CHAPTER 44

Mitosis and Cytokinesis

M itosis is the division of a somatic cell (a vegetative

Prophase Early prometaphase
cell in yeast) into two daughter cells. The daughters are CDK activity high
usually identical copies of the parent cell, but the process
can be asymmetrical. For example, division of some stem
cells gives rise to one stem cell and another daughter cell
that goes on to mature into a differentiated cell. See Box
41.2 for examples.
Traditionally, mitotic events are subdivided into six Late prometaphase Metaphase
phases: prophase, prometaphase, metaphase, ana-
phase, telophase, and cytokinesis (Fig. 44.1). The
dramatic reorganization of both the nucleus and cyto-
plasm during the mitotic phases is brought about by
activation of a number of protein kinases, including
Cdk1cyclin Bcks (abbreviated here as Cdk1 kinase;
Anaphase Telophase
see Chapter 40). After activation by Cdc25 phosphatase,
APC/C activity high
Cdk1 kinase accumulates in the nucleus, where it joins
Cdk1cyclin Acks, which was activated somewhat
earlier (see Fig. 43.6). These two Cdk1 kinase complexes
operate as both master controllers and workhorses that
directly phosphorylate many proteins whose functional
and structural status is altered during mitosis. Their
Early cytokinesis Late cytokinesis
progressive inactivation following the correct attach-
ment of the chromosomes to spindle microtubules drives
the orderly exit of cells from mitosis.
Mitosis is an ancient process, and a number of varia-
tions emerged during eukaryotic evolution. Many single-
celled eukaryotes, including yeast and slime molds,
undergo a closed mitosis, in which spindle formation FIGURE 44.1 OVERVIEW OF THE PHASES OF MITOSIS.
and chromosome segregation occur within an intact APC/C, anaphase-promoting complex/cyclosome.
nuclear envelope to which the spindle poles are
anchored. This chapter focuses on open mitosis, as
Prophase
used by most plants and animals, in which the nuclear
envelope disassembles before the chromosomes segre- Prophase, the transition from G2 into mitosis, begins
gate. Fig. 44.2 summarizes some of the important events with the first visible condensation of the chromosomes
during the various mitotic phases. and disassembly of the nucleolus (Fig. 44.3). In the

755
756 SECTION X n Cell Cycle

A. Interphase B. Prophase C. Prometaphase D. Metaphase

Nucleus
Centrosome
Chromosomes
NE

Microtubules

Centrosomes separate Begins with nuclear Chromosomes align

Chromosomes condense envelope (NE) break-down on spindle equator
Chromosomes attach
to spindle

E. Anaphase A F. Anaphase B G. Telophase H. Cytokinesis

NE

CS

Midbody
CF CF CS remnant
CS
Pole
Sister chromatids separate Organized central spindle (CS) Cleavage furrow (CF) constricts Chromosomes decondense
and move to poles assembles Nuclear envelope (NE) Interphase microtubule
Poles (arrows) separate reassembles network reforms
Cleavage Furrow (CF)
assembles Daughter cells separate

FIGURE 44.2 KEY EVENTS OF MITOSIS. AC, Prophaseprometaphase: Cdk1 kinase triggers condensation of replicated sister chromatids,
disassembly of the nuclear envelope and Golgi, and a dramatic reorganization of the cytoskeleton. As the barrier between the chromosomes and
cytoplasm is abolished, microtubules contact the condensed chromosomes and attach at the kinetochores (see Fig. 8.21). Interaction of kineto-
chores with dynamic microtubules culminates with the chromosomes aligned at the midplane of the bipolar spindle. DF, Metaphaseanaphase:
Once all chromosomes achieve a bipolar attachment to the spindle, an inhibitory signal is silenced, leading to activation of a proteolytic network
that destroys proteins responsible for holding sister chromatids together and also inactivates Cdk1 by destroying its cyclin B cofactor (see Fig.
40.15). Sister chromatids separate and move toward opposite spindle poles, which themselves move apart. GH, Telophasecytokinesis: Targeting
of nuclear envelope components back to the surface of the chromatids leads to the reformation of two daughter nuclei. In most cells, the two
daughter nuclei and the surrounding cytoplasm are partitioned by cytokinesis. (Micrographs courtesy William C. Earnshaw.)

cytoplasm, the interphase network of long microtubules
centered on a single centrosome (see Fig. 34.18) is
converted into two radial arrays of short microtubules
called asters. Most types of intermediate filaments disas-
semble, the Golgi apparatus and endoplasmic reticulum
fragment, and both endocytosis and exocytosis are
curtailed.
Nuclear Changes in Prophase
Chromosome condensation, the landmark event at the
onset of prophase, often begins in isolated patches of
chromatin at the nuclear periphery. Later, chromosome
condense into two threads termed sister chromatids
that are closely paired along their entire lengths. Although
chromosome condensation was first observed more than
a century ago, the biochemical mechanism remains a
mystery. Protein kinases trigger mitotic chromosome
condensation and onset of condensation is correlated
with phosphorylation of histones H1 by Cdk1 kinase and
H3 by Aurora-B protein kinase. However, chromosomes
still condense when both of these phosphorylation
events are blocked. It is possible that a combination of
histone modifications promotes mitotic chromatin con-
densation (see Fig. 8.3).
Two pentameric protein complexes, condensin I
and condensin II are major regulators of mitotic chromo-
some architecture. These complexes share the SMC2 and
SMC4 (structural maintenance of chromosomes) ABC
adenosine triphosphatases (ATPases), but have two dif-
ferent sets of three auxiliary proteins (see Fig. 8.18).
 CHAPTER 44 n Mitosis and Cytokinesis 757

A. Prophase Chromosome B
condensation
Phosphorylated begins
condensin enters nucleus
Histone H3 Duplicated
phosphorylation centrioles begin
begins to separate
Cell surface Microtubule half-
markers life decreases
internalized and asters form
Intracellular membrane DNA
Cell begins to Microtubules
networks remodeled round up Centrosomes

FIGURE 44.3 INTRODUCTION TO PROPHASE. A, Summary of the major events of prophase. B, Distribution of DNA (blue), microtubules
(red), and -tubulin (centrosomes [green]) in a prophase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the University of
Dundees School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)

Condensins are required to disassemble the topologically

associating domains (TAD) of interphase chromatin (see
Fig. 8.13) and promote the formation of the linear array
of loops that characterizes mitotic chromosomes (see
Fig. 8.14). The decisive experiment was to target SMC2
for rapid degradation, with the result that no well-
organized mitotic chromosomes formed. The chromatin
compacted but was less organized. Slower depletion of
condensins by RNA interference (RNAi) or conditional
gene disruption gave less-dramatic results.
Condensin complexes can encircle DNA and also
promote its supercoiling in vitro, but how these activi-
ties help them to orchestrate the changes in chromatin
architecture is not known. Condensin II is nuclear during
interphase, and has an important role in prophase chro-
mosome formation following its activation by phos-
phorylation. Condensin I acts both in prophase and
prometaphase in further compacting the chromosomes.
DNA topoisomerase II and other scaffold proteins also
function during mitotic chromosome formation, but
condensin appears to play the major role.
Cytoplasmic Changes in Prophase
Most of the cytoskeleton reorganizes during prophase.
The microtubule array changes from an extensive
network permeating the cytoplasm into two dense,
radial arrays of short, dynamic microtubules around the
duplicated centrosomes (see Fig. 34.17). Each of these
asters eventually becomes one pole of the mitotic
spindle. During prophase, the two asters usually migrate
apart across the surface of the nuclear envelope, signal-
ing the start of spindle assembly (Fig. 44.3).
Mitotic microtubules behave like interphase microtu-
bules in many ways (see Chapter 34). They are mostly
nucleated at their minus ends, they grow by addition of
tubulin subunits at their plus ends, and they undergo
random catastrophes during which they rapidly shorten.
To a large extent, the prophase changes in microtubule
organization can be explained by two simple biochemi-
cal changes: (a) increased microtubule-nucleating activ-
ity of centrosomes and (b) altered dynamic instability
TABLE 44.1 Comparison of Microtubule Dynamics
in Interphase and Mitotic Newt Lung Cells
Parameter Interphase Mitosis
Elongation rate 7m/min 14m/min
Elongation time before 71s 60s
catastrophe
Shortening rate 17m/min 17m/min
Probability of rescue 0.046/s 0
from catastrophe*
Length 100m 14m
*Most cellular microtubules grow constantly by addition of subunits to
their free ends but they occasionally stop growing and begin shrinking
rapidly (a catastrophe). Unless shrinking is reversed (a rescue), the
microtubule completely disappears.
Data from Gliksman NR, Skibbens RV, Salmon ED: How the transition
frequencies of microtubule dynamic instability regulate microtubule
dynamics in interphase and mitosis. Mol Biol Cell. 1993;4:10351050.
properties of the microtubules (Table 44.1; also see
Chapter 34). Interphase microtubules have a high prob-
ability of recovering from catastrophes, so they grow
quite long. Mitotic microtubules grow more rapidly
but exist only transiently, because rescues are rare fol-
lowing a catastrophe. Thus, they usually shorten all
the way back to the centrosome, with little chance of
rescue. These differences in dynamic instability can be
reproduced in vitro in mitotic and interphase cellular
extracts. They appear to arise, at least in part, from
counterbalancing interactions between microtubule-
associated proteins that promote microtubule stability,
and kinesin-13 (see Fig. 36.13), which promotes micro-
tubule disassembly.
Other cytoskeletal elements that disassemble during
prophase include many, but not all, classes of interme-
diate filaments (including the nuclear lamins) (see
Fig. 35.1A) and specialized actin filament structures,
such as stress fibers. However, the junctional complexes
between adjoining cells are maintained in epithelial
cells. As a result of the cytoskeletal reorganization, most
cells round up during prophase. This is particularly
evident for animal cells that are cultured on a flat
758 SECTION X n Cell Cycle

substrate, but cells in tissues also change their shape
dramatically during mitosis.
RNA transcription of the chromosomes stops during
mitosis except for highly specialized transcription at
centromeres. Phosphorylation of components of the
transcriptional machinery by Cdk1 kinase appears to be
responsible for this shutoff. Cdk1 kinase phosphoryla-
tion of ribosomal elongation factor 2a (EF2a) also stops
most (but not all) ongoing protein synthesis and assem-
Interphase Mitosis
Golgi Ministacks
Golgi stacking GM130
proteins
cyclin Bp9
p115
bly of new ribosomes. Phosphorylation of several nucleo-
lar proteins leads to disassembly of the nucleolus.
The Golgi apparatus and endoplasmic reticulum frag-
ment and disperse during prophase (Fig. 44.4). Several
kinases, including Cdk1 drives Golgi apparatus disas-
sembly, the first step being fragmentation into smaller
ministacks following phosphorylation of Golgi stacking
proteins and tethers. Later steps are still being investi-
gated. Many lines of evidence argue that Cdk1 phos-
phorylation of key components prevents the fusion of
transport vesicles back into Golgi stacks (see Chapter
21), the net result being that the Golgi buds away into
small vesicles that disperse throughout the mitotic cell
cytoplasm. Other evidence suggests that an imbalance of
vesicle flow between the Golgi and the endoplasmic
reticulum results in the Golgi being absorbed into the
endoplasmic reticulum during mitosis. Whatever the
Cdk1
mechanism of its disassembly, Golgi reassembly begins
again during late anaphase/early telophase, following
inactivation of Cdk1 kinase.

