indirect information on movement of materials in
axons in intact animals over time scales of hours to months whereas live-cell imag-
ing directly visualizes axonal transport over time frames of seconds to hours. Below,
I discuss contributions of these approaches to the current understanding of the
cargoes that undergo axonal transport and their transport behavior as seen at short
and long time scales.
1. PULSE-LABELING STUDIES OF AXONAL TRANSPORT

The pulse-labeling approach has revealed the kinetics of protein transport in axons
over long time scales and the identity of many transported proteins. Typically, radio-
active amino acids are injected into the environment surrounding the neuron cell
bodies under study. The amino acids are taken into the neurons and incorporated
into proteins, some of which are then transported into their axons. Because the
amino acids are cleared relatively rapidly by the circulation, this procedure produces
a pulse of labeling in vivo. To visualize the transport of the pulse-labeled proteins,
the nerve containing them is cut into consecutive pieces of a few millimeters in
length and the distribution of radioactivity along its length quantified. Also, the iden-
tity of specific radioactive proteins in the nerve segments has been determined using
biochemical procedures. As each animal provides a single time point for analysis,
multiple animals must be examined, each at different times after labeling.
Comparing the results at the various times yields a detailed, though indirect, picture
of the movement of proteins in axons.
This approach has been used with a variety of organisms and the essential
results obtained are consistent among systems. The transported pulse-labeled
1. Pulse-Labeling studies of axonal transport 3
proteins are distributed along the axons as waves with distinct crests and fronts
(Figure 1(A)). The positions and shapes of the waves change as a function of
time after injection based on the transport behavior of the proteins. At time frames
of hours, waves of pulse-labeled proteins are seen that advance at z50e400 mm/
day (0.6e5 mm/s) (reviewed in Grafstein & Forman, 1980). This corresponds to the
fast component (FC) of axonal transport. FC has both anterograde (soma toward
axon tip) and retrograde (axon tip toward soma) components. There is also a
slow component which moves at average rates of 0.2e10 mm/day (0.0002e
0.1 mm/s). Slow axonal transport consists of two subcomponents, slow component
a (SCa) and slow component b (SCb), that differ in specific protein composition and
transport rate. SCa moves at modal rates of 0.2e3 mm/day, while SCb moves at
2.0e10 mm/day (the range in rates reflects variations among different populations
of neurons). These three rate components provide most of the materials delivered to
the axon by axonal transport.
Cell fractionation and electron microscopic autoradiographic studies (Di
Giamberardino, Bennett, Koenig, & Droz, 1973; Droz, Koenig, Biamberardino, &
Di Giamberardino, 1973; Lorenz & Willard, 1978) showed that fast and slow axonal
transport deliver distinct materials to the axon. This result was confirmed by gel elec-
trophoretic analyses of the proteins comprising FC, SCa, and SCb (Tytell, Black,
Garner, & Lasek, 1981; Willard, Cowan, & Vagelos, 1974). FC and SCb each consists
of hundreds of proteins, whereas SCa transports comparatively few, and strikingly
very few proteins are present in more than one rate component (Figure 1(B) and
(C)). Thus, the underlying mechanisms of axonal transport prevent the mixing of pro-
teins as they move past each other in the axon. The structural hypothesis of axonal
transport was put forth to explain this and other differences between FC, SCa, and
SCb (Lasek, 1980; Lasek, Garner & Brady, 1984). This hypothesis posits that pro-
teins are actively transported in the axon either as integral parts of moving cytological
structures or in association with these structures. At the time, the strongest support
was for FC for which multiple criteria showed was associated with membrane-bound
organelles (Dahlström, Czernik, & Li, 1992; Droz et al., 1973; Di Giamberardino
et al., 1973; Goldman, Kim, & Schwartz, 1976; Lorenz & Willard, 1978).
The evidence for cytological correlates of slow axonal transport based on the
pulse-chase approach is much more limited. The principal proteins of SCa were
tubulin and neurofilament proteins, the subunits of microtubules and neurofilaments,
respectively (Black & Lasek, 1980; Hoffman & Lasek, 1975). Thus, it was hypoth-
esized that SCa represented the transport of these cytoskeletal polymers. Based on
the close similarity in transport kinetics of tubulin and neurofilament proteins, the
initial suggestion was that microtubules and neurofilaments moved as a network
of interacting polymers. However, as subsequent work revealed subtle differences
between tubulin and neurofilament protein transport (McQuarrie, Brady, & Lasek,
1986) and structural studies indicated limited interactions between neurofilaments
and microtubules (Brown & Lasek, 1993; Price, Paggi, Lasek, & Katz, 1988), the
view of SCa evolved to the independent movement of microtubules and
neurofilaments.
FIGURE 1
Axonal transport of proteins in hypoglossal and retinal ganglion cell axons of guinea pigs.
(These data are reprinted with permission from Tytell et al. (1981).) Panel (A). The
distribution of radioactive proteins in the hypoglossal nerves of guinea pigs 3 h (upper graph)
or 15 days (lower graph) after injecting radioactive amino acids into the hypoglossal nucleus
1. Pulse-Labeling studies of axonal transport 5
The only additional evidence to support the hypothesis that neurofilaments

moved in SCa was that neurofilament proteins were quantitatively assembled into
neurofilaments in axons (Black, Keyser, & Sobel, 1986; Morris & Lasek, 1982).
However, a small fraction of unassembled proteins could reasonably go undetected,
thus limiting the power of this observation. If tubulin is transported in the form of
microtubules, then microtubule-associated proteins should be cotransported with
tubulin. In this regard, minor proteins move with tubulin in SCa that have mobilities
similar to tau (Black & Lasek, 1980), a major axonal microtubule-associated pro-
tein. While an early study suggested that these may be tau (Tytell, Brady, & Lasek,
1984), subsequent analyses using two-dimensional gel electrophoresis indicated that
they are chartins (Oblinger & Black, unpublished data), a family of microtubule-
associated proteins distinct from tau. Thus, at least one microtubule-associated pro-
tein is cotransported with tubulin. However, other axonal microtubule-associated
proteins move faster than tubulin at rates in the range of SCb (Ma, Himes, Shea,
=
(the location of the neuron cell bodies whose axons form the hypoglossal nerve). Distance is
from the hypoglossal nucleus. At 3 h after injection, a well-defined wave which corresponds
to the FC is apparent, while at 15 days, two waves are apparent which correspond to SCa and
SCb. Panel (B). Comparison of the proteins comprising SCa, SCb, and FC of retinal ganglion
cell axons of guinea pigs using one-dimensional polyacrylamide gel electrophoresis.
Segments of the optic nerve and tract, which contain the retinal ganglion cell axons, were
obtained at 6 h, 6 days, or 38 days for proteins of FC, SCb, or SCa, respectively. FC and SCb
each consists of many polypeptides, whereas only five polypeptides account for the majority
of material transport in SCa. Even by one-dimensional gel electrophoresis, it is apparent that
any of the transported proteins appear in only one transport component (see the bands
highlighted by brackets). Note: the radioactive bands below tubulin in the SCa profile are not
transported in SCa but represent trailing proteins of SCb. Known polypeptides are indicated:
C ¼ clathrin, A ¼ actin, NFL, NFM, NFH ¼ low, middle, and heavy neurofilament subunits,
TUB ¼ tubulin. Apparent molecular weight is indicated on the left. Panel (C). Comparison of
the proteins comprising SCa, SCb, and FC of retinal ganglion cell axons of the guinea pig
using two-dimensional isoelectric focusingdpolyacrylamide gel electrophoresis. The
approximate pH gradient of each gel is indicated on the bottom and apparent molecule
weight is indicated on the left. This high-resolution technique shows that with very few
exceptions, each transported protein is present in only one rate component. The one
exception is the protein spot highlighted with parentheses in the samples of SCa and SCb.
Another protein present in more than one rate component is tubulin, which in peripheral
motor and sensory neurons, is transported in SCa and SCb; however, in retinal ganglion cell
axons, tubulin is only in SCa. Proteins of known identity when these data were originally
published are identified in the figures and include neurofilament subunits (NFH, NFM, NFL)
and tubulin (TUB), nerve-specific enolase (NSE), creatine phosphokinase (CPK), and actin
(A). Note: clathrin heavy chain is not identified because it forms a streak that is too faint to be
seen. The smearing of spots in the gel of FC is typical and is apparently due to the
carbohydrate and lipid modifications common to FC proteins. FC, fast component; SCa, slow
component a; SCb, slow component b.
& Fischer, 2000; Mercken, Fischer, Kosik, & Nixon, 1995). Interpretation of these
data is not straightforward. First, tau, MAP1a, and MAP1b have multiple interacting
partners in addition to tubulin, some of which (e.g., actin) move in SCb, and these
interactions can be expected to impact their movement in axons. Second, live-cell
imaging suggests that tau is cotransported with tubulin (Konzack, Thies, Marx,
Mandelkow, & Mandelkow, 2007). However, when tau dissociates from microtu-
bules, it diffuses quite rapidly, faster than the average rate of tubulin transport.
Thus, the population of tau moves faster than tubulin. While the pulse-chase studies
on transport of microtubule-associated proteins provide insights into the interactions
between tubulin and microtubule-associated proteins in axons, they do not effec-
tively address their transport form.
The structural correlates of SCb are unknown. This is in part due to its compo-
sitional complexity. Hundreds of diverse proteins move in SCb which include pro-
teins of the actin and membrane cytoskeletons, enzymes of intermediary
metabolism, proteins involved in membrane trafficking, and proteins that interact
with synaptic vesicles. Actin was one of the first proteins identified in SCb (Black
& Lasek, 1979; Willard, Wiseman, Levine, & Skene, 1979). It was suggested that
actin filaments form a scaffold to which other SCb proteins bind and the resulting
complex represents an SCb cargo. However, no direct data have been published to
support this possibility.
An early insight into SCb derived from the observations that SCb proteins move
together in a vectorial manner in axons and that they are also soluble components of
axoplasm. Such a result would be difficult to explain if the proteins were freely
diffusible. Thus, it was suggested that they existed as one or more assemblies that
were conveyed by the transport machinery (Garner & Lasek, 1982; Tytell et al.,
1981). This view is supported by cell fractionation analyses which show that
many SCb proteins behave as large multiprotein complexes (Lorenz & Willard,
1978; Scott, Das, Tang, & Roy, 2011). In addition, immunoprecipitation analyses
performed under nondenaturing conditions using antibodies specific for clathrin,
an SCb protein (Garner & Lasek, 1981), isolated a complex that included clathrin,
Hsc70, and several other minor SCb proteins (Black, Chestnut, Pleasure, & Keen,
1991). This complex may represent an SCb cargo. Finally, comparisons of the trans-
port behavior of several individual SCb proteins have revealed three distinct trans-
port profiles raising the possibility of three distinct cargoes (Garner & Lasek, 1982).
While these studies support the idea that SCb proteins form higher order assemblies
that undergo transport in axons, the identity of these complexes remains to be
discovered.
This selected review has discussed some of the history that led to the structural
hypothesis of axonal transport and the initial suggestions regarding structural corre-
lates of FC, SCa, and SCb. Many of the suggestions were controversial sparking
numerous studies using pulse-chase approaches that greatly enhanced knowledge
of axonal transport. However, these studies did not resolve the controversy because
they could not unambiguously reveal the identity of individual cargoes and the
moment-to-moment details of their movements. To move forward on these issues,
2. Live-Cell imaging of axonal transport 7
new approaches based on live-cell imaging have been developed that provide direct
visualization of the cargoes as they undergo transport in living axons. These new
methods have provided compelling support for the structural hypothesis of axonal
transport.
2. LIVE-CELL IMAGING OF AXONAL TRANSPORT

