Title: Actin and Cell Motility

Ryan Carroll
19496206

Introduction:
Actins are a group of proteins which form thin filaments within the cytoskeleton and muscle
fibrils. Actins are vital to the contractile mechanism of muscles and the movement of nonmuscle
cells via the formation of dynamic structure which exist in a state of constant disassembly and
reassembly.

In this review we will summarize the primary structures and functions of these structures and
finally analyse how these structures may be used in cell motility.

Mini review:

Structures:
Actin filaments:

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to
probe their environment
filopodia, in theory, can protrude indefinitely because actin filaments turn over continuously by
releasing monomers from the rear for reuse at the front.
Filopodia will buckle and retract after a period of elongation.
Actin bundles are best understood in the leading edge filopodia.
The Actins of the filopodia in the leading edge span the entire length of the filopodia, their
barbed end is at the tip of the filopodium.

Lamellipodia are a thin sheet-like branched network of actin filaments, which generates force for
advancing the leading edge.
Falt undulating cellular protrusions at the leading edge of the cell.
Lamellipodia can generate much greater protrusive forces than filopodia.
Lamellipodia serve as the major cellular engine to propel the leading edge forward.
Lamellipodia serve as navigation devices guiding the cell around obstacles it may detect the
chemical and physical properties of the substratum.
Lamellipodia can navigate both in 2D and 3D space.

Microspikes + retraction fibers:

These are rod-shaped actin filaments very similar to filopodia,
Filopodia, microspikes, and retraction fibers are capable of morphing into one another.
Microspikes are parallel actin bundles within the lamellipodium
Retraction fibers are long thin cellular processes that remain attached to the substratum after cell
withdrawal. Retraction fibers form during the retraction of the cell edge.

Microvilli:
These are nonmotile protrusions of the intestinal epithelial cells which increase the cell surface to
increase the efficiency at which nutrients are absorbed.
The bundle of actin within the microvillus is structurally similar to filopodia having
barbed/pointed ends acting as expected with one notable exception, the rate of the
treadmilling mechanism is several times slower than the treadmilling mechanism as observed
in the filopodia.
In microvilli the actin bundle is attached to the plasma membrane by the myosin 1-A gene.
Myosin 1-A is believed to regulate membrane tension in the microvilli.

Stereocilia:
These are specialized microvilli used for hearing and balance, ranging in size from 1 to 100
micrometers.
Stereocilia will have 10 times more actin filaments than microvilli, with these filaments playing a
very important structural role.
The stereocilia are cylindrical.
Fimbrin and espin are responsible for cross-linking actin filaments in stereofilaments.
The taper toward the stereocilia base allows them to pivot in response to vibration.

The Actin cytoskeleton in movement:

Protrusion:
Actin sub-units (G actin and F actin which is added?) are added to the barbed end of the
filopodium.
These sub-units then move away from the tip and form part of the filament lattice, and are finally
released at the rear of the filopodium.
This process is known as treadmilling.
As the filopodial tip extends forward the actin bundle slides backward.
This is a balancing act between protrusion (the extension) and retrograde (the retraction)
Retrograde may occur at a rate that counteracts protrusion.
Protrusion by branched networks:
The protrusion of lamellipodia at the leading edge of migrating cells is the best-studied
protrusion.
Branched networks promote various endocytic events, motility, and biogenesis of intracellular
organelles. [1]
Elongation:
Formation of long actin filaments, e.g filopodia is difficult due to capping protein. Capping
proteins will bind to the barbed end of barded ends of the filament which terminates the
elongation process. VASP /Ena & Formins act as competitors for the capping proteins,

Due to the long thin shape of the filopodia, it is difficult for actin sub-units to reach the active
filopodial tip where the sub-units are polymerized and added to the filament lattice via simple
diffusion and so other methods must be used.
Myosin X was found to promote elongation by delivering actin sub-units and VASP/Ena to
the filopodial tip through its motor activity.

Depolymerization:
The pointed ends of actin filaments release the actin subunits at the end of treadmilling.
The rate at which the actin subunits dissociate at the pointed end is slower than the rate at which
subunits are added to the barbed end, however, the rate of dissociation can be increased by
ADF and myosin II which act as severing promotors.