FIGURE 44.4 GOLGI APPARATUS DYNAMICS IN INTER-
PHASE AND MITOSIS. Disassembly in mitosis is driven by phos-
phorylation of components blocking fusion of Golgi membranes.
Prometaphase
In cells that undergo an open mitosis, prometaphase
begins abruptly with disassembly of the nuclear envelope
(Fig. 44.5). Microtubules growing outward from the
spindle poles penetrate holes in the nuclear envelope,
make contact with the chromosomes, and attach to them

A. Early prometaphase C. Late prometaphase E. Kinetochore attachments

to the spindle
2 4
1 Tension

3 5
(+) ends

1. Nuclear envelope disassembles 4. Chromosome slides rapidly poleward Amphitelic (correct)

2. Microtubules grow and shrink in aster
3. Kinetochore captures microtubule
along microtubule
5. Microtubule from opposite pole is
captured by sister kinetochore
6. Chomosome attached to both poles
congresses to middle of spindle

B D
Syntelic (errors)

DNA
Microtubules
Centrosomes Merotelic (errors)

FIGURE 44.5 INTRODUCTION TO PROMETAPHASE. A, Summary of the key events of early prometaphase. B, Distribution of DNA (blue),
microtubules (red), and -tubulin (centrosomes [green]) in early prometaphase human cells. C, Summary of the key events of late prometaphase.
D, Distribution of DNA, actin, microtubules, and centrosomes in late prometaphase PtK1 cells. E, Terms used to describe the orientation of
kinetochore attachments to the mitotic spindle. (B and D, Images were recorded by Dr. Melpomeni Platani on the University of Dundees School
of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
 CHAPTER 44 n Mitosis and Cytokinesis 759

at specialized structures called kinetochores (see Fig.
8.19). Interactions of the two opposing kinetochores of
paired sister chromatids with microtubules from oppo-
site poles of the spindle ultimately result in alignment of
the chromosomes in a group midway between the poles.
An important cell-cycle checkpoint (see Chapter 40)
known as the spindle assembly checkpoint (SAC)
delays the onset of chromosome segregation until all
kinetochores are attached to microtubules.
Nuclear Envelope Disassembly in Prometaphase
Nuclear envelope disassembly involves the removal of
two membrane bilayers coupled with disassembly of the
nuclear pores and the fibrous nuclear lamina mesh-
work that underlies the inner bilayer (Fig. 44.6). Phos-
phorylation causes the nucleoporin Nup98 to dissociate
from nuclear pores. This removes the permeability
barrier between nucleus and cytoplasm. Phosphoryla-
tion of other proteins causes the pore to disassemble to
soluble subcomplexes. Phosphorylation of the nuclear
lamins at two sites flanking the coiled-coil causes the
lamina network to disassemble into subunits. Interaction
between microtubules and dynein associated with the
nuclear envelope can rip holes in the envelope, although
this is not required for nuclear envelope disassembly.
Nuclear envelope membranes are dispersed in the
cytoplasm from prometaphase until telophase (Fig.
44.6), but the mechanism is not settled. Some experi-
ments suggest that the nuclear membranes break up into
small vesicles that disperse in the cytoplasm. Other
experiments suggest that the nuclear envelope is
absorbed into the endoplasmic reticulum, which remains
as an extensive tubular (or flattened cisternal network
another source of discussion) throughout mitosis.
Further experiments are required to answer this ques-
tion, and both mechanisms could contribute. Lamin B
remains associated with the dispersed nuclear envelope,
whereas lamins A and C and many proteins of the nuclear
pore complexes disperse as soluble subunits.
During prophase, kinetochores transform from non-
descript balls of condensed chromatin into structures on
the surface of the chromosomes. By early prometaphase,
the characteristic trilaminar disk structure (see Fig. 8.19)
can be seen. Each sister chromatid has a kinetochore.
Sister kinetochores are located on opposite faces of
the mitotic chromosome.
Organization of the Mitotic Spindle
The mature metaphase spindle is a bilaterally sym-
metrical structure with centrally located chromosomes
flanked by arrays of microtubules radiating from the
poles (Fig. 44.7).
Three predominant classes of microtubules are
present in the metaphase spindle (Fig. 44.12). Kineto-
chore microtubules have their plus ends embedded in
the kinetochore and their minus ends at or near the
spindle pole. They characteristically form bundles, called
kinetochore fibers, which contain anywhere from 1
microtubule in the budding yeast to more than 200
microtubules in some higher plants. Each human kineto-
chore binds approximately 20 microtubules. Up to
approximately 80% of the approximately 2200 spindle
microtubules in humans may be present in kinetochore
fibers, but not all microtubules in those fibers stretch all

A. Interphase B. Mitosis C D. Lamins A and C E. Lamin B

Nuclear Endoplasmic
membrane: reticulum
Outer
Inner
HO PO4 O PO4 O
Lamina
Interphase O O
disassembly C C
OCH3 O
Nuclear
lamina Cdk1 G2 / M G2 / M
cyclin Bp9
Condensing
chromosomes
PO4 PO4
O O
Eggs? Somatic C C
cells? Earliest OCH3 O
prometaphase OH O PO4 OH O PO4

Lamin B

+ or

Chromosomes Vesicles derived Lamin B

from the nuclear dispersed through the Soluble Subunits on
Metaphase Lamina Lamina
envelope endoplasmic reticulum subunits membrane vesicles

FIGURE 44.6 DISASSEMBLY OF THE NUCLEAR ENVELOPE DURING MITOSIS. AB, Two contrasting models to explain the fate of the
nuclear envelope during the transition from interphase to mitosis in a higher eukaryote. C, Micrographs showing solubilization of lamin A fused
to green fluorescent protein (GFP) (green) during mitosis. DNA is blue. Scale bar is 10 m. DE, Reversible disassembly of lamins A, C, and B
is driven by posttranslational modifications of the lamin polypeptides. (C, Courtesy William C. Earnshaw.)
760 SECTION X n Cell Cycle

A. Metaphaseforces balanced, spindle length stable B. Anaphaseforces elongating the spindle dominate

Cytoplasmic dynein moves

toward minus () end
Microtubule disassembly at kinetochores
Microtubule (+)
plus flux moves sister chromatids toward poles
(+) (+)
(+) (+)
(+) Flux
(+) (+)

(+)
Ordered central
(+) spindle assembles
() () () (+) () (+)
() () () ()
() ()
() () () () ()
(+) Kinesin-13
(+)
(+) ()
(+) ()
(+)

() (+)
() (+) NuMA
Bipolar plus-enddirected kinesin-5 dominates
Inward force from minus-enddirected kinesin-14 and microtubule to elongate spindle, pushing poles apart
disassembly at poles by kinesin-13 is balanced by outward force
from kinesin-5 plus dynein stretching poles apart
Microtubule assembly at kinetochores and disassembly at poles causes tubulin subunit flux along kinetochore microtubules

FIGURE 44.7 ROLE OF MOTOR PROTEINS IN SPINDLE DYNAMICS. Mitotic spindle structure depends on microtubule assembly/
disassembly plus balanced forces that slide microtubules relative to one another and to pull the poles together or push them apart. A, In
metaphase, the structure is at steady state. Forces that tend to elongate the spindle, including cytoplasmic dynein (which moves toward
microtubule minus ends, pulling the poles out toward the cell cortex) and bipolar kinesin-5 (which moves toward microtubule plus ends, pushing
the poles apart), are counterbalanced by cohesion between sister chromatids and kinesin-14, which moves toward microtubule minus ends (and
pulls the poles together) and microtubule disassembly at the spindle poles. Dynein and its associated protein, NuMA (nuclear mitotic apparatus),
also help to organize a focused spindle pole. B, In anaphase, sister chromatids separate, the balance of kinesin activity shifts, microtubule disas-
sembly at the poles declines, and the spindle undergoes a dramatic elongation. During anaphase, bipolar kinesin-5, chromokinesin KIF4A, and
protein regulated in cytokinesis 1 (PRC1) also have important roles in organizing the central spindle, which is essential for subsequent assembly
and function of the cleavage furrow.

the way from the kinetochores to the spindle poles.
Interpolar microtubules are distributed throughout
the body of the spindle and do not attach to kineto-
chores. Many interpolar microtubules penetrate between
and through the chromosomes and extend for some
distance beyond them. Thus, the central spindle contains
a large number of interdigitated antiparallel microtu-
bules. Tracking these spindle microtubules by electron
microscopy revealed a tendency for the interdigitated
microtubules of opposite polarity to pack next to one
another. During late anaphase, these antiparallel micro-
tubules bundle to form a structure called the central
spindle that plays important roles in signaling during
cytokinesis. Astral microtubules project out from the
poles and have a role in orienting and positioning the
spindle in the cell through interactions with the cell
cortex in somatic cells. All microtubules within each
aster have the same polarity, with their minus ends
proximal to the pole. Each unit of a spindle pole, with
its associated kinetochore and interpolar and astral
microtubules, is referred to as a half-spindle.
Spindle structure is largely determined by a combina-
tion of microtubule dynamics plus the action of at least
seven different types of kinesins and cytoplasmic dynein
(see Chapter 36). These motors often work in opposition
to one another. Furthermore, forces exerted by motors
can influence the dynamic assembly/disassembly of
microtubules. As a result, the highly dynamic spindle
changes shape as a delicate balance of forces shifts
between the various motors. For example, inactivating
one or more kinesins with drugs or switching a
temperature-sensitive mutant to the nonpermissive
temperature can cause the spindle to collapse rapidly
on itself.
Spindle Assembly
In metazoans, spindle assembly starts in prophase with
the separation of the asters. In most cells, each aster is
organized around a centrosome, consisting of a centriole
pair and associated pericentriolar material. -Tubulin
ring complexes in the pericentriolar material efficiently
nucleate microtubules (see Fig. 34.16), so each centro-
some acts as a microtubule organizing center
(MTOC). By the end of prophase, the spindle consists of
two asters linked by a few interpolar microtubules.
Cytoplasmic dynein at the cell cortex exerts an outward
force separating the centrosomes, whereas kinesin-14
motors (which move toward microtubule minus ends)
on the interpolar microtubules exert a counterbalancing
force holding the asters together.
This balance of forces changes when the nuclear
envelope breaks down. Bipolar kinesin-5 motors phos-
phorylated by Cdk1 kinase concentrate in the central
spindle, where they crosslink adjacent antiparallel