2.1 FC AND THE MOVEMENT OF VESICULAR CARGOES
Early studies using time-lapse optical imaging of living axons revealed the move-
ment of mitochondria and heterogeneous populations of roughly spherical objects
near the resolution limit of the light microscope (Forman, Padjen, & Siggins,
1977; Kirkpatrick, Bray, & Palmer, 1972). The rates of movement as well as their
sensitivity to metabolic inhibitors suggested that these were fast transport cargoes.
The introduction of video-enhanced contrast differential interference contrast mi-
croscopy revealed dramatically more movement than previously obtained because
of its ability to detect structures as small as 30 nm. Early studies on axoplasm
extruded from the squid giant axon revealed a large variety of structures moving
at rates corresponding to FC (Brady, Lasek, & Allen, 1982). Subsequent studies
using correlative electron microscopy identified many of the specific cargoes as a
variety of membrane-bound structures, thereby confirming the view derived from
pulse-chase studies (Miller & Lasek, 1985; Schnapp, Vale, Sheetz, & Reese,
1985). They also established that anterograde cargoes differed from those moving
retrogradely, with the former including Golgi-derived vesicles and the latter
including endocytic vesicles and prelysosomal structures. The squid axoplasm sys-
tem also led to the discovery of kinesin, a microtubule motor that powers fast anter-
ograde transport (Brady, 1985; Vale, Reese, & Sheetz, 1985) as well as the existence
of a distinct motor that powered fast retrograde transport (Vale, Schnaapp, et al.,
1985), which was later identified as cytoplasmic dynein. The reader is referred to
numerous reviews on fast axonal transport and the motors that power this motility
that have appeared in the intervening years.
Two points regarding FC will be highlighted. First, its anterograde and retrograde
cargoes typically move persistently and unidirectionally, pausing infrequently dur-
ing their transit in the axon. Second, while moving, their rates approximate both
the maximum rates reported for FC using pulse-chase methods and the maximum
rates reported for kinesin and dynein motors in vitro. Thus, fast axonal transport rep-
resents a system for efficiently moving vesicular structures between the cell body
and axon tip. While much remains to be learned about regulatory mechanisms
that control fast transport, the interactions of FC cargoes with the transport motors
are relatively stable and the motors interact processively with the microtubule tracks
upon which transport occurs.
Mitochondria, membrane-bound structures abundant in axons, exhibit very
different transport behavior from typical FC cargoes. Mitochondria have much
slower average rates of transport compared to fast transport cargoes (Hollenbeck &
Saxton, 2005). Live-cell imaging reveals that mitochondria pause frequently during
their transport in the axon often remaining stationary for extended times and they
can also undergo changes in direction (Saxton & Hollenbeck, 2012). Yet, mitochon-
dria transport is powered by the same kinesin and dynein motors that translocate FC
cargoes. Thus, differences in transport rate and behavior do not necessarily indicate
fundamental differences in mechanism. It is the differences in the regulation of the
transport machinery that allow the machinery to generate such distinctive transport
behaviors (Brown, 2003; Saxton & Hollenbeck, 2012). This same theme will come
up again in the discussion of slow axonal transport.
2.2 SLOW AXONAL TRANSPORT AND THE MOVEMENT OF

CYTOSKELETAL POLYMERS
The first studies attempting to reveal microtubule and neurofilament transport spe-
cifically tested the hypothesis that these structures moved slowly and steadily
from the cell body toward the axon tip at the modal rate of SCa as revealed by
pulse-chase studies (Lim, Edson, Letourneau, & Borisy, 1990; Okabe & Hirokawa,
1990; Okabe, Miyasaka, & Hirokawa, 1993). The results failed to show such slow
steady movement and were interpreted as evidence that microtubules and neurofila-
ments were not transported. However, given that these studies failed to reveal any
movement at all including that known to occur, a more conservative interpretation
would have been that microtubules and neurofilaments do not move in a slow steady
manner. As discussed below, hints already existed from pulse-chase studies on slow
axonal transport that the cargoes did not move in this manner.
2.3 NEUROFILAMENTS ARE TRANSPORTED IN AXONS

While pulse-chase studies showed that the bulk of neurofilament proteins moved
slowly and steadily at a modal rate of z1 mm/day, the wave is quite broad, indi-
cating that some SCa cargoes move faster and some slower than this. The SCa
wave also broadens substantially over time, further indicating that SCa cargoes
move at a distribution of rates. In a particularly detailed analysis of neurofilament
protein transport, rates ranged from <0.01 mm/day to several tens of mm/day
(Lasek, Paggi, & Katz, 1993). They suggested that the broad distribution of rates re-
flected a fundamental feature of the transport mechanisms in which neurofilament
proteins moved with brief but rapid translocation steps interrupted by pauses. This
is similar to the situation for mitochondria, but with pauses accounting for a
much greater percentage of the transport behavior to account for the slow average
rate of neurofilament protein transport. Although speculative at the time, this view
presaged the findings of subsequent studies directly visualizing neurofilament pro-
tein transport in living axons.
Wang, Ho, Sun, Liem, and Brown (2000) were the first to directly visualize neu-
rofilament transport in living axons, followed shortly thereafter by Roy et al. (2000).
Both groups expressed GFP-labeled neurofilament proteins in cultured sympathetic

neurons. These neurons contain a relatively sparse neurofilament array in their
axons, and many axons have regions along their length with no neurofilaments.
By focusing on these gaps which have near zero background fluorescence, GFP-
labeled neurofilaments, initially located outside of the gaps were observed to
move into and through them. Detecting these movements required the use of imag-
ing parameters to reveal fast but intermittent transport. The neurofilament proteins
moved with generally brief bouts of relatively rapid transport (z0.5 mm/s) interrup-
ted by prolonged pauses and unexpectedly, movement was bidirectional though the
majority moved anterogradely. The moving proteins comprised linear structures of
up to several tens of microns in length suggesting that they were moving as neuro-
filaments. Direct confirmation of this was subsequently provided by using correla-
tive electron microscopy to show that the moving structures were indeed
neurofilaments (Yan & Brown, 2005). Thus, the slow anterograde transport of neuro-
filament proteins in SCa actually reflects the average of brief episodes of rapid bidi-
rectional transport of neurofilament polymers interspersed with prolonged pauses of
little to no movement.
Subsequent studies showed that neurofilament transport is microtubule depen-
dent (Francis, Roy, Brady, & Black, 2005) and uses the same motors that power
fast axonal transport, with kinesin and dynein mediating anterograde and retrograde
neurofilament transport, respectively (He, Francis, Myers, Yu, Black & Baas, 2005;
Uchida, Alami, & Brown, 2009). Thus, neurofilament movement in slow transport
does not represent a novel mechanism, but instead reflects a variation on the theme
for the transport of vesicular cargoes. Specifically, fast motors propel neurofilaments
within the axon, but the movement is not processive. Specialized regulatory mech-
anisms generate prolonged pauses in this movement, resulting in a slow rate when
averaged over time. The specifics of this regulation are the subject of active
investigation.
In the years since these studies first appeared, Brown and colleagues have
continued to dissect neurofilament transport, revealing many novel details. One
goal has been to determine whether the transport behavior of individual neurofila-
ments as observed in cultured neurons imaged over short time frames can explain
the transport behavior of neurofilament proteins in axons observed over long time
frames in vivo with the pulse-chase approach (Brown, Wang, & Jung, 2005; Li,
Jung, & Brown, 2012). To address this, they developed computational models of
neurofilament transport employing the parameters for neurofilament transport rate,
directionality, and pausing observed in their studies. One essential feature of the
model is that neurofilaments move linearly and independently within axons, mostly
in the anterograde direction, but also retrogradely. In addition, individual filaments
cycle between distinct states of active transport and pausing, such that they spend
approximately 97% of their time pausing, while the remaining time, they move at
relatively fast rates. The model recapitulates the in vivo transport kinetics with
remarkable fidelity. Thus, the essential features of neurofilament protein transport
seen with the pulse-chase approach can be fully explained by the known properties
of neurofilament polymer transport seen by live imaging of cultured neurons. Over

the years, it has been suggested that neurofilament proteins may also undergo trans-
port in a form other than as neurofilaments. While this remains a formal possibility,
the available data indicate that neurofilaments constitute the principle transport form
of neurofilament proteins.
2.4 MICROTUBULES AND SLOW AXONAL TRANSPORT

Several studies have demonstrated that microtubules can redistribute within growing
neurons from the cell body into the axon and from the axon into the growth cone
(Ahmad & Baas, 1995; Slaughter, Wang, & Black, 1997; Yu, Schwei, & Baas,
1996). Though not directly observed, it was inferred that active transport accounted
for the redistribution. With the development of methods to reveal neurofilament trans-
port, it was natural to apply them to the issue of microtubule transport. The methods
clearly revealed tubulin moving in living axons, and like neurofilaments, the tubulin-
containing cargo moved rapidly but intermittently with an average rate in the range
reported for tubulin transport as seen in the pulse-chase studies (Hasaka, Myers, &
Baas, 2004; He, Francis, Myers, Black, & Baas, 2005; Wang & Brown, 2002). The
movement was bidirectional, though mostly anterograde, and during bouts of move-
ment the rate was typical of that seen with fast motors, z1e2 mm/s, but was interrup-
ted by pauses. Strikingly, the moving structures were short, z1e5 mm in length
(average ¼ 2.7 mm), and structures typical of the length of axonal microtubules
(many tens of microns long), were not observed to move. It was thus suggested
that short microtubules are conveyed rapidly but intermittently by slow axonal trans-
port while long microtubules are stationary (Baas, Nadar, & Myers, 2006).
Several observations support the view that long microtubules are not transported
in axons. For example, Chang, Svitkina, Borisy, and Popov (1999) used speckle mi-
croscopy to reveal individual axonal microtubules in living axons, and none of these
polymers was observed to move. In another approach, microtubule plus ends were
tagged with fluorescent tip-binding proteins and then imaged to see whether the
polymers moved. It is expected that the plus ends will advance as the microtubules
elongate. If they also undergo transport, then the rate of advance will exceed that due
to microtubule elongation alone. However, in no case was this observed (Kim &
Chang, 2006; Ma, Shakiryanova, Vardya, & Popov, 2004). As these studies imaged
large numbers of microtubules, if microtubule transport occurred, even infrequently,
it should have been detected. Thus, the conclusion that such transport does not occur
is reasonable. However, this needs to be qualified as the studies did not restrict
analyses to microtubules of particular length, but examined any polymer that could
be detected. As most axonal microtubules are many tens of microns in length
(Bray & Bunge, 1981), the findings reasonably apply to such long polymers.
Whether they apply to short microtubules is unknown, and given the results by
the Brown and Baas labs discussed above, they very well may not.
A key question in the studies by the Brown and Baas labs is whether the moving
tubulin-containing structures are in fact short microtubules. Given that tubulin
assembles into microtubules, this seems reasonable. However, as this has not been
directly tested by fixing tubulin-containing structures undergoing transport and im-
aging them by electron microscopy, uncertainty remains. The movement of tubulin-
containing structures that are not microtubules has been reported (Hollenbeck &
Bray, 1987). The majority move retrogradely, are spherical to oval in shape, and
are associated with membrane-bound structures. Thus, they seem unrelated to the
filamentous tubulin-containing structures of slow transport. Ma et al. (2004) have
also observed short filamentous tubulin-containing structures move in axons, but
have argued that these are not microtubules because they differ from microtubules
in fluorescence intensity. However, this conclusion is not supported by their
own data showing a transported tubulin-containing structure that is similar in fluo-
rescence intensity to microtubules elsewhere in the same images (see their
Figure 3(A)). Finally, it has been reported that brefeldin A blocks all slow axonal
transport, including the movement of tubulin (Campenot, Soin, Blacker, Lund,
Eng, & MacInnis, 2003). Because brefeldin A disrupts the Golgi complex and pre-
vents the formation of Golgi-derived vesicles that are the cargoes of FC, it was sug-
gested that slow axonal transport materials move by transient association with fast
transport cargoes. Recent support for this idea has been obtained for some SCb pro-
teins (see below), and thus it is a formal possibility for other slow transport cargoes
such as neurofilaments and tubulin. However, in these experiments, brefeldin A
treatment blocked the transport of all cargoes, including mitochondria. As mito-
chondria transport should not be affected by brefeldin A (Tang et al., 2013), the com-
plete block of transport in the experiments by Campenot et al. (2003) raises concern
of off target effects.
At present, the only independent evidence that these short tubulin-containing
structures are microtubules derives from studies of tau (Konzack et al., 2007).
These authors expressed various tau constructs in cultured neurons and examined
tau diffusion, tau association with microtubules, and tau transport. The studies
demonstrate that tau diffuses remarkably fast in axoplasm (D z 3 mm2/s) and
that tau association with microtubules exhibits a high exchange rate (t1/2 z 4 s).
Thus, diffusion is adequate to distribute tau throughout shorter axons (z1 mm).
However, as length increases beyond this, active transport is required to ensure de-
livery of tau to the distal axon. In terms of transport, the authors hypothesized that
tau was transported in association with microtubules, and used procedures similar
to that have revealed tubulin transport to visualize tau transport. Briefly, in neurons
expressing fluorescent tau, photobleaching was used to create a gap in the fluores-
cence of tau along the axon, and then the gap was imaged to determine whether
fluorescent tau located outside of the gap moved into and through the gap.
When tau with four microtubule-binding repeats was expressed, transport of
discrete structures was not observed. Given the short residence time of tau on mi-
crotubules combined with its rapid diffusion, this is expected; the fluorescent tau
would spend too little time associated with microtubules to detect its movement.
To increase the chances of detecting tau on moving microtubules, the authors
also expressed tau engineered to contain eight repeats. The eight-repeat tau resided
significantly longer on axonal microtubules and when used in the transport assay,
2- to 6-mm long filamentous structures were seen to move into and through the
gaps. The movement occurred in both anterograde and retrograde directions and
exhibited stop-and-go characteristics with brief bouts of fast transport (0.2e
2 mm/s) interrupted by pauses. The transport of these tau-containing structures
strikingly resembles that of tubulin-containing structures. It is noteworthy that
the manipulation that led to the detection of tau transport specifically involved
increasing the number of microtubule-binding domains on tau. Thus, the most
parsimonious explanation of the data on tubulin and tau transport is that tubulin
is transported as short microtubules and tau transport reflects its association with
transported microtubules.
It has been argued that microtubule transport is important for establishing the
microtubule polarity pattern of the axon, for the expansion of the axonal microtubule
array during growth and development and its maintenance in the adult (Baas, 2002;
Baas & Ahmad, 1993; Black, 1994). Impairment of microtubule transport in axons
may also be a factor in neurodegenerative diseases by compromising the axonal
microtubule array and thereby the various transport processes that depend upon it
(Baas & Mozgova, 2012). All of these ideas are based on the assumption that micro-
tubules are transported in axons, and while a strong case for this can be made, some
uncertainty remains. It is imperative to directly test whether these tubulin-containing
structures are indeed microtubules and hopefully move past this lingering uncer-
tainty. Methods are available for doing this using reagents that both fluoresce and
can be seen using the electron microscope. While such experiments pose technical
challenges, the effort will be worth the outcome because the issue will be resolved
once and for all, and the outcome will provide essential direction for the field to
move forward.
2.5 SCb AND THE MOVEMENT OF SOLUBLE PROTEINS OF