interpolar microtubules. Kinesin-5 moves toward the
plus ends of microtubules, so when attached to two
adjacent antiparallel microtubules, its action will cause
them to slide and push the spindle poles apart (Fig.
44.7). However, the two half-spindles do not separate
because they are physically linked via the chromo-
somes, with sister kinetochores attached to opposite
spindle poles.
Also at this time, the asters mature into focused
spindle poles. The pericentriolar material efficiently
nucleates the assembly of new microtubules with their
minus ends at the pole. Microtubule assembly at two
other sites also contributes to spindle morphology. At
kinetochores, a complex derived from interphase nuclear
pores recruits -tubulin. This locally nucleates microtu-
bules that grow by inserting subunits at the kinetochores,
pushing the minus ends with -tubulin outwards toward
the spindle poles. These preformed kinetochore fibers
are then incorporated into the spindle. In the central
spindle, microtubules are nucleated on the walls of other
microtubules by -tubulin recruited by the multi-subunit
augmin complex. The action of augmin creates branched
microtubules throughout the central spindle, contribut-
ing to an even fir tree-like distribution of microtubules.
The daughter microtubules may be released from their
nucleation site, free at both ends, and move toward the
spindle poles as part of the flux of tubulin from the
center of the spindle toward the centrosomes.
The microtubule array focuses at the poles partly
because centrosomes tether microtubules, and partly
due to the concerted action of various motors and micro-
tubule crosslinking proteins such as dynein and nuclear
mitotic apparatus (NuMA) protein. NuMA is released
from the nucleus when the nuclear envelope breaks
down, and it accumulates near the poles at the minus
ends of microtubules.
Large cells that lack centrosomes, such as eggs, form
spindles by an alternative pathway that also functions in
the background in cells with centrosomes (Fig. 44.8).
This mechanism hijacks the nuclear trafficking system to
Unstable microtubule
g radient
TP Microtubule
G
stabilized by
n-
Ra
Ran-GTP
gradient
Stabilized Motors sort
microtubules microtubules,
accumulate on which bind to
chromosomes kinetochores
FIGURE 44.8 ASSEMBLY OF A BIPOLAR SPINDLE IN THE ABSENCE OF CENTROSOMES. A gradient of Ran-guanosine triphosphate
(GTP) stabilizes microtubules around chromosomes, which contain high concentrations of bound Ran GEF RCC1. This releases spindle assembly
factors from importin and . Microtubules that accumulate around the chromosomes are sorted, organized, and focused to make poles by
motors and () end-binding proteins such as NuMA. These spindle poles lack prominent astral microtubules.
CHAPTER 44 n Mitosis and Cytokinesis 761
enable chromosomes to control the activity of key
spindle assembly factors. The nuclear import receptors
importin and bind these factors, as though they were
going to transport them into the nucleus. This blocks
their spindle assembly activity. During mitosis, chromo-
somes bind high levels of RCC1, the guanine exchange
factor for Ran (Ran-GEF in Fig. 9.18). This RCC1 creates
a gradient of the active guanosine triphosphatase
(GTPase) Ran-GTP (guanosine triphosphate) around the
chromosomes. During interphase, Ran-GTP is confined
to the nucleus, where it releases importins from their
cargo. In mitosis the chromosome-associated cytoplas-
mic Ran-GTP gradient locally liberates the spindle assem-
bly factors including motor proteins and NuMA from
sequestration by importins. They then stabilize nearby
microtubules and organize them into a bipolar spindle.
If centrosomes are removed or destroyed experimen-
tally in cells about to enter mitosis, somatic cells can also
use motor proteins to organize microtubules into bipolar
spindles that lack asters but are otherwise remarkably
normal. Most treated cells manage to complete mitosis
successfully but normal mammalian cells then either
arrest in the next cell cycle prior to replicating their DNA
or commit suicide. Both outcomes depend on the pres-
ence of the important tumor suppressor protein p53 (see
Fig. 43.11). Thus, centrosomes are not required to form
spindles, but they contribute to cell-cycle progression in
many cells. This dependence on centrosomes is not
universal; Drosophila, for example, can live without
centrosomes.
Chromosome Attachment to the Spindle
Dynamic microtubules of prometaphase asters scan the
cytoplasm effectively searching for binding sites that
will capture and stabilize their distal plus ends. Captured
microtubules are approximately fivefold less likely to
depolymerize catastrophically than free microtubules.
When catastrophes do occur, the microtubules depoly-
merize back to the pole, recycling tubulin subunits for
incorporation into other, growing microtubules.
Other motors and
() end-binding proteins
organize spindle poles
(note absence of asters)
762 SECTION X n Cell Cycle

Breakdown of the nuclear envelope makes the con-
densed chromosomes accessible to the microtubules.
Chance encounters allow kinetochores to capture
microtubule plus ends. Capture probably involves the
nine-component KMN network, which includes the rod-
shaped Ndc80 complex (see Fig. 8.21) that binds along
the sides of microtubules near their plus ends. Another
member of the complex, the scaffolding protein Knl1
(its name in vertebratesthe K of KMN; see later),
anchors Ncd80 in the kinetochore.
Historically, it was thought that forces generated by
bipolar attachment of the kinetochores of sister chro-
matids center chromosomes midway between the two
spindle poles. This hypothesis was based on the observa-
tion that when a kinetochore first attaches to a microtu-
bule, the chromosome moves along the side of that
microtubule toward the spindle pole (Fig. 44.9). Subse-
quent capture of a microtubule emanating from the
opposite spindle pole by the sister kinetochore would
provide a counterforce pulling the chromosome in the
opposite direction. Chromokinesin family motor proteins
distributed along the chromosome arms were also
thought to contribute to the gradual movement of the
chromosome toward the middle of the spindle. These
movements are accompanied by coordinated shrinkage
of the microtubules at the leading kinetochore and
growth of microtubules at the trailing kinetochore.
More recent studies revealed that chromosomes
attached to only one spindle pole can move away from
that pole if the unattached kinetochore associates with
the kinetochore fiber of a chromosome already aligned
at the spindle equator. In this case, the kinetochore of
the mono-oriented chromosome glides toward the
equator, where it is more likely to capture microtubules
emanating from the opposite pole. This motion of one
chromosome along the kinetochore fiber of another
chromosome requires the kinesin-7 motor centromere
protein E (CENP-E) (see Fig. 36.13) associated with the
kinetochore of the moving chromosome. Recognition of
a tubulin posttranslational modification leads CENP-E to
move the chromosome toward the spindle equator,
rather than out into the aster.
The attachment of microtubules to kinetochores can
be reconstituted in vitro from mixtures of chromosomes,
isolated centrosomes, and tubulin subunits. The plus
ends of microtubules grow out from centrosomes and

A E
Spindle
pole
Spindle pole
4 Microtubule
Kinetochore
Distance traveled toward

3
spindle pole (m)

B 1

1 Start of
movement
2
0 20 40 60 80
Time (seconds)

C D
Kinetochore Microtubule
10 m

FIGURE 44.9 INITIAL CHROMOSOMAL MOVEMENTS DURING PROMETAPHASE. AB, Capture of a microtubule by the kinetochore
results first in movement along the side of the microtubule toward the pole from which that microtubule originated. These images come from a
study in which living cells, observed by differential interference microscopy, were subjected to rapid chemical fixation just after a chromosome
had attached to the spindle (arrow). C, Attachment of the chromosome to the spindle was confirmed by indirect immunofluorescence staining
for tubulin and thin-section electron microscopy (D). E, The graph shows the movements of the chromosome before and after attachment. (From
Rieder CL, Alexander SP, Rupp G. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to
the spindle in newt lung cells. J Cell Biol. 1990;110:8195, copyright The Rockefeller University Press.)
 CHAPTER 44 n Mitosis and Cytokinesis 763

attach to the chromosomes. Surprisingly, chromosome-
bound microtubules can either lengthen or shorten at
the attached end without detaching from the chromo-
some. Similar experiments with kinetochores isolated
from budding yeast cells showed that kinetochores can
remain attached to a shortening microtubule plus end
even against an applied force of 9 pN (piconewtons).
Physiological levels of tension actually stabilize the
attachments of kinetochores to microtubules in vitro, as
in vivo. This tethering of kinetochores to disassembling
microtubules is essential for chromosome movements
during mitosis.
Correcting Errors in Chromosome Attachment to
the Spindle
The goal of mitosis is to partition the replicated chromo-
somes accurately between two daughter cells. Therefore,
all chromosomes must attach correctly to both spindle
poles (known as amphitelic attachment; Fig. 44.5)
before being segregated. Three other sorts of attachment
are seen: (a) chromosomes with one kinetochore lacking
attached microtubules (known as monotelic attach-
ment; this is a normal intermediate), (b) chromosomes
with both sister kinetochores attached to the same
spindle pole (known as syntelic attachment), and (c)
chromosomes with a single kinetochore attached simul-
taneously to both spindle poles (known as merotelic
attachment). Correcting monotelic and syntelic errors
takes time, and the SAC (see Finding Time to Fix
Chromosome Attachment Errors: The Spindle Assembly
Checkpoint below) delays mitotic progression to allow
the correction process to occur.
When syntelic attachments occur, one or both kineto-
chores must detach for the chromosome to achieve a
bipolar orientation. Chromosome attachment to oppo-
site spindle poles is more stable than attachment to a
single pole, because the tension generated by bipolar
attachment (where forces pull a chromosome simultane-
ously toward opposite spindle poles) preferentially sta-
bilizes microtubule connections to both kinetochores.
Merotelic attachments are more dangerous, as the kineto-
chore is under tension and the attachments are therefore
stable. Merotelic attachments are the most common
cause of chromosome segregation errors in cultured
mammalian cells.
Syntelic and merotelic chromosome attachments are
corrected through the action of Aurora B protein kinase,
which forms the chromosomal passenger complex
(CPC), along with inner centromere protein (INCENP),
survivin, and borealin (Fig. 44.10). The other subunits
target Aurora B to its various sites of action during mitosis
and regulate the kinase activity. The complex concen-
trates at inner centromeres (the heterochromatin beneath
and between the two sister kinetochores) during pro-
metaphase and metaphase. At anaphase onset, the CPC
moves to the overlapping interpolar microtubules of the
central spindle and to the cell cortex, where the cleav-
age furrow will form, ultimately winding up in the

A Borealin
B C

DNA
Survivin
Microtubules

D E. CPC Aurora-B F
INCENP

Borealin

Survivin

Histone H3 phosphorylation
Kinetochore targets
Central spindle targets
Cleavage furrow targets

FIGURE 44.10 CHROMOSOMAL PASSENGER COMPLEX (CPC) REGULATES MITOTIC EVENTS. The CPC (green) is present at
centromeres in prometaphase and metaphase (B), but transfers to the spindle midzone at anaphase (C) and midbody at anaphase (D).
E, Diagram of Aurora B protein kinase complexed with INCENP (inner centromere protein), survivin, and borealin with some key targets of the
CPC. F, If CPC function is inhibited (in this case by RNA interference [RNAi] depletion of borealin), chromosome attachment errors are common
and many chromosomes fail to segregate properly in anaphase. Distribution of DNA (blue), microtubules (red), and survivingreen fluorescent
protein (GFP) (green) in human mitotic cells. Inset in A, Distribution of kinetochores (red), and borealin (green) in a prometaphase cell.
(AD, Micrographs by Sally Wheatley and William C. Earnshaw. F and Inset in A, Micrographs by Ana Carvalho, Reto Gassmann, and William
C. Earnshaw. Insets in A and F, From Gassmann R, Carvalho A, Henzing AJ, etal. Borealin: a novel chromosomal passenger required for stability
of the bipolar mitotic spindle. J Cell Biol. 2004;166:179191, copyright The Rockefeller University Press. AC, From Wheatley SP, McNeish IA.
Survivin: a protein with dual roles in mitosis and apoptosis. Int Rev Cytol. 2005;247:3588.)
764 SECTION X n Cell Cycle

intercellular bridge during cytokinesis. The CPC regu-
lates mitotic events from prophase through cytokinesis.
Along the way it contributes to the correction of
chromosome attachment errors and to the operation of
the checkpoint that delays the cell cycle in response to
those errors.
Aurora B corrects chromosome attachment errors by
phosphorylating Ndc80 in the microtubule-binding KMN
complex (see Fig. 8.21). Aurora B phosphorylation
strongly inhibits Ndc80 binding to microtubules, causing
the kinetochore to release attached microtubules. When
a chromosome is correctly attached to both spindle
poles, tension stretches the kinetochore away from the
CPC buried in the chromatin beneath. This can stabilize
chromosomemicrotubule interactions by preventing
the kinase from phosphorylating Ndc80.
Finding Time to Fix Chromosome Attachment Errors:
The Spindle Assembly Checkpoint
Segregation of replicated chromosomes into daughter
cells is extremely accurate. For example, budding yeasts
lose a chromosome only once in 100,000 cell divisions.
The frequency of chromosome loss may be 20-fold to
400-fold higher for human cells grown in culture. To
achieve even this level of accuracy, most cells delay
entry into anaphase until all chromosomes have achieved
amphitelic attachment to the spindle. This delay is
caused by the spindle assembly checkpoint (SAC), which
senses the completion of microtubule binding to kineto-
chores at metaphase (Fig. 44.11). The spindle checkpoint
differs from DNA damage checkpoints in that its default
setting is on as cells enter mitosis. It is silenced only
when every chromosome is properly attached to the
spindle.
The SAC involves the products of the mitotic arrest
defective (MAD) genes, the budding-uninhibited-by-
benzimidazole (BUB) genes, the monopolar spindle
(Mps1) kinase and the CPC. The MAD and BUB genes
were identified in yeast genetic screens for cells that
continued to divide (and die) when the spindle was
disassembled by drugs. These genes are conserved
from yeast to humans. SAC proteins accumulate at

A D

C
23 minutes
Time

B
Seat
belt
N

Time Mad1 Mad2

Destroy free kinetochore 23 minutes
with blast of laser light
APC/C
substrate

C
APC/C APC/C
it

Go
Wa

MCC Cdc20APC/C
Kinetochore
Mad1 Mad2 Mad2
APC/C
Microtubule Cdc20APC/C

Checkpoint proteins
not on kinetochore BubR1

Cdc20MCC U Ub Ub
Go! Wait! Ub b

Mad2 MCC
Proteins not to scale relative to kinetochore Substrate

FIGURE 44.11 SPINDLE CHECKPOINT. Signaling by unattached kinetochores stops the cell from entering anaphase until all chromosomes
have made a proper bipolar spindle attachment. A, As long as there is a chromosome that is not properly attached to the spindle (beige cells),
the cell does not enter anaphase. The cell enters anaphase approximately 20 minutes after chromosome attachment is complete (green cells).
B, In a cell with a persistently maloriented chromosome, anaphase entry is delayed (beige cells). If the unattached kinetochore is destroyed with
a high-powered laser, the cell enters anaphase about 20 minutes later. This proves that the unattached kinetochore sends an inhibitory signal.
C, Overview of the spindle assembly checkpoint (see text for details). D, Structure of the Mad1/Mad2 complex.

kinetochores early in mitosis, when the checkpoint is
on (ie, during prophase or prometaphase), and are
gradually displaced as microtubules bind and the kineto-
chores come under tension.
The target of the SAC is the APC/CCdc20, the anaphase-
promoting complex/cyclosome (APC/C) ubiquitin-
protein ligase (an E3 enzyme; see Figs. 23.3 and 40.16)
with its substrate recognition factor Cdc20 bound, APC/
CCdc20 ubiquitylates target proteins to mark them for
destruction by proteasomes. Key APC/CCdc20 substrates
are proteins that must be degraded for the cell to move
from metaphase to anaphase, including cyclin B and
securin, an inhibitor of the enzyme that triggers separa-
tion of sister chromatids at anaphase (Fig. 44.16).
During mitosis, Cdk1 activates the APC/C by phos-
phorylating an auto-inhibitory loop, allowing Cdc20 to
bind. The SAC is an additional regulatory circuit that
inactivates APC/CCdc20 until all kinetochores attach to
spindle microtubules. Kinetochores without microtubules
attract proteins that assemble the mitotic checkpoint
complex (MCC), the inhibitor that inactivates APC/CCdc20.
Checkpoint activation starts when Aurora B in the
CPC activates Mps1 kinase, allowing it to phosphorylate
Knl1 in the kinetochore at several sites. Mps1 phos-
phorylation of Knl1 creates a binding site that results in
Mad1 recruitment to the kinetochore. Mad1 then recruits
Mad2 to form a stable complex (Fig. 44.11). A loop on
Mad2 wraps around Mad1 like a safety belt making
the complex particularly stable. This form of Mad 2 is
known as closed Mad2. Mad1/Mad2 can transiently
bind soluble Mad2 molecules (known as open Mad2),
load them onto Cdc20 in the closed safety belt conforma-
tion (this loading probably occurs at kinetochores), then
release them to form the soluble MCC of Mad2/Cdc20/
BubR1/Bub3. The MCC associates with the APC/CCdc20,
interfering with binding of cyclin B and other key sub-
strates. As each chromosome becomes attached to both
poles of the spindle it stops producing MCC. When the
last chromosome has achieved a proper attachment, the
last source of MCC is extinguished, and entry into ana-
phase can proceed.
A. Metaphase
Kinetochore
microtubules
Astral
microtubules
Interpolar
microtubules
Cyclin B and
securin degraded Chromosomes oscillate
FIGURE 44.12 INTRODUCTION TO METAPHASE. A, Summary of the major events of metaphase. B, Distribution of DNA (blue), microtu-
bules (red), and gamma tubulin (centrosomes [green]) in a metaphase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the
University of Dundees School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
CHAPTER 44 n Mitosis and Cytokinesis 765
Silencing of the checkpoint involves several pathways.
Protein phosphatase 2A (PP2A) and protein phosphatase
1 (PP1) are recruited to the kinetochore in feedback
loops involving the CPC and other checkpoint compo-
nents. They dephosphorylate Knl1 so that it releases
Mad1. When microtubules bind, cytoplasmic dynein
motors actively strip checkpoint components from the
kinetochore, dragging them away toward the centro-
somes. In yeast, access of Mps1 to its target sites on
Knl1 is physically blocked when microtubules bind.
Exactly how this interaction is regulated in metazoans is
still actively studied. In addition, the SAC appears to
crosstalk with the DNA damage response (see Chapter
43), since DNA damage response components activate
SAC components and vice versa. However, the network
of interactions is very complex and details are still being
worked out.
Experimental inactivation of the spindle checkpoint
causes a catastrophic, premature entry into anaphase,
regardless of the status of chromosome alignment. This
leads to an unequal distribution of sister chromatids and
genetic imbalance between daughter cells known as
aneuploidy. Yeasts can live without the checkpoint
genes, but their loss is lethal for mice, which die early
during embryogenesis. Mice heterozygous for various
checkpoint components show increased aneuploidy.
Humans with mutations in BubR1 have mosaic variegated
aneuploidy syndrome (extra copies or loss of various
chromosomes in a variety of tissues), which is associated
with microcephaly (decreased brain size) and an
increased cancer risk.
Metaphase
When all the chromosomes have attained amphitelic
orientations and moved to positions roughly midway
between the two spindle poles, the cell is said to be in
metaphase (Fig. 44.12). The compact grouping of chro-
mosomes at the middle of the spindle is referred to as
the metaphase plate. In many cells, even though chro-
mosomes remain, on average, balanced at the middle of
B
DNA
Microtubules
Centrosomes
766 SECTION X n Cell Cycle

A B C D

Spindle pole
E F G

P AP

AP P

2 m
2 min
Spindle pole

FIGURE 44.13 KINETOCHORE OSCILLATIONS BETWEEN P (POLEWARD) AND AP (AWAY FROM THE POLE) MOVEMENT DURING
LATE PROMETAPHASE AND ANAPHASE IN PTK1 (RAT KANGAROO) CELLS. AD, Images showing the movements of several pairs of sister
kinetochores, labeled with green fluorescent protein (GFP)-Cdc20 (green), combined with phase-contrast images of the cell (red). E and G, Higher-
magnification views of sister kinetochores (marked with dashed lines) in prometaphase and anaphase, respectively. F, Kymograph (collage of images
of a vertical strip showing the same two kinetochores at various time points during the movie) showing the movements of these two kinetochores.
Movements toward (P) and away from (AP) spindle poles are indicated. P movement involves microtubule shrinkage at the leading kinetochore and
microtubule growth at the trailing kinetochore (which is undergoing AP movement away from its associated kinetochore). Spindle poles are near
the top and bottom of panels E to G. (Micrographs courtesy E.D. Salmon, University of North Carolina, Chapel Hill.)

the spindle, they jostle one another and undergo numer-

ous small excursions toward one pole or the other
throughout metaphase (Fig. 44.13).
Metaphase can also be defined biochemically as the
time of destruction of cyclin B and securin (Fig. 44.16),
because this begins as soon as the last chromosome
achieves amphitelic orientation. Degradation of cyclin A
begins earlier, at the entry into prometaphase, and is
largely complete before metaphase. Loss of securin initi-
ates a process leading to the separation of sister chroma-
tids and the onset of anaphase.
Microtubule Flux Within the Metaphase Spindle
Although the average length of the kinetochore micro-
tubules is roughly constant during metaphase, the
microtubules change continuously in three ways. First,
there is constant net addition of new tubulin subunits
(approximately 10 subunits per second) to the plus end
of the microtubules, where they are attached to the
kinetochore. Second, a comparable number of tubulin
subunits is continuously lost from the minus end of
the kinetochore tubules at the spindle poles. There
fore, tubulin subunits slowly migrate through kineto-
chore microtubules from the kinetochore to the pole
(Fig. 44.14). This subunit flux or treadmilling in kineto-
chore microtubules is caused by microtubule depolymer-
ization at the poles driven by kinesin-13 family members.
In addition, tubulin moves toward the poles as by micro-
tubules are transported towards the pole (many nucle-
ated by the augmin complex) within the kinetochore
fiber. All microtubules attached to each kinetochore
A B C
0 sec
Fluorescent tubulin
10 sec speckles move
toward poles
20 sec
P P
P P P P
30 sec
FIGURE 44.14 MICROTUBULE FLUX IN METAPHASE. A, Cells
entering mitosis were injected with tubulin subunits modified chemi-
cally by attachment of a caged fluorescent dye. This dye becomes
fluorescent after being irradiated with UV light. When cells entered
metaphase, the spindle was illuminated with a narrow stripe of UV
light, activating a narrow band of fluorescent tubulin subunits. With
time, these subunits approach the spindle poles (P). Because the
length of kinetochore microtubules is constant during this time, the
labeled tubulin molecules must migrate along the microtubules toward
the pole (arrows). This can occur if new subunits are added to the
microtubule at the kinetochore and old subunits are removed at the
pole. B, Microtubule flux at metaphase in a Drosophila embryo visual-
ized by fluorescence speckle microscopy. Embryos were injected with
very low levels of fluorescent tubulin, which appears as speckles dis-
tributed along the microtubules. If a very sensitive camera is used,
these speckles can be seen to move toward the poles, reflecting the
flux in the underlying microtubules. Scale bar is 5m. C, Movement
of labeled tubulin speckles toward the spindle poles. (A, Courtesy
Arshad Desai and the MBL Cell Division Group, Marine Biology Labo-
ratory, Woods Hole, MA; from Mitchison TJ, Salmon ED. Mitosis: a
history of division. Nat Cell Biol. 2001;3:E17E21. B, Courtesy Paul
Maddox and Arshad Desai, University of California, San Diego.)
 CHAPTER 44 n Mitosis and Cytokinesis 767