AXOPLASM
In some respects, little progress has been made in deciphering SCb, whereas in other
respects great strides have been made. With regard to the former, to explain the
movement of the 200þ soluble proteins of SCb, it was hypothesized that they
assemble into multiprotein complexes that are the cargoes of SCb (Garner & Lasek,
1982; Lorenz & Willard, 1978). While recent studies have provided further support
for the hypothesis that SCb proteins assemble into multiprotein complexes (Scott
et al., 2011; Tang, Das, Scott, & Roy, 2012), the identity of the complexes and their
possible relationship to cytological structures of the axon remain unknown. On the
other hand, substantial progress has been made in dissecting the transport mecha-
nisms for these proteins. As described below, the theme of rapid but infrequent
movements also figures important for SCb.
Initial studies expressed GFP-tagged SCb proteins (a-synuclein, synapsin-1,
glceraldehyde-3-phosphate dehydrogenase) in cultured hippocampal neurons, and
used imaging parameters to detect rapid but intermittent movements (Roy, Winton,
Black, Trojanowski, & Lee, 2007; Roy, Winton, Black, Trojanowski, & Lee, 2008).
These studies focused on thin axons in which the GFP-tagged SCb proteins appeared
as occasional discrete puncta above a more diffuse distribution. While many puncta
remained stationary during imaging, some moved at rates comparable to FC (z1e
2 mm/s). Such movements were relatively infrequent and they were interrupted by
pauses of variable duration. Furthermore, while most puncta moved anterogradely,
some moved retrogradely. These results showed that SCb proteins can move bidirec-
tionally in a stop-and-go manner. It was argued that the overall slow rate of transport
reflected the average for the population of the time spent moving rapidly and the
time spent pausing.
The key findings of these studies not fully appreciated at the time derived from
direct comparisons of the transport behavior of SCb and FC cargoes in individual
axons. Neurons coexpressing red-tagged a-synuclein (and later synapsin-1 (Tang
et al., 2013)) and green-tagged synaptophysin, an integral membrane protein of
FC, were imaged simultaneously to reveal their transport. Synaptophysin appeared
as small puncta and exhibited typical FC behavior as reported by others, with syn-
aptophysin puncta moving frequently and highly persistent. This was in marked
contrast to the infrequent and less persistent movements of SCb puncta. However,
SCb and synaptophysin puncta exhibited nearly identical transport velocities during
bouts of movement, and furthermore, in dual imaging analyses, all moving SCb
puncta moved together with synaptophysin. This latter finding was striking and sug-
gestive of a linkage between the movement of SCb proteins and FC cargoes. Subse-
quent work by Roy and colleagues demonstrated the importance of this linkage to
the transport of at least some SCb proteins.
To further dissect the mechanisms of SCb transport, Roy and colleagues devel-
oped an assay for SCb transport in cultured neurons using photoactivatable vectors
in which bulk cargo movement and particle dynamics could be visualized with high
resolution (Scott et al., 2011; Tang et al., 2012, 2013). These studies focused on three
SCb proteins, synapsin-1a, calmodulin-dependent kinase IIa, and a-synuclein.
While these proteins have distinctive transport kinetics, I will focus on their com-
monalities. The results obtained show that the bulk of these proteins moves with
an anterograde bias at a rate of z0.01 mm/s, which is similar to the rates reported
for SCb proteins based on pulse-labeling studies. The transport requires microtu-
bules, microtubule motors, and ATP.
It has not been possible to visualize discrete movements within the bulk popula-
tion of SCb proteins presumably because individual movements are too brief to cap-
ture and/or the vectorially moving proteins do not stand out from their neighbors that
are just diffusing. However, a minor subpopulation of these SCb proteins appears as
discrete particles that exhibit intricate transport kinetics. During their movement,
transport rates are relatively fast, z1e2 mm/s, but the duration of movement is var-
iable, ranging from a few seconds to a few tens of seconds (the original live-cell im-
aging studies by Roy et al. (2007) focused on this minor subpopulation of SCb
cargoes). It is assumed that movement within the wave exhibits transport kinetics
similar to these particles, but for much shorter durations. Simulations were
developed to test specific mechanisms that could explain both the slow advance of
the bulk of SCb proteins as well as the more persistent particle movements, focusing
specifically on synapsin-1 transport. The model that best fit the data involved tran-
sient association of synapsin-1 with mobile units that moved persistently with a
range of rates typical of microtubule motors and with an anterograde bias. The as-
sociation of synapsin-1 with the mobile units occurred with a range of interaction
strengths, such that most movements were of short duration (1 s) and distance
(1 mm), although a minor fraction persisted for many seconds and moved many
microns. Given the biochemical evidence suggesting that synapsin-1 along with
other SCb proteins exist as multiprotein assemblies, this suggests a model in which
complexes of SCb proteins containing synapsin-1 transiently engage with motors,
either directly or indirectly, resulting in an overall slow anterograde advance within
the axon.
Given the dual imaging analyses showing that SCb cargoes are cotransported
with synaptophysin (Roy et al., 2007; Scott et al., 2011), the vesicular cargoes of
FC that contain synaptophysin are logical candidates for the mobile units. Direct
support for this possibility was obtained by showing that manipulations that sup-
pressed FC similarly suppressed synapsin-1 transport (Tang et al., 2013). In addition,
the transport of synapsin-1 was dependent on its domains that interact with vesicular
structures. Refinements of the simulation parameters suggest a model in which syn-
apsin-1 assembles into multiprotein complexes that have an affinity to vesicular
cargoes of fast transport. The synapsin-1 complexes and vesicles interact stochasti-
cally, with most synapsin-1 complexes interacting transiently and thus advancing
slowly within the axon, whereas a minor subset interacts for longer periods and
so moves with FC.
While the extent to which this model applies to other SCb proteins is unknown,
the finding that some of the synapsin-1 moves together with two other SCb proteins,
a-synuclein and glceraldehyde-3-phosphate dehydrogenase (Roy et al., 2007), sug-
gests some generality. However, SCb is compositionally very complex, containing
200þ different proteins, and these are likely organized into multiple cargo com-
plexes (Black et al., 1991; Garner & Lasek, 1982; Lorenz & Willard, 1978; Roy
et al., 2007). A number of SCb proteins are able to interact directly or indirectly
with membranes (for example, a-synuclein, spectrin, actin, clathrin) and so may
move via transient associations with FC cargoes in a manner resembling that of syn-
apsin-1. However, it is also possible that SCb complexes are transported directly by
molecular motors in a manner that results in an overall slow transport within the
axon. Since its initial description as a discrete component of axonal transport (Black
& Lasek, 1979; Garner & Lasek, 1982; Willard et al., 1974), SCb has been a mys-
tery. Many of its proteins still remain to be identified and the current understanding
of their organization in the axon is limited and has not advanced much beyond those
of the early pulse-chase studies. However, the work of Roy and colleagues has pro-
vided a mechanistic understanding of the transport of select SCb proteins and the
next several years promise to reveal many new insights into this still enigmatic
component of axonal transport.
References 15
3. SUMMARY
In 1980, Raymond Lasek published an article on axonal transport entitled “Axonal
Transport: A Dynamic View of Neuronal Structures” in which he emphasized the
close relationship between axonal transport and the fine structure of the axon. He
argued that the structures observed in axons by electron microscopy are the cargoes
of axonal transport. In this view, studies of the fine structure of the axon and of
axonal transport provide highly complementary information. Specifically, cytolog-
ical studies provide a snapshot in time of the organization of axonal structures,
whereas studies of axonal transport provide information on the orderly motion of
these structures over time scales ranging from seconds to days, months, and longer.
Combining this information provides a dynamic view of axonal structure. Based on
information available at that time, specific hypotheses were proposed regarding the
relationship between axonal transport and axonal structures. As new technologies
were developed that provided increasingly higher resolution information on the
motility of axonal components, it became possible to directly test these hypotheses,
and some were proven correct, though often in very different ways from what was
initially envisioned, whereas others were not. The contemporary picture of axonal
transport is very different from that of three decades ago, but the fundamental prem-
ise that axonal transport provides dynamic information on axonal structures has been
fully validated by contemporary studies. Indeed, this perspective is still at the heart
of many studies of axonal transport. In many cases, the transported structures are
well defined and the studies are aimed at more subtle issues of the regulation of
transport. In other cases, the connection between the transported cargoes and axonal
structure is still being defined. The increasing resolution with which these issues can
now be examined promises answers to many of the currently outstanding questions
and with these an increasingly sophisticated understanding of how axonal transport
contributes to the elaboration and maintenance of axonal structure and function.
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CHAPTER
Live-cell imaging of
neurofilament transport in
cultured neurons 2
Atsuko Uchida, Paula C. Monsma, J. Daniel Fenn, Anthony Brown1
1
Corresponding author: E-mail: brown.2302@osu.edu
CHAPTER OUTLINE
Introduction .............................................................................................................. 23
1. Culturing Neurons ................................................................................................ 25
1.1 General Considerations ........................................................................ 25
1.2 Culture Media ..................................................................................... 26
1.2.1 Media for superior cervical ganglion neuron cultures ....................... 26
1.2.2 Media for cortical neuron cultures................................................... 27
1.2.3 Media for dorsal root ganglion neuron cultures ................................ 28
1.3 Coverslips and Culture Dishes .............................................................. 29
1.3.1 Glass-bottomed dishes ................................................................... 30
1.3.2 Sterilizing and coating coverslips and glass-bottomed dishes ........... 32
1.4 Superior Cervical Ganglion Neuron Cultures........................................... 34
1.5 Cortical Neuron Cultures...................................................................... 36
1.5.1 Preparation of coverslips for glial sandwich cultures ........................ 37
1.5.2 Preparation of a suspension of cortical neurons and glia.................. 37
1.5.3 Preparation of cortical neuron cultures............................................ 40
1.5.4 Preparation of glial sandwich cultures ............................................. 40
1.6 Dorsal Root Ganglion Neuron Cultures................................................... 42
1.6.1 Long-term myelinating DRG cocultures ........................................... 42
1.6.2 Short-term nonmyelinating DRG cultures ........................................ 44
2. Neurofilament Fusion Proteins............................................................................... 44
2.1 Choice of Neurofilament Subunit .......................................................... 44
2.2 Choice of Fluorescent Protein............................................................... 45
2.3 Design of Fusion and Expression Construct............................................ 46
2.4 Testing the Constructs for Assembly Competence................................... 48
3. Transfecting Neurons............................................................................................ 49
3.2 Plasmid Purification ............................................................................ 49
Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.07.001 21