change coordinately in length during chromosomal
oscillations.
Anaphase
The separation of sister chromatids at anaphase is one of
the most dramatic events of the entire cell cycle (Fig.
44.15). Sister chromatids move to opposite spindle poles
(anaphase A), and the poles move apart (anaphase B).
Anaphase is also the time when the mitotic spindle
activates the cell cortex in preparation for cytokinesis.
Two forms of the APC/C (see Fig. 40.15) trigger the
transition from metaphase to anaphase by degrading key
proteins. APC/CCdc20 targets cyclin B for degradation,
causing Cdk activity to fall (see Fig. 40.17). This decline
in Cdk activity allows for activation of APC/CCdh1, because
Cdh1 phosphorylated by Cdk1 kinase cannot bind to the
APC/C. APC/CCdh1 targets polypeptides whose destruc-
tion by the proteasome is required for the cell to exit
from mitosis and return to interphase. APC/CCdh1 remains
active during G1 phase, where it is essential for the
licensing of DNA replication origins (see Fig. 42.28).
Biochemical Mechanism of
Sister Chromatid Separation
Separation of sister chromatids is regulated by the chro-
mosomes themselves, not by the mitotic spindle. Under
certain circumstances, sister chromatids can separate in
the absence of microtubules, ruling out a requirement
for forces from the spindle in the process.
Three factors regulate sister chromatid separation: a
protein complex known as cohesin, a protease known
as separase, and an inhibitor of separase known
as securin (Fig. 44.16). This system is conserved from
yeast to human. Chapter 8 discusses the functions of
cohesin in interphase.
Cohesin is a complex of four proteins that resembles
the condensin complex (see Fig. 8.18). Like condensin,
cohesin has two large subunits from the SMC ATPase
family. These proteins, SMC1 and SMC3, are complexed
with proteins called Scc1 (which has other names
omitted here for simplicity) and Scc3. Additional proteins
are required to stabilize the loading of this complex onto
DNA. Cells with mutations in cohesin components sepa-
rate sister chromatids prematurely in mitosis, resulting
in chaotic chromosome missegregation. This system is
very ancient, as bacteria depend on an SMC-related
protein for orderly chromosome segregation.
A variety of evidence suggests that cohesin forms a
ring with a diameter of 35nm, large enough to encircle
two sister chromatids like a lasso. In yeast, the complex
functions only if it binds chromosomes during DNA
replication. Cohesin accumulates at preferred sites on
the chromosomes, often near centromeres in budding
yeast or in regions of heterochromatin in fission yeast.
In vertebrates, most cohesin dissociates from the chro-
mosome arms by late metaphase, owing to the action of
the protein kinases Plk1 and Aurora B. Importantly, a
critical fraction remains associated with heterochromatin
flanking centromeres where it is protected from cleav-
age by shugoshin until the onset of anaphase (see follow-
ing paragraphs and Chapter 45).
Sequential cleavage of two key proteins triggers sister
chromatid separation at anaphase. This proteolysis makes
anaphase onset an irreversible transition. The first target,
securin, inhibits the separase protease. After the last
chromosome forms an amphitelic attachment to the
spindle, the spindle checkpoint is silenced. This allows
APC/CCdc20 to tag securin with ubiquitin, leading to its
destruction by proteasomes throughout metaphase. When
securin levels fall below a critical threshold, separase is
unleashed to cleave the Scc1 subunit of cohesin. Cleavage
of Scc1 breaks the cohesin ring, allowing the sister
chromatids to separate triggering the onset of anaphase
(Fig. 44.16B).
Efficient Scc1 cleavage requires that the protein be
phosphorylated near its cleavage site. This allows a
mode of regulation where shugoshin (Japanese for
guardian spirit) recruits PP2A to centromeres. PP2A
keeps Scc1 dephosphorylated. This inhibits its cleavage
and protects cohesin until shugoshin is released follow-
ing amphitelic attachment of the chromosome. This

A. Anaphase B
Sister chromatids separate
Cohesin Anaphase A: Chromatids approach
degrades poles (APC/CCdc20 active)
Interdigitated interpolar
microtubules bundled by PRC1
and stem body material to
form central spindle
DNA
Anaphase B: Spindle poles Microtubules
migrate apart (APC/C Cdh1active) Centrosomes

FIGURE 44.15 INTRODUCTION TO ANAPHASE. A, Summary of the major events of anaphase. B, Distribution of DNA (blue), microtubules
(red), and gamma tubulin (centrosomes [green]) in a mid-anaphase human cell. APC/C, anaphase-promoting complex/cyclosome; PRC1, protein
regulated in cytokinesis 1. (B, Images were recorded by Dr. Melpomeni Platani on the University of Dundees School of Life Sciences Imaging
Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
768 SECTION X n Cell Cycle

Hinge
A. S phase

Replication
fork
Cohesin
complex

Smc3

Smc1
Scc1

SA1

B C D E
Separase
Chromatin
loops Securin
APC/C
Ubiquitin Sister
Cohesin chromatids
Proteasome separate
Sister
chromatids

Active
Mitotic separase
chromosome cleaves Scc1
Metaphase/
anaphase
S phase Early mitosis transition Anaphase

FIGURE 44.16 REGULATION OF SISTER CHROMATID PAIRING BY THE COHESIN COMPLEX. AB, The cohesin complex forms a
35-nm diameter ring that links sister chromatids during DNA replication. At anaphase onset, degradation of its securin inhibitor liberates active
separase enzyme. CE, Separase then cleaves cohesin subunit Scc1, and the two sister chromatids are freed to separate from one another and
move toward opposite spindle poles.

mechanism is absolutely essential during meiosis, as
without it, it would not be able to segregate homologous
chromosomes from each other (see Fig. 45.12).
Securin can act as an oncogene in cultured cells
and is overexpressed in some human pituitary tumors.
Overexpression of securin may disrupt the timing of
chromosome segregation, leading to chromosome loss
and, ultimately, contributing to cancer progression.
Mitotic Spindle Dynamics and Chromosome
Movement During Anaphase
Anaphase is dominated by the orderly movement of
sister chromatids to opposite spindle poles brought
about by the combined action of motor proteins and
changes in microtubule length. There are two compo-
nents to anaphase chromosome movements (Fig. 44.15).
Anaphase A, the movement of the sister chromatids to
the spindle poles, requires a shortening of the kineto-
chore fibers. During anaphase B, the spindle elongates,
pushing the spindle poles apart. The poles separate
partially because of interactions between the antiparallel
interpolar microtubules of the central spindle and par-
tially because of intrinsic motility of the asters. Most cells
use both components of anaphase, but one component
may predominate in relation to the other.
Microtubule disassembly on its own can move chro-
mosomes (see Fig. 37.8). Energy for this movement
comes from hydrolysis of GTP bound to assembled
tubulin, which is stored in the conformation of the
lattice of tubulin subunits. Microtubule protofilaments
are straight when growing, but after GTP hydrolysis
protofilaments are curved, so they peel back from the
ends of shrinking microtubules (see Fig. 34.6). Several
kinesin motors influence the dynamic instability of the
spindle microtubules. Members of the kinesin-13 class,
which encircle microtubules near kinetochores and at
spindle poles, use adenosine triphosphate (ATP) hydro-
lysis to remove tubulin dimers and promote microtubule
disassembly rather than movement.
Kinetochores are remarkable in their ability to hold
onto disassembling microtubules. In straight (growing)
microtubules, the Ndc80 complex is mostly responsible
for microtubule binding. It binds to the interface between
and tubulin subunits. This interface bends in curved
(shrinking) microtubules, so Ndc80 cannot bind. This
could allow it to redistribute onto straight sections of
the lattice and thereby move away from the curved
protofilaments at the disassembling end. In metazoans
the Ska complex in the outer kinetochore binds and
tubulin subunits away from the interface, so it can bind

to curved (disassembling) protofilaments. At yeast kineto-
chores the Dam1 ring (green in Fig. 8.21) couples the
kinetochore to disassembling microtubules.
Anaphase A chromosome movement involves a com-
bination of microtubule shortening and translocation of
the microtubule lattice that result from flux of tubulin
subunits (Fig. 44.14). The contributions of the two
mechanisms vary among different cell types. When living
vertebrate cells are injected with fluorescently labeled
tubulin subunits, the spindle becomes fluorescent (Fig.
44.17). If a laser is used to bleach a narrow zone in the
fluorescent tubulin across the spindle between the
chromosomes and the pole early in anaphase, the chro-
mosomes approach the bleached zone much faster than
the bleached zone approaches the spindle pole. This
shows that the chromosomes eat their way along the
A of the poles involves interaction of the astral microtu-
Photobleached
zone
Time
Microtubule
disassembly
B 42 70 212 422
5 m
C 40 92 210 436
FIGURE 44.17 CHROMOSOMES MOVE ON SHRINKING
MICROTUBULES DURING ANAPHASE. A, Mitotic cells were
injected with a fluorescently labeled tubulin that was incorporated into
the spindle. Just after anaphase onset, a laser was used to photo-
bleach a stripe (white) across the spindle near the upper pole. The live
cell was monitored over time by fluorescence (B) and phase-contrast
(C) microscopy. In this mammalian cell, the chromosomes approach
the bleached stripe much faster than the stripe approaches the spindle
pole. In other organisms with higher rates of microtubule flux in their
spindles, the bleached zone would also move appreciably toward the
pole. The numbers are time in seconds. (BC, From Gorbsky GJ,
Sammak PJ, Borisy GG. Microtubule dynamics and chromosome
motion visualized in living anaphase cells. J Cell Biol. 1988;106:1185
1192, copyright The Rockefeller University Press.)
CHAPTER 44 n Mitosis and Cytokinesis 769
kinetochore microtubules toward the pole. In these
cells, subunit flux accounts for only 20% to 30% of
chromosome movement during anaphase A, and this flux
is dispensable for chromosome movement. In Drosophila
embryos, in which subunit flux accounts for approxi-
mately 90% of anaphase A chromosome movement, the
chromosomes catch up with a marked region of the
kinetochore fiber slowly, if at all.
Anaphase B appears to be triggered at least in part by
the inactivation of the minus-enddirected kinesin-14
motors, so that all the net motor force favors spindle
elongation. Four factors contribute to overall lengthen-
ing of the spindle: release of sister chromatid cohesion,
sliding apart of the interdigitated half-spindles, microtu-
bule growth, and intrinsic motility of the poles them-
selves (Fig. 44.7). During the latter stages of anaphase B,
the spindle poles, with their attached kinetochore micro-
tubules, appear to move away from the interpolar
microtubules as the spindle lengthens. This movement
bules with cytoplasmic dynein molecules anchored at
the cell cortex.
Anaphase B spindle elongation is accompanied by
reorganization of the interpolar microtubules into a
highly organized central spindle between the separat-
ing chromatids (Fig. 44.15). Within the central spindle,
an amorphous dense material called stem body matrix
stabilizes bundles of antiparallel microtubules and holds
together the two interdigitated half-spindles. Proteins
concentrated in the central spindle help regulate cytoki-
nesis. One key factor, PRC1 (protein regulated in cytoki-
nesis 1), is inactive when phosphorylated by Cdk kinase
and functions only during anaphase when Cdk activity
declines and phosphatases remove the phosphate groups
placed on target proteins by Cdks and other mitotic
kinases. PRC1 directs the binding of several kinesins to
the central spindle. The kinesin KIF4A targets Aurora B
kinase to a particular domain of the central spindle,
where phosphorylation of key substrates then regulates
spindle elongation and cytokinesis.
How can protein kinases such as Aurora B continue
to function during anaphase while protein phosphatases
are removing phosphate groups placed there by Cdks
and, indeed, Aurora B during early mitosis? One answer
is that the phosphatase activity is highly localized, con-
trolled by specific targeting subunits. Cdk phosphoryla-
tion can inhibit targeting subunits such as the exotically
named Repo-Man (recruits PP1 onto mitotic chromatin
at anaphase) from binding protein phosphatase 1 or
localizing to targets, such as chromatin in early mitosis.
When Cdk activity drops, Repo-Man (and other similar
targeting subunits) is dephosphorylated, and now targets
PP1 to chromatin, where it removes phosphates placed
there by Aurora B in the CPC. As long as phosphatases
are not specifically targeted to the cleavage furrow,
Aurora B can continue to control events there during
770 SECTION X n Cell Cycle

mitotic exit by phosphorylating key target proteins
required for cytokinesis.
Telophase
During telophase, the nuclear envelope reforms on the
surface of the separated sister chromatids, which typi-
cally cluster in a dense mass near the spindle poles (Fig.
44.18). Some further anaphase B movement may still
occur, but the most dramatic change in cellular structure
at this time is the constriction of the cleavage furrow and
subsequent cytokinesis.
Reassembly of the Nuclear Envelope
Nuclear envelope reassembly begins during anaphase
and is completed during telophase (Fig. 44.19). As in
spindle assembly, Ran-GTP promotes early steps of
nuclear envelope assembly at the surface of the chromo-
somes by releasing key components sequestered by
importin . These include several nuclear pore com
ponents, and one of the earliest events in nuclear enve-
lope reassembly involves binding of the nuclear pore
scaffold protein ELYS to chromatin. ELYS can recognize
DNA regions rich in A:T base pairs, so it is likely to
bind directly to the DNA. ELYS then recruits other
components of the nuclear pore scaffold and nuclear
pore trans-membrane proteins. The pore subsequently
matures as various peripheral components and elements
of the permeability barrier are added.
The mechanism of nuclear membrane reassembly is
debated. In cells where nuclear membranes fragments
into vesicles during mitosis, a Ran-GTPdependent
pathway directs at least two discrete populations of
vesicles to chromatin where they fuse to reform the
nuclear envelope. In cells where the nuclear membrane
is absorbed into the endoplasmic reticulum during
mitosis, reassembly involves lateral movements of mem-
brane components within the membrane network and
their stabilization at preferred binding sites at the periph-
ery of the chromosomes.
Lamin subunits disassembled in prophase are recycled
to reassemble at the end of mitosis. Lamina reassembly
is triggered by removal of mitosis-specific phosphate
groups and methyl-esterification of several COOH side

A. Telophase B
Cleavage plane
specified
Nuclear envelope
reassembles around Organized
chromosomes central spindle
assembles

Poles DNA
continue Microtubules
to separate Centrosomes

FIGURE 44.18 INTRODUCTION TO TELOPHASE. A, Summary of the major events of telophase. B, Distribution of DNA (blue), microtubules
(red), and -tubulin (centrosomes [green]) in a telophase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the University of
Dundees School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)

A B C

0 min 810 min 333 nm 25 min

FIGURE 44.19 SCANNING ELECTRON MICROSCOPY OF THE STAGES OF ASSEMBLY OF MEMBRANE VESICLES ON THE
SURFACE OF CHROMOSOMES IN A XENOPUS EGG CYTOSOLIC EXTRACT. A cell lysate containing membrane vesicles was added to
isolated chromatin from Xenopus sperm, fixed, and then imaged by scanning electron microscopy. Each panel shows the time of incubation prior
to fixation. (Micrographs courtesy of K.L. Wilson, Johns Hopkins Medical School, Baltimore, MD. A and C, From Wiese C, Goldberg MW, Allen
TD, etal. Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent envelope smoothing event.
J Cell Sci. 1997;110:14891502.)
 CHAPTER 44 n Mitosis and Cytokinesis 771

chains on lamin B (Fig. 44.6). Together with ELYS, B-type

lamins are among the earliest components of the nuclear
envelope to target to the surface of the chromosomes
during mid-anaphase. Either at this time or shortly there-
after, other proteins associated with the inner nuclear
membrane, including BAF, LAP2, and lamin B receptor
(see Fig. 9.10), join the forming envelope. Later during
telophase when nuclear import is reestablished, lamin
A enters the reforming nucleus and slowly assembles
into the peripheral lamina over several hours in the
G1 phase. If lamin transport through nuclear pores is
prevented, chromosomes remain highly condensed fol-
lowing cytokinesis, and the cells fail to reenter the next
S phase.
Cytokinesis
Cytokinesis divides a mitotic cell into two daughter cells
(Fig. 44.20). Cytokinesis depends on signals to specify
the cleavage plane (Fig. 44.21), assembly and constric-
tion of the contractile apparatus, specific alterations of
the cell membrane, and the final separation (abscission)
of the two daughter cells.
In animals, protozoa, and most fungi, a contractile
ring of actin filaments and myosin-II guides the separa-
tion of daughter cells at the end of mitosis (Fig. 44.2).
Myosin-II pulls on the ring of actin filaments, applying
tension to the plasma membrane, much like contraction
of smooth muscle (see Figs. 39.23 and 39.24). Because

A. Early cytokinesis B. Late cytokinesis C

Chromatin decondenses
New membrane Actomycin
inserted Nuclear substructures
contractile reform
Actomycin ring forms
Interphase microtubule
Midbody array reassembles
begins
to form Midbody

ESCRT III action leads to

separation (abscission) of the two cells

FIGURE 44.20 INTRODUCTION TO CYTOKINESIS. AB, Summary of the major events of cytokinesis. C, Distribution of DNA (blue),
microtubules (red), and -tubulin (centrosomes [green]) in a human cell undergoing cytokinesis. ESCRT, endosomal sorting complexes required
for transport. (C, Images were recorded by Dr. Melpomeni Platani on the University of Dundees School of Life Sciences Imaging Facility OMX
3DSIM Microscope and stored and processed in OMERO.)

A. Evidence that the cleavage furrow is positioned B. An organized central spindle is required for
midway between asters in eggs cleavage furrow formation and/or function
TOP VIEW
Glass rod pushed
down into egg

90

Sand dollar egg Microtubules

Chromosomes

Ectopic furrow Actin

ring

Profilin
Metaphase 1 Cytokinesis 1 Metaphase 2 Cytokinesis 2 Wild type mutant

FIGURE 44.21 IN EGGS, A CLEAVAGE FURROW FORMS MIDWAY BETWEEN SPINDLE ASTERS. IN ANIMAL CELLS, THE CENTRAL
SPINDLE IS IMPORTANT. A, A classic experiment in which a sand-dollar egg is caused to adopt a toroid shape. At cytokinesis 2, the egg
cleaves into four cells, and a furrow forms between the back sides of the two spindles. (For a description of this and other classic experiments
in cytokinesis, see the book by Rappaport in the Selected Readings list.) B, Left, A wild-type Drosophila spermatocyte undergoing cytokinesis,
with the contractile ring stained in yellow. Right, In a profilin mutant, no central spindle forms, and the cell fails to form a contractile ring.
(Micrographs courtesy Professor Maurizio Gatti, University of Rome, Italy. B, From Giansanti MG, Bonaccorsi S, Williams B, etal. Cooperative
interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev. 1998;12:396410.)
772 SECTION X n Cell Cycle
the contractile ring is confined to a narrow band of
cortex around the equator, it forms a cleavage furrow,
constricting the plasma membrane locally like a purse
string (Fig. 44.20). Signals from the mitotic spindle and
cell cycle machinery control the position of this ring (ie,
the relative sizes of the two daughter cells) and the
timing of its constriction.
Protozoa, animals, fungi, and plants use an evolution-
arily conserved set of components to implement differ-
ent strategies to separate daughter cells. For example,
both fission yeast and metazoan cells use signals from
polo kinase and a Rho-GTPase to direct the assembly of
a contractile ring of actin, myosin-II, and other conserved
components, even though the yeast has a closed mitosis
and the metazoans have an open mitosis. In animal
cells, contractile ring constriction provides the force that
remodels the cortex to generate the two daughter cells.
In contrast, in yeasts, which have a cell wall, contractile
ring constriction is thought to guide the orderly centrip-
etal growth of the cell wall septum, which contributes
force to overcome turgor pressure and invaginate the
plasma membrane. Plants lack myosin-II, so they divide
by targeted fusion of membrane vesicles to build a new
cell wall rather than constricting a cleavage furrow (Box
44.1 and Fig. 44.26). These differences reflect the fact
that widely divergent eukaryotes use variations of similar
themes for cytokinesis. Cytokinesis in prokaryotes is
genuinely different, since completely different proteins
are involved (Box 44.2 and Fig. 44.27).
Although cytokinesis has been studied for more
than 100 years, it has posed a number of challenges due
to its complexity at the molecular level. For example
genetic analysis of fission yeast revealed more than
150 genes that contribute to cytokinesis. RNAi-based
protein knockdown and molecular replacement analy
sis indicates that similar proteins participate in cytoki-
nesis of Caenorhabditis elegans, Drosophila, and
vertebrate tissue culture cells. Cytokinesis research typi-
cally employs living cells, although progress is being
made toward reconstituting some aspects of the process
in cell-free systems.
Signals Regulating the Position of
the Cleavage Furrow
Elegant experimental data from classic studies on fertil-
ized echinoderm eggs suggest that a cleavage stimulus,
emitted by the mitotic spindle, specifies the position of
the cleavage furrow midway between the poles and
perpendicular to the long axis of the spindle, thereby
ensuring that the cleavage process separates the daugh-
ter nuclei (Fig. 44.21). In fertilized eggs, the poles, with
their large astral arrays of microtubules (see Fig. 6.4B),
were regarded as the source of the cleavage stimulus, as
furrows can be induced to form midway between two
poles, even when no chromosomes are present. In addi-
tion, a signal emitted by the bundled microtubules of the
central spindle appeared to modulate the behavior of the
furrow signaled by the poles. We now know that the
central spindle does emit a positive signal directing a
cleavage furrow to form above it, while the poles con-
tribute by focusing that furrow at a point on the cortex
midway between them.
The molecular nature of the cleavage stimulus is now
beginning to be understood in animals. The following is
a simplified scenario:
1. During anaphase, overlapping microtubules between
the separating chromatids establish an ordered array
known as the central spindle. A key protein compo-
nent of this array is a protein heterodimer known as
centralspindlin. Centralspindlin is normally seques-
tered in the cytoplasm, but phosphorylation by the
CPC enables it to target to the central spindle. Dro-
sophila mutants that fail to form a central spindle
cannot initiate cytokinesis (Fig. 44.21B). In contrast,
C. elegans embryos that lack a central spindle can
initiate but not complete the process.
2. One of the components of centralspindlin recruits a
GEF (guanine exchange factor; see Figs. 4.6 and 4.7)
for the small GTPase RhoA. This Rho-GEF, Ect2, also
has a motif for targeting to the inside surface of the
equatorial plasma membrane.
3. Membrane associated Ect2 locally activates RhoA,
which then stimulates localized actin filament assem-
bly and activation of myosin-II to begin assembly of
the contractile ring.
Signals from the poles of the mitotic spindle contribute,
particularly in large invertebrate embryos, by confining
the zone of active RhoA to a narrow equatorial band
between the separating sister chromatids.
Assembly and Regulation of the Contractile Ring
Exposure of the cell cortex to the cleavage stimulus
culminates in the assembly of a contractile ring consist-
ing of a very thin (0.1 to 0.2 m) array of actin filaments
attached to the plasma membrane at many sites around
the equator (Fig. 44.22). Polymerization of the actin fila-
ments depends on formins (see Fig. 33.14). Small, bipolar
filaments of myosin-II are interdigitated with actin fila-
ments. The plasma membrane adjacent to this actin-
myosin ring undergoes alterations in its lipid composition
that may help recruit proteins important for the function
of the contractile ring.
Membrane furrowing requires actin and the motor
activity of myosin-II (see Fig. 36.7). In animals, the small
GTPase RhoA regulates actin polymerization by formins
as well as constriction of the ring. Many other proteins
are required for cytokinesis to go to completion. In their
absence, furrowing begins, but the cleavage furrows
ultimately regress, producing binucleated cells. These
supporting proteins include anillin, actin filament cross-
linking proteins, the CPC (Fig. 44.10) and the central-
spindlin complex, among many others. Anillin helps
 CHAPTER 44 n Mitosis and Cytokinesis 773