© 2016 Elsevier Inc. All rights reserved.
22 CHAPTER 2 Live-cell imaging of neurofilament transport in cultured neurons
3.3 Electroporation ................................................................................... 49

3.4 Lipofection ......................................................................................... 52
3.5 Magnetofection ................................................................................... 52
3.6 Nuclear Injection ................................................................................ 53
4. Strategies for Observing Movement ....................................................................... 57
4.2 Naturally Occurring Gaps ..................................................................... 58
4.3 Fluorescence Photobleaching ............................................................... 61
4.4 Fluorescence Photoactivation ............................................................... 61
5. Live-Cell Imaging ................................................................................................. 65
5.2 Imaging Media .................................................................................... 66
5.3 Controlling Temperature, CO2, and Humidity ......................................... 66
5.3.1 Stage-top incubators....................................................................... 66
5.3.2 Imaging chambers.......................................................................... 67
5.3.3 Air stream incubators ..................................................................... 68
5.3.4 Objective heaters............................................................................ 68
5.4 Choice of Microscope .......................................................................... 68
5.5 Choice of Objective ............................................................................. 69
5.6 Choice of Camera ................................................................................ 70
5.7 Selection of Cells and Axonal Regions for Imaging ................................. 71
5.8 Time-Lapse or Streaming Image Acquisition .......................................... 72
5.9 Focus Drift ......................................................................................... 73
5.10 Minimizing Photobleaching and Photodamage ....................................... 74
5.11 Methods for Spatially Selective Illumination .......................................... 74
5.11.1 Iris diaphragm method ................................................................... 75
5.11.2 DMD method ................................................................................. 75
5.11.3 Laser scanning method .................................................................. 76
5.12 Fluorescence Photobleaching Experiments ............................................ 76
5.13 Fluorescence Photoactivation Experiments ............................................ 77
6. Software and Analysis .......................................................................................... 78
6.1 Software............................................................................................. 78
6.2 Fixed-Field Tracking ............................................................................ 79
6.3 Multifield Tracking .............................................................................. 81
6.4 Kymograph Analysis ............................................................................ 82
6.5 Pulse-Escape Fluorescence Photoactivation Analysis .............................. 83
7. Summary ............................................................................................................. 86
Acknowledgments ..................................................................................................... 87
References ............................................................................................................... 87
Introduction 23
Abstract
Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling
cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their
structural role, neurofilaments are cargos of axonal transport that move along microtubule
tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10 nm
in diameter, which is well below the diffraction limit of optical microscopes, these
polymers can reach 100 mm or more in length and are often packed densely, just tens of
nanometers apart. These properties of neurofilaments present unique challenges for studies
on their movement. In this article, we describe several live-cell fluorescence imaging
strategies that we have developed to image neurofilament transport in axons of cultured
neurons on short and long timescales. Together, these methods form a powerful set of
complementary tools with which to study the axonal transport of these unique intracellular
cargos.
List of Abbreviations
DIC Differential interference contrast
DMD Digital multimirror device
DRG Dorsal root ganglion
FBS Fetal bovine serum
GFP Green fluorescent protein
HBSS Hank’s balanced salt solution
MEM Minimum essential medium
NFH Neurofilament protein H
NFL Neurofilament protein L
NFM Neurofilament protein M
NGF Nerve growth factor
NT3 Neurotrophin-3
PAGFP Photoactivatable GFP
PBS Phosphate buffered saline
SCG Superior cervical ganglion
INTRODUCTION
Neurofilaments, which are the intermediate filaments of nerve cells, are long flexible
rope-like cytoskeletal polymers composed of internexin, peripherin, and neurofila-
ment proteins L, M, and H (low, medium, and high molecular mass) (Laser-Azogui,
Kornreich, Malka-Gibor, & Beck, 2015; Perrot & Eyer, 2013). Like other interme-
diate filament proteins, these proteins all share a conserved alpha-helical central
domain, called the rod domain, that assembles to form the filament backbone,
flanked by variable amino- and carboxy-terminal domains which regulate subunit
assembly and filament interactions. However, neurofilament proteins can coassem-
ble in various combinations and stoichiometries so the precise composition of neuro-
filaments depends on which of the neurofilament proteins are expressed and on their
relative level of expression, both of which vary depending on the neuronal cell type
and stage of development.
The principal known function of neurofilaments is to expand axon caliber, which
is a major determinant of axonal conduction velocity (Hoffman, 1995). The space-
filling properties of these polymers are maximized by the carboxy-terminal domains
of the subunit proteins, particularly of neurofilament proteins M and H, which proj-
ect outward from the filament backbone. The extended and unstructured nature of
these highly charged domains forms a dense polyelectrolyte brush border that ap-
pears to define a zone of exclusion around the backbone of the polymer much
like the bristles on a bottle brush, increasing the spacing between neurofilaments
and neighboring structures (Mukhopadhyay, Kumar, & Hoh, 2004).
In addition to their structural role, neurofilaments are cargos of axonal transport
that move along microtubule tracks, propelled by microtubule motor proteins (Brown,
2000, 2009; Wang, Ho, Sun, Liem, & Brown, 2000). The observed rate of movement
depends on the time frame of observation. Radioisotopic pulse-labeling experiments
in laboratory animals on a timescale of days or weeks have revealed that neurofila-
ments are conveyed in the slow component of axonal transport at average rates of
approximately 0.3e3 mm/day. In contrast, live-cell fluorescence imaging in cultured
neurons on a timescale of seconds or minutes has revealed that neurofilaments move
at average rates of w1e3 mm/s, but these rapid bouts of movement are intermittent
and highly asynchronous. Thus, the axonal transport of neurofilaments is a “stop-
and-go” motility characterized by rapid infrequent movements interrupted by pro-
longed pauses. The velocity is slow on long timescales because the polymers spend
most of their time pausing. It is now generally assumed that other cargos of slow
axonal transport are transported in a similar stop-and-go manner, but this has yet
to be proven.
Neurofilaments are also of clinical interest because mutations in neurofilament
protein L (NFL) can cause peripheral neuropathy and because neurofilaments accu-
mulate abnormally in many different neurodegenerative diseases and toxic neurop-
athies (Lariviere & Julien, 2004). In extreme cases, the neurofilament accumulations
lead to large balloonlike swellings of the affected axons. These accumulations are
thought to be caused by an impairment of the axonal transport of these cytoskeletal
polymers, but how this occurs is poorly understood.
In spite of considerable progress over the past 15 years, there are still many unan-
swered questions regarding the mechanism of neurofilament transport. For example,
how do motors interact with neurofilaments, how do these motors coordinate bidi-
rectional motility, how is the organization of neurofilaments in axons established
and maintained, what determines when neurofilaments move and pause, what regu-
lates the accumulation of neurofilaments in developing axons, and what causes neu-
rofilaments to accumulate abnormally in axons in neurodegenerative disease?
Cultured neurons offer researchers a special opportunity to address these questions
because of their amenability to observation by high-resolution live-cell fluorescence
imaging. However, a major challenge is that these polymers measure just 10 nm in
diameter and are often packed close together, just tens of nanometers apart. Since the
1. Culturing neurons 25
diameter and spacing of these polymers are well below the optical diffraction limit
of light microscopes, the movement of single neurofilament polymers can be diffi-
cult to detect. A further challenge is that the movement is very infrequent, with sin-
gle neurofilaments capable of pausing for hours between successive bouts of
movement. In this article, we describe fluorescence imaging strategies that we
have developed to overcome these and other challenges. Each of these approaches
has its strengths and weaknesses, but taken together they form a set of powerful
and complementary tools with which to investigate the kinetics of neurofilament
transport in a variety of neuronal cell types.
1. CULTURING NEURONS
1.1 GENERAL CONSIDERATIONS
We prefer to use primary neuronal cultures, which are cultures of differentiated
nerve cells isolated from neuronal tissue. Since these neurons are postmitotic differ-
entiated cells, each batch of cultures yields a fixed number of neurons. Typically, we
establish cultures on a weekly or biweekly basis depending on our needs. For our
live-cell imaging studies of neurofilament transport we have used autonomic motor
neurons from superior cervical ganglion (SCG), sensory neurons from dorsal root
ganglion (DRG), or neurons from cerebral cortex (“cortical neurons”). We establish
these cultures from either rats or mice. We purchase timed pregnant rats or mice
from Harlan (Indianapolis, IN) or Taconic Biosciences (Cambridge City, IN). If
we have a choice we generally prefer to use rats, but in general the procedures
are identical for mice. We focus here on our own current methods. Similar or alter-
native methods for culturing these neuronal cell types, which include detailed
descriptions of the tissue dissection and culturing procedures, can be found else-
where (Goslin, Hannelore, & Banker, 1998; Hawrot & Patterson, 1979; He &
Baas, 2003; Higgins, Lein, Osterhout, & Johnson, 1991; Johnson & Argiro, 1983;
Johnson, Iacovitti, Higgins, Bunge, & Burton, 1981; Kaech & Banker, 2006;
Kleitman, Wood, & Bunge, 1998; Mahanthappa & Patterson, 1998).
To establish and maintain primary neuronal cultures, we use fine dissecting in-
struments, a binocular dissecting microscope capable of 8e25x magnification, a
horizontal laminar flow hood, and a tissue culture incubator with temperature and
CO2 control as well as active or passive humidification. The fine dissection and
all subsequent steps are performed under sterile conditions in the hood to avoid bac-
terial or fungal contamination (Freshney, 2010, 796 pp.). Hoods with a vertical flow
design are not suitable because they cannot accommodate a dissection microscope.
With the exception of myelinating cocultures (Section 1.6), we generally prefer
to plate the cells at low densities. At higher densities, the axons tend to fasciculate,
which makes axon tracing more challenging. In addition, axons in dense cultures
often do not lie directly against the glass coverslip, making it hard to keep moving
neurofilaments in focus. Higher cell densities also result in more nonneuronal cells
and more cell debris, leading to higher background fluorescence. However, since
neurons fare much better in high-density cultures, culturing them at low density
requires that extra care and attention be paid to the culture procedures and condi-
tions. We maintain the cultures in an incubator at 37 C and 95% relative humidity
in an atmosphere of either ambient or 5% CO2, depending on the culture medium.
1.2 CULTURE MEDIA

1.2.1 Media for superior cervical ganglion neuron cultures
For short-term cultures (up to 1 week or so), we use a medium based on the formu-
lation of Bray (1991) which contains Leibovitz’s L-15 culture medium (phenol
red-free) supplemented with 10% (v/v) adult rat serum, 0.6% (w/v) glucose,
2 mM L-glutamine, 0.5% (w/v) hydroxypropylmethylcellulose (MethocelÔ), and
50 ng/mL nerve growth factor (NGF; 2.5S subunit purified from mouse salivary
glands) (L-15 culture medium; Table 1). L-15 medium is designed to buffer its
pH at atmospheric CO2 so it is more convenient for observation and microinjection
of cells on the microscope stage than media with bicarbonate buffering systems. An
additional benefit is that nonneuronal cells proliferate far less in this medium than in
Table 1 Composition of SCG Neuron Culture Media. The L-15 culture medium is
maintained at atmospheric CO2 and the DMEM/F12 culture medium is maintained at
5% CO2. DMEM/F12 is a 1:1 (V/V) mixture of Dulbecco’s Modified Eagle and Ham’s
F-12 nutrient media. 100 mg/mL bovine transferrin can be substituted with 20 mg/mL
rat transferrin.
Component Source
L-15 Culture medium
Leibovitz’s L-15 medium, phenol red-free Gibco Life Technologies
10% (v/v) adult rat serum Harlan Laboratories
0.6% (w/v) D-glucose (33.3 mM) Sigma
0.29 mg/mL L-glutamine (2 mM) Sigma
0.5% (w/v) MethocelÔ (F4M premium grade) Dow Chemical Company
50 ng/mL NGF, 2.5S subunit BD Biosciences
DMEM/F12 Culture medium
DMEM/F12 medium, phenol red-free Gibco Life Technologies
100 mg/mL apo-transferrin, bovine Sigma
10 mg/mL insulin, bovine Sigma
5 ng/mL sodium selenite (30 nM) Sigma
0.2 mg/mL L-glutamine (1.4 mM) Sigma
0.5% (w/v) bovine serum albumin, fraction V EMD Millipore
0.5% (w/v) MethocelÔ (F4M premium grade) Dow Chemical Company
50 ng/mL NGF, 2.5S subunit BD Biosciences
bicarbonate-buffered media, thereby eliminating the need for the addition of antimi-
totic agents. NGF is required to support the survival of the neurons. For longer term
cultures, we have used a serum-free DMEM/F12-based medium based on the N2
formulation of Bottenstein and Sato (1979) as modified by Higgins et al. (1991)
(DMEM culture medium; Table 1). For low-density cultures, this medium can be
supplemented with 10% adult rat serum. A defined serum-free L-15-based culture
medium can also be used (Hawrot & Patterson, 1979; Mahanthappa & Patterson,
1998). With good sterile technique it is not necessary to use antibiotics, which
can have deleterious side effects on neurons.
Adult rat serum can be prepared by the method of Hawrot and Patterson (1979)
or purchased from a commercial source. We sterilize the serum by syringe filtration
with a 0.2 mm filter and store it frozen in aliquots to minimize repeated freezing and
thawing. Often the serum forms a fine precipitate after a few days at 37 C. This pre-
cipitate can be mistakenly identified as microbial contamination, but it is innocuous
and does not harm the cells. With serum from some commercial sources, the precip-
itate can become so dense that it obscures the cells. In this case, switch to a different
commercial source or prepare the serum yourself. Fetal bovine serum (FBS) can also
be used, but it is our impression that both rat and mouse SCG neurons are healthier
and attach better to the substrate in the presence of adult rat serum.
The function of the MethocelÔ is to increase the viscosity of the culture me-
dium. It is not critical, but the cells appear to attach better to the substrate when it
is present. Since MethocelÔ solutions are too viscous to sterilize by filtration, the
powder must be autoclaved. We weigh out 200 mg aliquots of MethocelÔ powder
in 50 mL disposable polypropylene centrifuge tubes and then autoclave with the
caps loosely attached. After cooling, the caps can be tightened, and the tubes can
be stored indefinitely at room temperature. To dissolve the MethocelÔ, we add
40 mL sterile L-15 and shake the tube overnight at 37 C. The resulting solution
contains insoluble particulate material, which can be removed by filtration using a
5 mm syringe filter (Millex-SV, EMD Millipore).
1.2.2 Media for cortical neuron cultures

The cortical neurons are cultured with a glial feeder layer. To expand and maintain the
glia, we use a medium consisting of Minimum Essential Medium (MEM) supple-
mented with 10% (v/v) horse serum, 0.7% (w/v) D-glucose, and 0.5 mg/mL gentamicin
(Glia MEM culture medium; Table 2). The neurons are cultured in NbActiv4Ô
medium (BrainBits LLC, Springfield, IL) with or without additional salt depending
on the desired osmolarity (NbActiv4Ô culture medium; Table 2). NbActiv4Ô is
identical to Neurobasal/B27Ô culture medium except for three additional supple-
ments: creatine, estrogen, and cholesterol. NbActiv4Ô medium has been reported
to yield hippocampal neuron cultures with more synapses, increased electrical activ-
ity, and less metabolic stress compared to Neurobasal/B27Ô medium (Brewer et al.,
2009). In our experience, we obtain improved cell health and viability using this
medium. To enhance neuronal recovery improve cell attachment and minimize the
risk of bacterial contamination arising from the initial dissection, we supplement
Table 2 Composition of Cortical Neuron and Glia Culture Media. These media are
maintained at 5% CO2.
Component Source
Glia MEM Culture medium
Minimum Essential Medium (MEM) Gibco Life Technologies
10% (v/v) horse serum Gibco Life Technologies
0.7% (w/v) D-glucose (39 mM) Sigma
5 mg/mL gentamicin Sigma
NbActiv4Ô Plating medium
NbActiv4Ô medium BrainBits
5% (v/v) fetal bovine serum (FBS) Hyclone, GE Healthcare Life Sciences
5 mg/mL gentamicin Sigma
NbActiv4Ô Culture medium
NbActiv4Ô medium BrainBits
37.5 mM NaCl (included w1 week after Sigma
plating)
the NbActiv4Ô medium with 5% (v/v) FBS and 0.5 mg/mL gentamicin for plating
(NbActiv4Ô plating medium; Table 2). On the next day, we replace it with
NbActiv4Ô culture medium (which lacks FBS and gentamicin) and then maintain
the cultures in this medium. With good sterile technique the gentamicin can be
omitted. Around 1 week after plating, we increase the osmolarity of the medium
from w230e245 mOs to w280e295 mOs by addition of NaCl to a final concentra-
tion of 37.5 mM, which we have found to improve long-term cell viability (Section
5.2). While it is possible to culture cortical neurons in this medium for at least
1 month, all of our own work has been performed within 2 weeks of plating.
1.2.3 Media for dorsal root ganglion neuron cultures

For long-term myelinating cocultures of DRG neurons and Schwann cells, we use
NbActiv4Ô myelination medium consisting of NbActiv4Ô (phenol red-free) sup-
plemented with 100 ng/mL NGF or 25 ng/mL neurotrophin-3 (NT-3; Table 3). As
with SCG neurons, a neurotrophic factor is required to support neuron survival.
NGF supports the survival of cutaneous sensory neurons, which have relatively
small axon diameters, whereas NT-3 supports the survival of the proprioceptive
sensory neurons that innervate the skeletal muscles. The NT-3-dependent neurons
contain more neurofilaments, have larger axons, and myelinate more readily and
more continuously in culture. Five days after plating the cells, the medium is
replaced with fresh medium containing a one-time supplement of MatrigelÔ at a
1:100 dilution (BD Biosciences). Three days later, half the medium is removed
and replaced with fresh medium lacking MatrigelÔ and containing 50 mg/mL
Table 3 Composition of Culture Medium for Myelinating DRG Cultures. This

medium is maintained at 5% CO2. Note that to prevent gelation and clumping, the
MatrigelÔ should be thawed on a slurry of ice and water and then diluted into ice-cold
NbActiv4Ô medium. After mixing, the medium can then be warmed and the other
components can be added.
NbActiv4Ô Myelination Medium
Component Source
NbActiv4Ô medium, phenol red-free BrainBits
Either 100 ng/mL NGF, 2.5S subunit Or 25 ng/mL NT-3 BD Biosciences (NGF)
PeproTech (NT-3)
1% (v/v) MatrigelÔ, phenol red-free (one time application BD Biosciences
w5 days after plating)
50 mg/mL ascorbic acid (initiated w8 days after plating) Sigma
ascorbic acid. Ascorbic acid is an essential cofactor in collagen biosynthesis, which

is required for Schwann cells to form a basal lamina, which is in turn required for
efficient myelination (Eldridge, Bunge, & Bunge, 1989; Eldridge, Bunge, Bunge,
& Wood, 1987). The cultures can be maintained for up to 3 months, with a half-
medium change every 2e3 days, and a full-medium change once each week. For
short-term nonmyelinating cultures, ascorbic acid can be omitted or the cultures
can be maintained in the L-15 culture medium described above for SCG neurons
(Alami, Jung, & Brown, 2009) (Table 1).
1.3 COVERSLIPS AND CULTURE DISHES

Since the highest quality microscope objectives have large numerical apertures and
short working distances, it is preferable to perform live-cell imaging using an
inverted microscope, i.e., one in which the objective is positioned beneath the cul-
ture dish focusing up on the cells through the bottom of the dish. For high resolution
imaging, it is necessary to culture the cells on glass coverslips that match the optical
correction of high numerical aperture microscope objectives. Usually these objec-
tives are corrected for imaging through a thickness of glass equal to 170 mm, so
the optimal coverslips are ones designated #1.5, which corresponds to a thickness
of 0.16e0.19 mm.
We routinely acid-wash all coverslips for cell culture regardless of whether they
were acid-washed by the manufacturer. To do this, we soak the coverslips in concen-
trated nitric acid for at least 18 h and then rinse extensively with deionized water
over a period of several days. To dry the coverslips, we dip them in ethanol and
then flame them.
To culture the cells on coverslips, we use one of two approaches. The simplest is
to culture the cells in glass-bottomed plastic Petri dishes so that the cells can be
imaged in the same dish in which they were cultured. Alternatively, we culture
the cells on loose coverslips in plastic Petri dishes and then transfer the coverslips
to an imaging chamber prior to live-cell imaging. Each of these strategies has pros
and cons, which will be discussed later (Section 5.3).
For glass-bottomed dishes, we use 22 22 mm #1.5 square coverslips (Fish-
erfinestÔ premium brand). When culturing cells on loose coverslips, the shape
and dimensions of the coverslip that is required depend on the shape and dimensions
of the imaging chamber that will be used (40 mm #1.5 round coverslips for the Bio-
ptechs FCS2 chamber, 22 22 mm #1.5 square coverslips for the Warner Instru-
ments RC-21B or RC-30HV chambers; Section 5.3).
1.3.1 Glass-bottomed dishes

Glass-bottomed dishes are plastic Petri dishes with a glass coverslip base (Bray,
1991) (Figure 1). The cells are plated and cultured on the coverslip and can be
imaged by placing the dish on the stage of an inverted microscope. There are
many commercially available options (e.g., MatTek Corporation, Ashland, MA),
but they are expensive so we make our own.
Materials required for drilling the holes:
• 35 mm plastic Petri dishes. We use NuncÔ brand tissue culture dishes with
vented lids (Nalge Nunc International, Rochester, NY), but any similar dish
should be suitable.
• A drill press with a depth stop.
• A 1/800 e1/200 step drill bit with 1/3200 step increments (McMaster-Carr, Elm-
hurst, IL).
• Though not essential, it is helpful to have a jig to hold the dish so that the holes
are centered. We use a specially built jig milled out of aluminum to hold the dish
in position on the drill press.
• A source of clean compressed air to remove the plastic shards from the drill bit,
jig, and dishes.
FIGURE 1
Drawing of a glass-bottomed culture dish. A hole is drilled in the bottom of a 35 mm plastic
Petri dish, and a glass coverslip is affixed to the base of the dish using wax or silicone
adhesive. The cells are plated onto the coverslip in the shallow well created by the hole in the
bottom of the dish.
Drilling the holes:

1. Mount the drill bit in the drill press and the custom jig on the drill press table.
2. Adjust the depth stop to ensure a 13/3200 hole in the dishes.
3. Remove the lids from the dishes and store for later use.
4. Place a dish in the jig and hold it firmly against the base to prevent it from
rotating
5. Slowly lower the spinning drill bit down onto the dish to cut the hole.
6. Remove the dish and blow away any plastic shards using a source of clean
compressed air.
7. Repeat for each dish. We usually drill a batch of 500 dishes at a time, which takes
about 8 h.
Notes:
• Handle the drill bit and dishes throughout with gloves to keep them clean.
• The drill bit should be cleaned thoroughly prior to use because it contacts the
culture dish. We use hot soapy water and then rinse with water and alcohol.
• In step 5, it is important to use slow and steady pressure and a sharp drill bit.
Support the dish from underneath to avoid cracking the plastic. The jig facili-
tates this. The dish may crack during the initial penetration of the drill bit but
this does not matter as long as the cracks do not extend beyond the final
diameter of the hole. When the drill bit becomes dull, it should be replaced.
• With a different jig, holes as large as 1/200 can be drilled.
• In step 6, the drill often leaves a slight burr, which must be removed by gently
scraping around the holes with a scalpel blade in order to allow coverslips to be
attached flush against the plastic (see below).
We attach the coverslips to the dishes using either paraffin wax or a platinum-
cured medical-grade silicone adhesive (A-103 Medical Grade Elastomer, Factor II
Inc., Lakeside, AZ). The advantage of paraffin wax is that it is easy to remove the
coverslips later using a razor or scalpel blade. This is helpful after fixing and immu-
nostaining the cells because it allows the coverslip to be mounted on a glass slide
using hardening mounting medium. However, a disadvantage of using paraffin is
that it softens at 37 C and is not a strong adhesive so sometimes the coverslip
can detach.
Materials required for attaching coverslips using paraffin wax:
• A microelectric hot plate (we use a Thermolyne Aluminum-Top Micro Hot Plate;
Thermo Fisher Scientific, Waltham, MA)
• Retort stand and large retort stand hose clamp.
• Round-tipped artist’s paint brush for applying the molten wax.
Attaching the coverslips using paraffin wax:
1. Invert the hot plate and mount it on the retort stand using the large retort stand
hose clamp.
2. Invert the dishes on a clean work surface so that the bases with the holes drilled in
them are facing upward
3. Using the paint brush, apply a ring of hot molten paraffin wax in a circle around
the hole on each dish, taking care to keep the wax at least a few millimeters
away from the edge of the hole.
4. After the paraffin has cooled, use forceps to place a clean 22 22 mm square
#1.5 acid-washed glass coverslip on the base of each inverted dish so as to cover
the hole and the wax surrounding it.
5. Raise the inverted dish with the coverslip laying on it to bring it close to the
inverted hot plate and hold it in place until the wax melts and spreads out to fill
the space between the coverslip and the base of the inverted plastic dish.
6. Move the dish away from the hot plate and cool it in a gentle stream of air to
solidify the wax before it flows into the well.
7. Repeat for all the dishes.
Notes:
• In step 3, be careful not to apply the paraffin too close to the hole or to use too
much paraffin. Insufficient paraffin may result in gaps or bubbles between the
coverslip and the base of the dish. Too much paraffin may result in molten wax
flowing into the hole or over the edge of the dish in step 5, or may result in the
coverslip not laying evenly.
• In step 5, it is best to avoid melting the paraffin too quickly. For best results, set
the temperature of the hot plate to 60 C.
• After step 6, incubating the dishes overnight at 37 C before storing them at room
temperature appears to improve the seal and reduce the likelihood of future
leaks.
• Getting steps 3, 5, and 6 just right takes some trial and error but with practice the
procedure becomes routine.
• The dishes can be stored indefinitely before use.
To prepare dishes using silicone adhesive, mix the silicone elastomer and cross-
linker according to the manufacturer’s instructions and then dispense onto the bases
of the inverted dishes using a syringe with a narrow plastic tip. Apply the coverslips
using gentle pressure, and then cure for 24 h at room temperature or 5 h at 40 C.
The adhesive is transparent.
1.3.2 Sterilizing and coating coverslips and glass-bottomed dishes