A. Early anaphase B. Late anaphase C

INCENP
Myosin II

Actin pointed ends

INCENP
Myosin II

Actin barbed ends

E. Myosin confocal

Central optical section

H. Contractile mechanism I. Equatorial section

G Actin
Myosin II

J. Grazing saggital section

FIGURE 44.22 ORGANIZATION OF THE CONTRACTILE RING. A, Organization of actin at the cell cortex prior to cytokinesis. B, Distribution
of actin and myosin at the start of ring contraction. C, INCENP (inner centromere protein) (red) concentrates at the site where the cleavage furrow
will form just before myosin (green). D, INCENP and myosin concentrate in the contractile ring during contraction. E, Confocal micrograph shows
the distribution of myosin in an optical cross-section contracting contractile ring. FG, Dividing invertebrate egg with DNA (blue) and actin (red)
in the contractile ring. H, Organization of actin and myosin filaments during cytokinesis. IJ, Electron micrographs showing actin filaments in
the contractile ring. Note the thick filaments that are thought to be myosin-II filaments (red arrowheads) and the thinner actin filaments (yellow
arrows). (CD, Courtesy William C. Earnshaw. E, I, and J, Courtesy P. Maupin, Johns Hopkins Medical School, Baltimore, MD. FG, Courtesy
Professor Issei Mabuchi, University of Tokyo, Japan. For reference, see Maupin P, Pollard TD. Arrangement of actin filaments and myosin-like fila-
ments in the contractile ring and actin-like filaments in the mitotic spindle of dividing HeLa cells. J Ultrastruct Res. 1986;94:92103; Maupin P,
Phillips CL, Adelstein RS, etal: Differential localization of myosin-II isozymes in human cultured cells and blood cells. J Cell Sci. 1994;107:30773090;
and Eckley DM, Ainsztein AM, MacKay AM, etal. Chromosomal proteins and cytokinesis. J Cell Biol. 1997;136:11691183.)

keep active myosin-II focused into an organized contrac-
tile ring throughout cytokinesis.
The CPC and the centralspindlin complex are both
required for animal cells to assemble the central spindle.
Consequently, if either of the two complexes is elimi-
nated in C. elegans, a contractile ring fails to form. Thus,
they appear to contribute to the cleavage stimulus, and
indeed, both require microtubules to localize to the site
of cleavage furrow formation as originally shown for the
cleavage stimulus. In addition, the CPC regulates the
timely completion of cytokinesis by blocking premature
activation of the ESCRT (endosomal sorting complexes
required for transport) complex, which has a key role in
the final separation of daughter cells (see later).
In fission yeast, with closed mitosis, the nucleus
determines the position of cleavage. Fission yeast assem
ble a contractile ring along a well-defined pathway by
recruiting proteins from cytoplasmic pools (Fig. 44.23).
During interphase, assemblies of proteins called nodes
form on the inside of the plasma membrane around the
middle of cell. Prior to mitosis, an anillin-like protein
leaves the nucleus and joins these nodes. During pro-
phase, myosin-II, a formin and other contractile ring
proteins join the nodes. When the formin polymerizes
774 SECTION X n Cell Cycle

Interphase
-60 Mid1p (anillin-like actin patches
protein) exits nucleus
Anillin-like
Cell wall

Formin Myosin-II
-10 Nodes containing
anillin, myosin-II and formin Profilin
assemble around equator

0 SPBs separate Nodes condense into

a contractile ring of
+5 Anaphase A actin filaments and myosin-II
Time (min)

+10 Anaphase B
elongates mitotic Contractile ring
spindle matures by addition of
actin binding proteins

+30 End Anaphase B

Signal from cell cycle via SIN

pathway triggers constriction of
+40 Constriction begins
contractile ring and deposition of
cell wall material to form a septum

Plasma membrane fusion

completes cytokinesis
+70 Constriction ends

FIGURE 44.23 CYTOKINESIS IN FISSION YEAST SCHIZOSACCHAROMYCES POMBE. During interphase, microtubules (red) position
the nucleus in the middle of the cell. Actin filaments concentrate in small patches (yellow) in the cortex at the two growing ends of the cell (see
Fig. 33.1). The mitotic spindle is inside the nucleus, as the nuclear membrane does not break down during mitosis. As the cell enters mitosis,
an anillin-like protein moves from the nucleus to the equatorial cortex, where it sets up nodes of proteins, including myosin-II and a formin. The
formin grows actin filaments (yellow), and myosin-II pulls the nodes together into a continuous contractile ring. At the end of anaphase a signaling
system consisting of a GTPase and three protein kinases (the septation initiation network [SIN]) triggers constriction of the contractile ring and
associated synthesis of new cell wall to form a septum. The septum is a three-layered structure, with the primary septum flanked by two secondary
septae. Digestion of the primary septum separates the daughter cells. (For reference, see Wu J-Q, Kuhn JR, Kovar DR, etal. Spatial and temporal
pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev Cell. 2004;5:723734.)

actin filaments, myosin-II pulls the nodes together into a

ring around the equator of the cell (Fig. 44.23).
Contractile ring assembly in animal cells shares many
properties with fission yeast, but is less completely
understood. The decline in the activity of cell cycle
kinases at the onset of anaphase is part of the trigger,
since they inhibit centralspindlin components through
metaphase. INCENP and anillin move from the inter-
phase nucleus to the cortex around the cell equator in
early anaphase (Fig. 44.22). Formins and profilin polym-
erize some new actin filaments, but preexisting actin fila-
ments are recruited into the contractile ring from
adjacent areas of the cortex. Myosin-II is dispersed
throughout the cytoplasm until anaphase, when it con-
centrates in the cortex, especially around the equator
where the furrow forms. The myosin-II is derived from
various interphase structures including stress fibers (see
Fig. 33.1) that break down during prophase.
Constriction of the Cleavage Furrow
Contractile rings of echinoderm eggs produce enough
force to invaginate the plasma membrane and form the
cleavage furrow, although many details are still being
studied. Constriction of the ring probably involves a
sliding filament mechanism similar to muscle (see Figs.
39.9 and 39.23), but little is known about how the
contractile ring is attached to the plasma membrane.
During the early stages of furrowing, contractile rings
maintain a constant volume, but then disassemble as
they constrict further.
The role of myosin-II as the motor for cytokinesis was
established by microinjection of inhibitory antibodies
into echinoderm embryos and confirmed by genetic
inactivation in the slime mold Dictyostelium. Slime mold
amoebas lacking the myosin-II heavy chain round up
during mitosis and complete nuclear division but cannot
 CHAPTER 44 n Mitosis and Cytokinesis 775

form a normal cleavage furrow. Mutant cells accumulate

A
many nuclei, because the mitotic cycle continues. Mutant
cells can divide on a substratum using pseudopods to
pull themselves apart into smaller cells.
Constriction of the contractile ring is regulated so that
it does not begin until after the onset of anaphase B,
when sister chromatids are well separated. In fission Midbody ring
yeast, a signaling pathway called the septation initiation B. Abscission (centralspindlin, anillin, cep55)
ESCRT I and ESCRT II
network (SIN) initiates constriction. Much less is known ESCRT III
Midbody
in other cells. Vps4
Microtubules