Prior to plating neurons it is necessary to coat the coverslips, whether loose or in
glass-bottomed dishes, to promote cell adhesion and axon outgrowth. For cortical
neuron cultures, we coat with poly-D-lysine. For DRG and SCG neurons, we coat
first with poly-D-lysine and then with extracellular matrix proteins. We like to use
MatrigelÔ (BD Biosciences), which is a heterogenous basement membrane extract
containing laminin, Type IV collagen, entactin, and heparan sulfate proteoglycans
secreted by Engelbreth-Holm-Swarm mouse tumor cells. It is an excellent substrate
for SCG and DRG neurons, but it tends to form sticky clumps that can increase back-
ground staining in immunofluorescence applications. If this problem is encountered,
laminin is a good substitute. Both MatrigelÔ and laminin are stored in aliquots at
80 C. To prevent gelation, they are thawed on a slurry of ice and water on or
before the day of the dissection, and diluted into ice-cold L-15 medium. After dilu-
tion, the L-15 is allowed to warm to room temperature.
Procedure for sterilizing and coating glass-bottomed dishes:
1. On the day before the dissection, place the required number of glass-bottomed
culture dishes and lids in a laminar flow hood and sterilize them by filling them
to the brim with 70% (v/v) ethanol and letting them stand for 45 min.
2. Aspirate the ethanol and allow the dishes and lids to dry.
3. Treat the coverslips with 1 mg/mL poly-D-lysine hydrobromide (Mw 70e150,000,
Sigma) in 0.1 M sodium borate buffer, pH 8.5, for 3 h (150 mL per well) as
described by Higgins et al. (1991).
4. After the poly-D-lysine treatment, rinse the coverslips six times with sterile
water (w5 min per rinse) to remove poly-D-lysine that is not bound to the
glass.
5. For SCG and DRG neuron cultures and for glial cultures, add a few milliliters
of sterile water to each dish, cover with a lid, and store overnight at room
temperature. For cortical neuron cultures, use NbActiv4Ô plating medium
(Table 2) and store overnight in an incubator with 5% CO2.
6. For SCG neuron cultures, on the morning of the dissection, treat the poly-D-
lysine coated coverslips with 10 mg/mL phenol red-free MatrigelÔ or 10 mg/mL
mouse laminin (BD Biosciences) in L-15. We apply 150 mL of MatrigelÔ or
laminin solution to each coverslip well and incubate for 4 h in the incubator
(37 C, 95% relative humidity, atmospheric CO2).
7. For myelinating DRG cultures, treat the poly-D-lysine coated coverslips with
MatrigelÔ diluted to w50 mg/mL in L-15 medium and leave the lids off the
dishes in the hood until the MatrigelÔ has dried (4e5 h). The dried dishes can
be stored at 4 C for up to 2 months, so the dishes can be prepared well in
advance of the dissection if required.
Notes:
• For step 3, we make up a 10 mg/mL stock solution poly-D-lysine in borate buffer
and freeze it in aliquots. Prior to use, the aliquots are diluted in borate buffer and
sterilized by syringe filtration with a 0.2 mm filter.
• In step 5, the coverslips can be rinsed three times and then stored in the fourth
rinse for up to 1 week. Rinse them an additional three times immediately before
use.
• In step 6, the dissection is performed during the 4-h incubation period and then
the coverslips are rinsed once with L-15 before plating.
• In step 7, drying the MatrigelÔ reduces the chance that it peels off the coverslip,
which sometimes happens after several weeks in culture.
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painted in alternate bands of black and white, occasionally red and
white, and resembles a barber’s pole more than anything. The
“Djundagalla” stands in the centre of the cleared space and the rites
are performed around it. In the northern Kimberleys, we find a stone
phallus taking the place of the pole.
It is not every tribe that submits its young men to these mutilations
at the initiation ceremonies. There are some which institute great
graduation-festivals without the infliction of bodily harm to the virile
aspirants. Notably among these are the Larrekiya, Melville Islanders,
and the tribes living along the coast from the King River to the heads
of the Roper and East Alligator Rivers.
As an illustration of a tribe which celebrates the coming of
manhood without resorting to operative measures, the Larrekiya
perhaps serve best. The boy, when definite signs of adolescence
manifest themselves, is decorated with the kapok of the silk cotton-
tree (Bombatt malabaricum) and birds’ down. A straight band passes
below his eyes from ear, and the ends thereof are connected by
means of a horseshoe-shaped figure traversing the cheeks and
having its closed end at the chin. Another horizontal band extends
from shoulder to shoulder, above the nipples, and from this two
symmetrical lines are constructed down the abdomen and on to the
thighs, where each terminates in a circular band around the knee. A
white line is also drawn down the outer surface of each upper arm
and is made to end in the plaited armlets worn above the elbow. His
forehead is decorated with a broad band consisting of a number of
parallel strands of opossum fur thickly besmeared with white
pipeclay; in the middle of this is stuck a plume of emu or heron
feathers, and fur-tassels pend from either side of it. He also wears a
coiled bark belt and, over it, a human hair girdle supporting a large
pubic tassel.
The initiates are made to sit in a row before the old men and are
instructed to keep their eyes closed with their hands. The old men
stamp the ground wildly and brandish their spears poised in the
spear-throwers. Every now and then they utter harsh cries of “Arr-re!
Arr-re!” and “Gora!” Whilst this pandemonium is in full swing, the
boys are ordered to open their eyes and behold their elders
performing; then they are led away into the bush and have to wait on
the men, having especially to collect for them many things that are
good to eat. During this period they are often cowed by being struck
between the shoulder-blades, and threatened with violence if at any
time they talk publicly about anything that has transpired or in any
way betray the trust which the old men have placed in them. Upon
their return to camp, the young men have additional scars cut into
the skin of their chest and are then entitled “Böllier” which signifies
that the first stepping stone to maturity has been passed.
A second ceremony takes place some years later. Each youth is
then under the individual charge of an old man and is decorated
much the same way as the Böllier candidate described above, with
the distinguishing features of four red ochre stripes across the white
forehead band and an extra plume of white cockatoo feathers stuck
into his hair. The proceedings start soon after sundown and last till
about midnight; they include much gesticulation and vociferation. At
the solemn moment when the “conferring” of the maturity-degree
takes place, the youth, still tended by the old man, remains
motionless, with downcast eyes, and listens to the melancholy chant
rendered by the old men in low lagging accents:
“Makolär manga, malolär, ä, är, maklär, immanga.”
No beating of sticks or clapping of hands accompanies this tune,
and no further ceremonial dance follows.
The youth has now been elevated to the status of “Mollinya” which
qualifies him to the full rank and privileges of manhood. Further
cicatrices may now be added to either side of his abdomen. The cuts
are horizontal but do not extend right up to the median line.
During the period intervening between Böllier and Mollinya
festivals, bustard, flying-fox, and yam are forbidden articles of diet,
but after the latter event the fledgelings are invited to eat with the old
men. They honestly believe that if any of the young men, while
undergoing initiation, ate one of the forbidden articles secretly, the
medicine man would be able to detect the food in his stomach; and
having thus disobeyed, the medicine man would be justified in
running a spear through the offender, or at any rate compel him to
swallow certain things which would poison him. These rules are
strictly observed, and, whenever some of the privileged members
have eaten flying-fox or bustard, they take the precaution to collect
the bones and burn them.
The tribes on Nullarbor Plains will tell you that the initiation
ceremonies originated in the following way. Many, many years ago,
the emu and the kangaroo were more or less human in appearance
and possessed of mighty powers. One day the emu caught the
kangaroo with the object of making a man of it. But the great
struthious bird had no hands wherewith it might have performed an
operation; all it possessed was a “finger” on each side of its body. It
might be explained that the emu, because it cannot fly, is not
regarded as a “bird” in the generally recognized sense, and
consequently the wings are looked upon as “fingers.” In most of the
vocabularies, indeed, no distinction is made between “finger” and
“hand,” the south-western tribes of central Australia referring to one
or the other as “marra.” Nothing daunted, however, the emu removed
the præputium from the kangaroo by clutching it between its wings
and pulling it off. Thereupon the emu said to the kangaroo: “Will you
make me a man?” And the kangaroo replied, “Yes.” The kangaroo
had the advantage over the emu because it possessed five “fingers,”
with which it could perform the operation the right way. The animal
caught hold of the bird and circumcised it with a sharp splinter of
flint. But the emu requested to be further operated upon and so it
came about that the kangaroo decided upon a subincision. To the
present day the emu retains the marks of this operation. Some while
after these happenings, the tribal fathers ran across the sacred emu
and noted the change in its anatomy; they forthwith mutilated each
other in a similar way, and only then did they realize that they were
men.
Not boys alone are required to submit to the various initiation
ceremonies here mentioned, but in most tribes young women are
“made” marriageable by having to submit themselves to ordeals
which are quite similar to those of manhood’s approbation.
While discussing the female breast, we noted that when it begins
to develop a girl is taken away by the men and the breast anointed
and sung to, to stimulate its growth. This procedure is the forerunner
of initiation. The girl’s development is forthwith watched with care,
and when the unmistakable signs of ripening are detected the event
is celebrated with dance and song.
Men and women attend, and the items rendered are more or less
of the nature of an ordinary corrobboree, although occasionally some
special feature characterizes the performance. For instance the
Larrekiya and Wogait tribes pass the girl through a “smoking”
ceremony after the following fashion. An old gin places herself
behind the girl and lays her hands upon the latter’s shoulders. Then
all the other women taking part form a continuous chain by standing
in a single row behind each other and “linking up” in a similar way.
They begin to sing “Ya, Ya, Ya,” in a long-drawn melancholy note,
and the old-gin immediately stamps her feet, and, moving forwards,
pushes the girl along in front of her. All the other performers follow
her, stamping in unison and holding on to the shoulders of the
person in front. Quite unexpectedly the monosyllabic “Ya” is changed
to “Yen da min,” and at this the old gin stops short and strikes the
girl’s back thrice with her hand. The same performance is repeated
time after time during the night. Early in the morning of the next day,
the girl is led to the sea, and the whole party wades out to about
hips’ depth. Here a grotesque dance is started during which they
strike their arms, bent in the elbows, against the sides of their bodies
under water, the splash producing a peculiar hollow-sounding note.
The process reminds one of a goose flapping its wings while
enjoying a bath. At this stage, the wording of the song sounds like
“A-lö-lö-lö,” and when its final syllable has resounded, all bathers
duck under the surface of the water.
Next a fire is kindled upon the shore, and, when a good blaze has
been obtained, a heap of grass and leaves previously steeped in
water, is piled upon it. Upon this the old gin seats herself and makes
the girl sit upon her lap facing her and with her legs astride. The
volumes of smoke which are generated completely hide the two from
view. The idea is to allow the smoke to thoroughly play upon the
parts of the novice, the process being facilitated by the manipulation
of the old gin. When the ceremony is concluded, the girl is led into
the bush by the old women and for some time to follow she is not
allowed to partake of certain articles of diet, such as for instance
snake, dugong, and goanna.
Several of the northern and north-eastern coastal tribes mutilate
the hand of a young gin during the period of her initiation by
removing two joints from a finger. The forefinger of either hand is
generally chosen by the former tribes, the latter favouring the small
finger. The Ginmu at the mouth of the Victoria River make the
amputation with a stone knife. In this district a singular case came
under my notice which is of considerable interest from an evolutional
point of view since it suggests a phenomenon usually only met with
in crustations, reptiles, and other creatures whose position is very
much lower in the animal kingdom. A young girl had had two end
phalanges of a finger imperfectly removed, and yet upon the
mutilated stump a horny growth resembling a diminutive finger-nail
had formed anew. The Daly River tribes remove the bones by tying a
ligature of cobweb which they find in the mangroves very tightly
around the joint. The end phalanges of the finger, thus deprived of
the circulation, gradually mortify and drop off. Occasionally the joints
may be bitten off by a parent of the child.
As a general rule, it may be said that wherever mutilations of the
male are undertaken during initiation ceremonies, a corresponding
operation is performed upon the female; and, vice versa, where the
former practice is not indulged in, the latter is also unknown.
Generally speaking, too, the female mutilation ceremonies are much
the same wherever practised in Australia, but the implements or
devices employed for the actual mutilation vary in different localities.
Invitations to the event are sent by special messengers to
adjoining groups and neighbouring friendly tribes. These
messengers are of mixed sexes and are decorated by having their
bodies covered with ochre. The common method is to make the
ground colour of the body a rich red and to draw upon it concentric
circles of white and black. The men carry a “female” tjuringa, whilst
the women, apart from numerous necklaces and armlets which they
wear, are unaccoutred. The latter are near group-relatives of the
young woman concerned. Their mission is readily understood by the
people they look up during their walk-about, and, without much
interchange of words, acceptance is indicated by the recipients of
the message by resorting to an intimacy with the feminine
emissaries. Although considerable liberality is shown during this
indulgence, the privilege is by no means stretched to beyond the
bounds of a tolerable promiscuousness, even though the
messengers may be entertained at the distant camp for two or three
days before they return home.
The celebrating camp in the interim has been busily preparing for
the approaching event. Nightly corrobborees have been held at the
chosen spot by both the men and the women, and the novice has
repeatedly appeared before the performing crowd richly decorated
and besmeared with emu-fat and ochre. At no time, however, even
after the invited guests have arrived, does the excitement become
anywhere near as great as during the initiation ceremonies of the
opposite sex; in fact, at its best, the performance is extremely dull
and monotonous.
When at length it becomes apparent that even the principal actors
themselves are tiring, it seems as though the moment had arrived
when only a desperate decision could revive the enthusiasm. A
number of men, who stand in the same group-relationship to the
novice as her future husband, lead the girl away without any ado,
except perhaps that the remaining members slightly spur their
acting. This stage is mostly reached at daylight, as often as not early
in the morning, after the whole night has been spent in dancing and
singing.
Away from the din of her tribespeople’s celebration in honour of
the occasion of her stepping from girlhood to womanhood, the silent
victim is told to squat on the ground whilst the men surround her. Her
oldest “group-husband” produces a flat, wooden tjuringa, of the
“male” type, with which he several times touches her person, whilst
he mutters incoherent and garbled words. This is done to dispel from
her all possible pain and likely loss of blood during the operation she
is about to be submitted to.
Then she is requested to lie flat on her back, and her head is
placed upon the lap of one of the men who squats to keep it there. It
follows the act which is destined to make her marriageable; her
virginity is doomed to mechanical destruction.
The instruments, if any, which are used for the operation vary
according to locality. In the central areas (Aluridja, Wongapitcha,
Kukata), an ordinary stone-knife with resin haft is used. The Victoria
desert tribes employ cylindro-conical stones from six to eight inches
long, and from one and a half to two inches in diameter. Among the
tribes of the northern Kimberley districts of Western Australia no real
instrument is used at all, but the operator winds the index and middle
fingers of his right hand together with a long piece of fur-string; and
this device answers the same purpose as the above-named
instruments.
The tribes indulging in this practice admit that their action is
prompted by a desire to offer the girl’s pudicity to one of her spirit-
husbands. We might indeed look upon this rite as the equivalent of
sacrificing the jus primae noctis to a mythical or legendary tribal
relative who is supposed to be living in the astral form and who is
likely to come back to earth at any day.
PLATE XXXII
An episode of the great fire ceremony, Kolaia tribe.
“Presently the music starts again, and the spirit known as ‘Ngardaddi’ is seen to be
stealthily creeping towards the fire, his body lying flat upon the ground and his legs
dragging behind.”
CHAPTER XXVII
RELIGIOUS IDEAS
Religious instincts of aboriginal—Nature worship—Fire ceremony—Fire legends—

Mythical fire thief called “Ngardaddi”—Water legends and ceremonial—Sun
worship—Sun myths—The moon man—The mythical serpent—The kobong
and totem—The tjuringa—Tjuringa legend—Ancestor worship—“Knaninja” or
“Totem” deities—The significance of the tjuringa—Sacred tjuringa caves
—“Totemic” diet restrictions—Gradation of sacred ceremonial—Great emu
ceremony—The “Altjerringa”—The sacred yam or “Ladjia” ceremony—The
“Etominja” design—Sex worship—The phallus—Mythical origin of phallus—
Ideas concerning procreation—Grey hairs blackened artificially—A phallic
monolith known as “Knurriga Tjilba Purra”—Foetal elements or “Rattappa”—
The “Tjilba Purra” embodied in the headgear—“Waraka,” a phallic stone on
the Roper River—Similar Kukata legend—Phallic ceremonial on Cambridge
Gulf—Cylindro-conical stones of phallic significance—Matronal chasm of
Killalpaninna—“Arrolmolba,” a sacred stone possessing stimulating principles
—Phallic drawing of “Mongarrapungja”—Evil spirits—Disenchanted
enclosures—Aboriginal belief in Supreme Being—Etymology of His name—
The eternal home of all deities and spirit ancestors.
It has often been written that the Australian aboriginal is without

religious ideas and without religious ceremonies. Such assertions
are grossly incorrect and by no means portray the psychological side
of the primitive man in its true light. He has, on the contrary, religious
institutions and obligations which verge on the basis of all modern
conceptions and recognition of divine supremacy. If we can class
Nature-worship, Ancestor-worship, and Sex-worship as the
beginnings of all religious teachings, then the Australian aboriginal
has certainly inherited by instinct and tradition a very solid foundation
from which we might trace the origin of many, if not most, of our most
sacred beliefs in Christianity. At the same time, it must not be
forgotten that it is really a difficult matter to distinguish clearly
between mythological beliefs and what we class as religion.
Religious thought has fluctuated with the advance of civilization and
science to such a degree that, even within the short space of time
covered by the more reliable records of our history, several
revolutionary modifications have come about. As time advances,
man becomes more sceptical and more exacting in his demand for
proofs, and in his despair over finding nothing tangible to worship, he
resorts to the recognition, by instinct or persuasion, of a God who is
a Spirit. But all the while, as this secular metamorphosis is
proceeding, he keeps his innermost feelings and faith alive by
appealing to his knowledge of the gospel or his belief in salvation, in
the manner it was presented to him by myth, by legend, or by the
Scriptures. His principal guide is his intellect; the less it is trained the
stronger his inherited conviction; the more scientific it becomes, the
greater his desire to probe the truth.
The modern man has so accustomed himself to an artificial
environment that he takes the so-called “elements” of Nature,
especially water and fire, in a strictly matter-of-fact sort of way. But
the primitive man, who realizes that his very existence is dependent
upon these factors, has learned to respect, preserve, and worship
them as legacies he imagines to have been left him by some of his
illustrious forbears who, he supposes, have gone to an unknown
realm where they live in peace and can only return temporarily to
their former haunts in the invisible form or through the medium of
some other object which is related to the individual in some
mysterious way.
The aboriginal looks upon fire as one of the great indispensible
quantities of his social existence; it is the element which dispels the
evil spirits from his camp; it is the means by which comfort and
friendship are made accessible to him; it is his universal companion.
More than this, it is the fire, with its warmth and its light, which draws
individuals, families, groups, and tribes together and through its
agency and influence that social concourse is established which lies
at the bottom of all conviviality, oracular discussion, and ceremony.
How well this sentiment agrees with the knowledge we possess of
the origin of civilization! Indeed the appreciation of fire together with
the knowledge of its preservation is perhaps the mightiest factor
responsible for making our species human. Once man learned to
nurse an original flame he found through accidental cause and kept
it constantly by his side, his progress became an established fact.
His crude camp-fire talks developed into discussions which he
further expanded by means of drawings on the walls of caves he
occupied. The free exchange of thought brought about by
congregation round the cheerful flame could not fail to incite the
intellect; and thus he ascended to the high road of civilization and
gathered the fruits of culture he now enjoys.
The Aluridja, Wongapitcha, and some of the north-western coastal
tribes believe that many years ago, a party of ancestral creatures,
more animal than human, came down from the sky through the
branches of tall gum-trees to confer with the spirits which roam about
at night and conceal themselves in inanimate objects during the day.
These monsters brought a fire-stick with them and when they
reached the earth, they lit a fire to cook some grubs which they had
taken from the bark of the trees during their descent. As they were
feasting, the spirits called them and they went with them to a cave
where the bones of the persons rested, originally occupied by the
spirits themselves. Whilst they were away, the fire which had been
left unguarded, decided to run into the bush and, being in a
mischievous mood, started an enormous blaze which burned down
much of the forest and the tall gum-trees as well. The spirit-
ancestors and the heavenly monsters beheld the disaster with
consternation and called upon the fire to come back. This it did. But
it so happened that some of the tribes’ fathers were hunting in the
area, and when they saw the fire, which was strange to them, they
snatched portion of it away and ran with it to their camp, where they
kept it and fed it with dry grass and sticks. The spirits and their
visitors were very angry and never left the fire out of their sight, lest it
might abscond again; they were compelled to live on earth for a very
long time until the trees grew up again to their lofty domain. The
hunters, on the other hand, zealously guarded their prize fearing that
it might run away from them. Even to the present day, this belief
exists among the older folks, and they always take great care that
the ground is cleared of inflammable matter to stop the fire from
bolting; to be on the safe side, they invariably carry or keep near to
them a fair-sized, glowing fire-stick.
Among the Minning this legend is circulated in a slightly modified
form. Two ancestral spirits had their fires burning in the sky at points
represented by the pointers of the Southern Cross constellation,
when one day they decided to come down to the earth to hunt
opossum. They took their fires with them, but while engaged in the
chase they left them at their camp. When they had obtained a
sufficient number of opossums to make a good meal, they returned
to their camp, where they noticed six young men sitting around the
fires, who immediately made off, and, in doing so, each took a fire-
stick away with him. The spirits gave chase and re-captured five of
the thieves, but the sixth, who was named “Warrupu,” reached the
camp of his tribe and handed the fire-stick to his mother, “Wenoinn.”
The woman ran with it to the white sand hills about Eucla in which
she intended hiding it. But the spirits had noticed her and came
towards her from above with a spear. In her predicament, the woman
threw the fire-stick away, which immediately set the whole of the
country ablaze between Eucla and Israelite Bay. All the tribes were
thus enabled to seize some of the fire which they have carefully
watched over ever since.
PLATE XXXIII
Ceremonial venesection, Arunndta tribe.
1. The median basilic vein is being slit. Note ligature above the biceps.
2. The blood which is spurting from the incision is being collected on a shield.
A similar tradition is perpetuated by the north-western tribes

referred to and affords the motive of one of the most earnest and
sacred fire-ceremonies known in Australia. The performance takes
place during the night. It is introduced by two men; the one
represents a mischievous spirit trying to steal back the sacred fire
which is being carefully guarded by a number of men impersonating
the ancestral tribesmen who originally discovered it; the other is a
warrior who has accidentally come upon the would-be thief and
overpowered him. The spirit crouches at the feet of the warrior,
sitting upon his heels, with his head drooping upon his chest and his
hands hanging loosely between his thighs. The warrior stands erect
behind his supposed captive, with his legs apart, and continues
striking the fellow with small bundles of brushwood, one of which he
holds in either hand. The beating is done regularly, both hands rising
simultaneously, high above the warrior’s head, and falling together
upon the spirit’s head.
Some two chains away, the tribal ancestors are grouped by the
fire-side and are chanting the following lines:
“Wai dang bunnai,