Abscission
As the contractile ring pulls the cell membrane inward, ESCRT I

the single cell that entered mitosis is gradually trans-

formed into two daughters joined by a thin intercellular
bridge (Fig. 44.20). This process requires a significant
net increase in the surface area of the cell. New plasma
FIGURE 44.24 ABSCISSION IN ANIMAL CELLS. Proteins
membrane is inserted adjacent to the leading edge of associated with overlapping bundles of microtubules form the midbody.
the furrow. The source of the new membrane appears The midbody ring links the midbody to the membrane, and nucleates
to be recycling endosomes (see Chapter 22), so addition the formation of ESCRT (endosomal sorting complexes required for
of membrane to the cleavage furrow is a specialized form transport) III filaments that constrict the membrane to divide the
daughter cells.
of exocytosis. Fusion of vesicles providing the new
membrane depends on specific syntaxins, t-SNAREs
(soluble N-ethylmaleimide-sensitive factor attachment
protein receptors) (see Fig. 21.15) that promote vesicle
fusion along the secretory pathway.
The plasma membrane in the cleavage furrow has a
discrete composition. In budding yeast, this compart-
ment is delineated by rings made from polymers of
septins, a family of GTP-binding proteins recruited by
anillin. Septins are essential for cytokinesis in Saccharo-
myces cerevisiae but not fission yeast.
In most animal cells, constriction of the cleavage
furrow ultimately reduces the cytoplasm to a thin inter-
cellular bridge between the two daughter cells. The
FIGURE 44.25 INCOMPLETE CYTOKINESIS IN A DRO-
intercellular bridge contains a highly ordered, antiparal-
SOPHILA EGG CHAMBER LEAVES CELLS JOINED BY RING
lel array of microtubules derived from the spindle with CANALS. Colocalization of actin (red) and the ring canal protein
a dense knob, the midbody, at its center (Fig. 44.20). HtsRC (green) in the ring canals makes them appear yellow. In the
Isolated midbodies contain more than 160 proteins, with Drosophila egg chamber, ring canals connect nurse cells to each other
approximately one-third involved in various aspects of and to the oocyte. Late in oocyte development, nurse cell contraction
forces their cytoplasmic contents through the ring canals and into the
membrane trafficking.
oocyte. This helps the oocyte gain the stockpile of components that
The midbody is encircled by a dense ring of proteins is needed for early development of the fly embryo. (Courtesy Andrew
that includes centralspindlin, anillin, and a centrosomal Hudson and Lynn Cooley, Yale University, New Haven, CT.)
protein known as Cep55. Interactions between anillin
and membrane-associated septin filaments tether the
membrane to the ring. Cep55 recruits the ESCRT (endo- Ultimately, disassembly of the ESCRT filaments leads to
somal sorting complexes required for transport) III separation of the two daughter cellsabscission.
complex, proteins with important roles in vesicle In some tissues, intercellular bridges remain open as
budding events such as formation of multivesicular ring canals. After several rounds of nuclear division
bodies (see Chapters 22 and 23). In the intercellular with incomplete cytokinesis, the network of cells main-
bridge, ESCRT III forms a helical filament that spirals tains cytoplasmic continuity as each former contractile
around the inner surface of the membrane, becoming ring matures into a larger ring canal. During Drosophila
more and more constricted as it grows away from the oogenesis, four rounds of nuclear division with persis-
midbody (Fig. 44.24). ESCRT III also recruits factors that tent ring canals creates 15 nurse cells, all in continuity
disassemble the bundled microtubules in the intercellu- with the oocytes (Fig. 44.25). The cytoplasmic continu-
lar bridge, allowing the membrane to constrict further. ity through ring canals allows nurse cells to transfer
776 SECTION X n Cell Cycle
BOX 44.1 Cytokinesis in Plants
Chromosome segregation is similar in plants and animals, but
cytokinesis is very different because plants lack myosin-II
and do not form a conventional contractile ring (Fig. 44.26).
Myosin-II appeared during evolution in the common ances-
tor of amoebas, fungi and animals, after branching from
plants (see Fig. 2.4B). Plants also lack dynein, so microtubule
dynamics in mitosis are regulated by some of the more than
20 different plus-end and minus-enddirected kinesins that
are expressed in mitotic cells. In a further difference from
animals, plants also lack centrosomes, and during interphase,
microtubules radiate out from the surface of the cell nucleus
in all directions. In mitosis, the spindle does not focus to
sharp poles at metaphase; instead, it assumes a barrel shape
with broad, flat poles. Early in mitosis, a band of microtu-
bules and actin filaments forms around the equator of the
cell adjacent to the nucleus. This so-called preprophase band
disassembles as cells enter prometaphase. Because the entire
cell cortex is covered by a meshwork of actin filaments,
disassembly of the preprophase band actually leaves an actin-
poor zone in a ring where cytokinesis will ultimately occur.
This is called the cortical division site, and it is marked by
the tethering of specific kinesin motors. In late anaphase,
two nonoverlapping, antiparallel arrays of microtubules
form over the central spindle. This structure, the phragmo-
plast, gradually expands laterally until it makes a mirror-
symmetric double disk of short microtubules oriented
parallel to the spindle axis with their plus ends abutting the
plane of cell cleavage. Golgi vesicles, containing cell wall
Chromosomes Cytokinesis in higher plants Early phragmoplast
Cortical
actin
Preprophase Cortical
band actin-
(microtubules) depleted
zone
Prophase Metaphase
FIGURE 44.26 CYTOKINESIS IN HIGHER PLANTS. See the text for details.
their cytoplasm into the developing egg, thus greatly
increasing its stockpile of proteins and messenger RNAs
available for use in early development. In mammals,
incomplete cytokinesis in the testis results in ring canals
connecting several hundred developing sperm cells.
Exit From Mitosis
To exit from mitosis, cells must inactivate the Cdk1
kinase. This reverses the biochemical and structural
changes that are characteristic of mitosis and prepares
the cell for proliferation in the next cell cycle.
materials (see Fig. 32.13), move along phragmoplast micro-
tubules to the equator, where they fuse due to the action of
cytokinesis-specific soluble N-ethylmaleimide-sensitive factor
(NSF) attachment protein receptor (SNARE) proteins (see
Fig. 21.15), forming a membrane network that becomes the
new plasma membrane and laying down the material that
will become the new cell wall. Dynamin-related proteins also
participate in shaping the newly forming plasma membrane.
Thus, the membrane fusion machinery used for cytokinesis
by eukaryotes likely came from the last eukaryotic common
ancestor. Actin filaments polymerized by formins and
myosin-VIII help position the phragmoplast in the cell. As
the zone of newly deposited membrane expands radially, the
ring of microtubules surrounding it similarly expands. Even-
tually, the new membrane reaches the lateral cell periphery,
and fusion with the plasma membrane separates the two
daughter cells. The cortical division site, not the spindle,
determines the site of cleavage. This was shown by centri-
fuging mitotic cells to displace the spindle from the central
location where it initially formed. Late in mitosis, the phrag-
moplast formed at the midzone of the displaced spindle, but
this phragmoplast then migrated to the plane of the prepro-
phase band, where cytokinesis occurred. Since plant cells
have cell walls and do not move, the orientation of cleavage
planes critically determines the morphology of the organism.
The hormone auxin can influence cleavage, giving rise to
asymmetric division of daughter cells, but the underlying
mechanism is not yet known.
Late phragmoplast
Golgi
vesicles
Early cytokinesis Late cytokinesis Daughter cells
Yeast cells use a signaling pathway to terminate
mitosis, promote contraction of the contractile ring, and
initiate septation. These pathways, called the mitotic exit
network (MEN) in budding yeast and the SIN in fission
yeast, involve a small GTPase and protein kinases. Cdk
kinase activity suppresses the pathway until anaphase,
when Cdk activity drops sharply. The MEN GTPase is
associated with one spindle pole body (the yeast version
of the centrosome), while its key regulator, a GTP
exchange factor, is located in the bud. Elongation of the
mitotic spindle during anaphase B moves the GTPase
into the bud, where it is activated.

BOX 44.2 Cytokinesis in Bacteria
The strategy for cytokinesis in bacteria is similar to that in
animal cells (Fig. 44.27), but the molecules are completely
different. Cleavage of most bacterial cells depends on
a ring of the FtsZ protein (filamentous temperature-sensitive;
mutants in fts genes cannot divide and make long filaments
on cells). This is called the Z ring. FtsZ is the prokaryotic
homolog of eukaryotic tubulins, but it assembles into fila-
ments rather than tubules. As for tubulins (see Fig. 34.4),
FtsZ polymerization requires bound GTP and hydrolysis of
this GTP destabilizes the polymers. Although purified FtsZ
forms rings that use energy from GTP hydrolysis to deform
lipid vesicles, the main function of the Z ring seems to be to
coordinate the assembly of a complex of proteins (divisome)
including an actin homolog FtsA and number of transmem-
brane proteins. The transmembrane proteins synthesize cell
wall materials to form the cleavage furrow.
The Z ring is positioned at the cell equator of Escherichia
coli by the action of three gene products: MinC, MinD, and
MinE (minicell mutants divide at inappropriate locations and
give birth to tiny cells). MinD is an enzyme that recruits MinC
Min C/D Min E Cytokinesis in E. coli
inhibitor Nucleoid
2 minutes
FIGURE 44.27 CYTOKINESIS IN THE BACTERIUM ESCHERICHIA COLI. See the text for details.
The MEN kinases downstream of the GTPase activate
the phosphatase Cdc14p by releasing it from sequestra-
tion in the nucleolus. Cdc14p inhibits Cdk kinase activity
in two ways: (a) it inhibits the degradation of a Cdk
inhibitor protein, and (b) it dephosphorylates Cdh1,
which binds the APC/C and triggers the degradation of
B-type cyclins and other proteins. Cdc14p also triggers
other events during anaphase, including the transfer of
chromosomal passenger proteins to the central spindle.
In metazoans mitotic exit is triggered by the inactiva-
tion of Cdk1 and other mitotic kinases. This transition
is irreversible, in part because cyclins and Aurora
(and other kinases) are degraded. PP2A and its inhibitory
kinase Greatwall (see Chapter 40) replace Cdc14
in mitotic regulation in metazoans. Greatwall activity
requires Cdks, so when Cdk activity declines, PP2A
is released from inhibition. When directed to targets
CHAPTER 44 n Mitosis and Cytokinesis 777
to the cell cortex, where it inhibits Z-ring formation. MinE
is an antagonist of MinC/MinD action. This system works in
a truly remarkable way. MinE forms a ring at the cell equator
that migrates along the inner surface of the cell membrane
until it reaches the end of the cell, at which point it disas-
sembles. The ring then reforms in the center of the cell and
sweeps toward the other end of the cell. As it moves, MinE
inactivates the MinC/MinD inhibitory complex on the cell
cortex. The inhibitory complex rapidly reestablishes itself on
the cell cortex behind the moving MinE ring. It takes
approximately 2 minutes for each sweep of the MinE ring
along half of the cell, and this cycle is repeated continuously
until the FtsZ ring assembles at the cell center. Bacillus
subtilis uses an alternative mechanism to position the Z ring
for cytokinesis.
Chloroplasts use a homolog of FtsZ for their division, and
FtsZ has been detected in mitochondria of certain primitive
eukaryotes. Mitochondria of higher eukaryotes appear to use
another GTPase, dynamin, to coordinate their fission (see
Biogenesis of Mitochondria in Chapter 19).
FtsZ ring
Zone of minimal
Min C/D
throughout cells by their specificity, determining sub-
units PP2A and PP1 remove many of the phosphates
placed on target proteins by the mitotic kinases. Targets
include chromatin, where phosphorylation during
mitosis had displaced factors involved in both gene
activation and repression. Removal of those phosphates
allows the interphase regulation of gene expression to
resume. Dephosphorylation of other targets allows
intermediate filaments to reform, nuclear envelope reas-
sembly plus the resumption of RNA transcription, protein
translation, and membrane trafficking.
ACKNOWLEDGMENTS
We thank David Burgess, Iain Cheeseman, Per Paolo
DAvino, Arshad Desai, Tatsuo Fukagawa, Gary Gorbsky,
Karen Oegema, Jonathon Pines, and Graham Warren for
778 SECTION X n Cell Cycle
their suggestions on revisions to this chapter. We thank
the Dundee Imaging Facility for access to the OMX and
help with microscopy.
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