Inna dinna dulla ngai.”
The men sit in a row at the back of the fire, with their thighs asunder
and their legs bent in the knees; their chins are resting upon their
chests whilst they beat the backs of their heads with small bundles of
brushwood, keeping time with their song and with the performance of
the warrior.
When, after a while, the music ceases, the warrior is seen to be
lying asleep beside his captive. The ancestors become restless and
begin to move sideways, first in a body to the left and then to the
right; then they move backwards and forwards. This movement is
peculiarly weird since the performers do it by shuffling over the
ground in the sitting posture, with their arms held erect, but bent in
the elbow.
Presently the music starts again, and the spirit known as
“Ngardaddi” is seen to be stealthily creeping towards the fire, his
body lying flat upon the ground and his legs dragging behind. He
advances very slowly, turning his face towards the ground, in search
of the fire which escaped from heaven. He wears a tall head-dress
quite thirty-two inches long, which consists of a tightly fitting
hemispherical cap carrying a column in its centre, at the top of which
a bundle of split black-cockatoo feathers is attached. The feathers
are from the male bird’s tail, and the brilliant red patches in them are
representative of fire. The whole structure is made of paper-bark and
human hair-string, the outer surface being decorated with ochre,
pipeclay, charcoal, and vegetable-down. Vide Plate XXXII.
All the time the men at the fire-side are beating time with their
hands and simultaneously turn their heads from side to side, to all
intents and purposes quite unconcerned about the Ngardaddi who is
gradually crawling near to them. This is done to entice the thief
nearer and lead him to believe that he is unobserved. All of a
sudden, however, when the spirit is about to touch the fire and is in
the act of snatching it from the tribesmen, one of the group on either
side of the fire throws a handful of dry grass upon the smouldering
heap. The flame responds immediately and casts a bright light all
around.
Alarm is raised by the tribesmen by clapping their hands together
violently. The spirit collapses and lies flat upon the ground at full
length. Two or three of the men nearest by seize some of the burning
grass and hit the prostrate figure over the head. The spirit jumps to
his feet and treads the ground as if endeavouring to make his
escape. Seeing this, the men at the fire rise quickly and treat their
victim most unmercifully with bundles of burning grass and twigs.
Eventually each of them seizes a fire-brand and digs the burning end
deeply into the spirit’s back and the unfortunate fellow eventually
decamps into the darkness amidst the bellowing whoops of his
victors.
The air is fouled for some distance around by the smell of the
burned skin, reminding one of the stench in a smithy when horses
are being shod. The back of the spirit-impersonator is naturally
severely scored by the cruel treatment it is subjected to, but the
fellow takes it all in good faith and without flinching.
The object of the ceremony is twofold. Firstly all members of the
community who are present, men, women, and children, are taught
to appreciate the value of fire, and secondly it is believed that the
exemplification of so harsh and drastic a treatment for attempted
theft will tend to make abortive any schemes of the evil spirits.
The Arunndta are quite convinced in their own minds that in the
days of their tribal fathers there was no water on the surface of the
ground they occupied; their ancestors in those times were compelled
to live on grass and succulent plants, no consideration being given to
the fact, as we have learned, that the vegetation derives its moisture
from outside sources. But it happened one day, when their
forefathers were out hunting, that they met with a number of strange-
looking men who were sitting around a pool of pure water from which
they were drinking. At the sight of the men, the strangers fled,
leaving the water behind. The hunters gave chase but all except one
disappeared and he made for a cave in the hills. The hunters closed
the mouth of the cave with a big stone and went back to the pool of
water to quench their thirst, but when they reached the spot, the
water had turned into a massive, round stone. The men made back
to the cave and removed the obstruction, but imagine their surprise
when they found the cave empty. Upon making a careful search,
however, they discovered a long cylindrical stick which had some
peculiar markings on it. They took the stick and walked once more
towards the petrified pool, and, lo, they beheld the stranger they
were looking for walking in the sky. When he saw the stick in the
hands of the hunters, he took the form of a cloud, and as he bent his
body towards the stick, his long matted hair fell forwards and from it
water poured upon the earth beneath. The hunters drank freely of
the precious fluid and when they looked skywards again the
cloudman had vanished.
From that day onwards the Arunndta medicine men (“Nangarri”)
have kept that spot sacred and taboo to the women and children;
they call the big stone “Imbodna” which means “the hailstone.” The
man who fled to the cave and then escaped from the hunters as a
vapour they call “Nangali,” the name for a cloud. The tribe has never
since been without water because Nangali left his magic wand in the
hands of their ancient sorcerers and whenever the country was
suffering from drought they could call upon him to appear in the sky
and bring forth rain.
Nangali is one of a group of celestial beings who have been
termed “Atoakwatje,” that is Water-Men; they are now looked upon
as Demigods who control all terrestrial supplies of water from their
abode in the clouds. The Atoakwatje are believed to have certain
mysterious connections with some of the tribal sorcerers who in a
sense parade on earth as their disciples and attend to the rain-
making ceremonies through which they are able to commune with
each other.
When the people are in need of water, the rain-makers assemble
around the Imbodna and one or two of them produce the sacred
stick, known to the Arunndta as “kwatje-purra,” literally meaning “the
reproductive organ of water,” and to the Aluridja as “kapi-wiyinna.”
Nowadays these sticks, which strictly speaking are of phallic
significance, are flat and more like a tjuringa in shape, and have a
number of peculiar markings on them. For a time the stick is laid
beside the great water-stone, and the sorcerers kneel while they
chant with a barely audible voice. They rise to their feet and the most
influential individual who is decorated with stripes of yellow
vegetable-down and wears a dog-tail tassel on his belt, lifts the stick
towards the sky and continues mumbling. The other members kneel
again and all present chat together. The man who is standing poises
the stick horizontally between his hands and rocks it one way, then
another; and this performance is frequently repeated.
When at length the principal performer sits down, the other men
leave the spot and run in a single file towards the camp, loudly crying
“kurreke ta ta” in imitation of the call of the spur-winged plover.
In the evening a general corroboree is indulged in; and all grown-
up persons, male and female, are allowed to join in. Several refrains
are forthcoming which are connected with ordinary rain or water
festivals. The principal rainmaker does not attend but joins the camp
again during the night. It appears that in the interim he has visited
the sacred cave, in company of one or two of his brother-sorcerers,
to hide the magic stick and preserve it for future use. Any
representative of the Atoakwatje group inherits the power to fashion
and use the rain-stick, but it is imperative that he learns the art under
the direction of a senior and duly qualified nangarri.
A ceremony directly connected with sun-worship belongs to the
old Arunndta people and is known as “Ilpalinja.” When the weather
has been and continues to be unpleasantly cold, and the mating
season of birds and animals has on that account been long delayed,
the men construct a large colored design upon the selected
ceremonial ground. Radiating from a point upon a cleared space,
many lines are drawn with red and white vegetable-down to
represent the rays of the sun; and these are intersected at different
distances from the central point by a number of concentric circles
which represent the fathers of the tribe. The centre of the design is
occupied by a stick which is supposed to incorporate some mystical
and sacred sun-creature known as “Knaninja Arrerreka.” The same
Ilpalinja-design is occasionally carved as the crest of the Knaninja
upon a sun-tjuringa. Vide Fig. 6.
Fig. 6. Sacred sun-design of the “Ilpalinja” ceremony (× 1/20).
A most impressive function might occasionally be witnessed on

the north coast, which is associated with the setting sun; it is known
to at least two tribes, the one living on the upper reaches of the
Victoria River and the other on the western shores of Carpentaria
Gulf, including some of the islands. It is usually performed in
conjunction with demonstrations calling upon a fabulous being which
lives in the sky to fecundate certain species of plants and animals
necessary for their daily life. The Carpentaria tribes, moreover, keep
their sacred poles, akin to the tjuringas of central Australia, not in
caves but in special huts which they construct upon chosen spots
absolutely taboo to the general public. These slabs of wood are up to
five feet long and are covered with peculiar carvings and markings;
they are of the two sexes. Ordinarily they are kept “asleep” by laying
them on the floor of the hut side by side, and covering them with
sand. When the hour of the ceremony arrives, they are brought out
by the “Sun-Men” and stuck in the ground in the full light of the
sinking sun. Just as the orb is about to touch the horizon, the tenders
of the sacred implements kneel, with their faces turned towards the
sun and, lifting their hands, bend their bodies to the ground much
after the fashion of an Eastern salaam. We have before us a true
form of worship recognizing the supreme powers of the sun, but
aimed primarily at calling upon a demigod or Deity in supplication for
making a needed article of diet, animal or vegetable, fruitful or
prolific.
Mythologically the sun is regarded as a female having human form
and a fiery exterior, who walks daily across the firmament and
returns at night to rest at her sacred haunts on earth. Some of the
central tribes, like the Aluridja, split the sun’s identity into an
indefinite number of such women, a different one of which makes the
journey every day.
The moon on the other hand is thought to be a man who originally
inhabited the earth but was one day chased off it by a gigantic dog
the Aluridja call “Tutrarre.” The man jumped into space and walked
among the clouds until he reached the earth again. His long walk
had made him so hungry and thin that he ravenously ate a great
number of opossums which he found in the trees at night. In
consequence he swelled out, and became fat and round. Then it was
his bad fortune to fall in with the dog again, and this time his obesity
prevented his escape. The dog tore him to pieces and swallowed
him, bone and all. But it so happened that one of his arm-bones flew
from the dog’s jaws and found its way to the sky. There it floated
from east to west as a luminous sickle and gradually swelled until it
was perfectly round. The dog stood looking up at the bone and
howled in anger, but the moon-man reappeared in the sky and
converted the dog into stone.
The Kakatu natives believe in a moon-man who lives in the sky
and controls the clouds. On a certain day, very long ago, this man
was seen by the ancestors of the tribe. It happened thus: Just about
dusk, a cloud was observed descending from the sky which came to
rest upon the summit of a hill; it was glowing red. A big man, a
woman, and two girls stepped upon the earth, and the man took a
fire-stick from the cloud which then became black and ascended
again. It was the moon-man and his family. The party walked down
on to the plain and camped, the old man making a fire with his torch
whereby his feminine escort could warm themselves. The moon-man
left, taking a new fire-stick with him. In a deep, green water-hole
lived a monstrous snake whose colour was much like that of the
slime which covered the surface of the water. A lengthy and secret
interview took place between the moon-man and the snake on the
bank of the lagoon, and the snake produced many tubers of water-
lily, and mussels also, for the moon-man to eat. Then the two heard
a rustling noise. The snake exclaimed: “What is that? Who dares
approach our trysting place?” The moon-man snatched a fire-brand
and held it high in the air; this made it light as day. The moon-man’s
daughters could be seen creeping towards the men to hear the
secret discussion! With a curse upon his mouth, the angry father
hurled the fire-stick at his deceitful daughters. The stick struck the
ground and sent a shower of sparks over the girls. In an instant
everything became dark as night, but every now and again there
came from the spot the girls had last been seen at long-drawn
growls; from the same spot flashes of light shot forth and illumined
the clouds. The snake and the moon-man had disappeared, but the
daughters remained just where they had last been seen, for they had
been turned to stone and had assumed the rigid form of a dog
whose head was directed skywards as if to rebuke the moon-man for
the curse he had brought upon them. For a long time the clouds
remained dark; then the moon-man re-appeared among them and
cast a mournful beam upon the canine image of his daughters. From
then till now he has continued to appear periodically in the sky, and
his repentant daughters gaze at him; but at times, when the sky is
covered with heavy black clouds, the daughters become angry and
growl aloud. At these times, too, bright flashes dart from their eyes

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Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.001 1

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Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.07.001 21

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An episode of the great fire ceremony, Kolaia tribe.

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Ceremonial venesection, Arunndta tribe.

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Fig. 6. Sacred sun-design of the “Ilpalinja” ceremony (× 1/20).

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