Shape maintenance + membrane interactions:

The actin filament bundle acts as a scaffold that maintains the shape of the filopodia.

Interaction with the extracellular environment:

Filopodia act as probing fingers which may sense, reach and grab a target. Filopodia may
establish communication with another cell or extracellular molecule and in doing so guide
cellular locomotion.
Filopodia can reach out and grab pathogens and internalize them e.g phagocytes. The leading
edge of filopodia has an array of adhesion and signaling proteins.

Dynamics

Structure & Dynamics:

Lamellipodium is filled with a network of long diagonal actin filaments orientated with their
barbed ends towards the leading edge.
We can find branched actin filaments that are formed when the pointed end of an actin
filament binds to the side of another filament with a formation angle of approximately 70°.
These branched actin networks may be referred to as dendritic actin networks.
They vary in density, length, and frequency depending on cell types and protrusion.
A low density of long-branched actin filaments will allow for a faster rate of protrusion
however due to the low amount of cross-linkage these protrusions will be prone to buckling.
A high density of long-branched actin filaments will limit the rate at which protrusions can
advance but will be less prone to buckling due to the greater about of cross-linkages.

Actin subunits in lamellipodia are added near the plasma membrane and move away from the
membrane similarly to filopodia, however, the rate at which actin subunits are swapped out is
much higher.
In contrast to filopodia, Lamellipodia replace the actin subunits throughout the entirety of the
filament.
This mechanism of actin swapping results in the creation of new daughter filaments from the
mother filaments, this process is known as dendritic nucleation.
After the daughter filament spends a short period of time elongating, its barber end is capped
and the elongation is terminated. These daughter filaments are then disassembled by
debranching and severing of actin filaments, which are then depolymerized. Very different
from the treadmilling of filopodia.

The dendritic nucleation mechanism has several distinct advantages for the generation of pushing
forces in protrusion,
Short stiff filaments allow for a higher density of cross-linkages which prevent buckling.
As the daughter filament is connected to the mother filament throughout the entire lamellipodia
the filaments can convert the energy from polymerization into useful work easier.
The repetitive branching of the actin network in the lamellipodia allows the lamellipodia to
expand easier as it can increase the amount of site for nucleation and decrease the rate of
capping.
According to Brownian ratchet model, these angled actins will also push against the membrane
more effectively when compared to straight actin filaments.

Bundling + Cross-linking:
During elongation the actin filament will buckle if they exceed a length of 0.7 micrometers, this
limits its pushing power.
The issue of buckling may be corrected by cross-linking multiple filopodial filaments.
The main protein responsible for bundling is Fascin, a small bivalent monomeric protein.
Fascin cross-links F-actin microfilaments into tight parallel bundles.
Depolymerization:
The pointed ends of actin filaments release the actin subunits at the end of treadmilling.
The rate at which the actin subunits dissociate at the pointed end is slower than the rate at which
subunits are added to the barbed end, however, the rate of dissociation can be increased by
ADF and myosin II which act as severing promotors.

Molecular machinery:
There are a large number of accessory molecules that assist the actin in creating healthy
dendritic networks.
Arp2/3 complex, capping protein and ADF/Cofilin and an Arp 2/3 activator is responsible for
the treadmilling mechanism.

Arp2/3 complex is responsible for the nucleation of the daughter filaments from the mother
filaments in lamellipodia and the capping of the pointed ends of daughter filaments.
The Arp2/3 complex exists in an inactive state until a nucleation-promoting factor (NPF) binds to
it which triggers actin nucleation in the lamellipodia.
Another important protein is the Wiskott-Aldrich syndrome protein (WASp), WASp stimulates
the actin nucleation activity of the Arp2/3 complex.
The capping of the barbed ends of the actin filaments is performed by heterodimeric capping
protein (CP/CapZ) which blocks actin polymerization. CP/CapZ is made up of alpha and beta
subunits. [5]
CP collaborates with Arp2/3 and the WASp proteins in order to create a dense highly regulated
network of short branched filaments.

ADF/Cofilin family of proteins are responsible for the severing of capped filaments in the
lamellipodia, it does this through a process of ATP hydrolysis. [4]
ADF/Cofilin is generally restricted to the actin rich lamellipodia in motile cells.
ADF greatly improves the speed at which treadmilling may occur for the actin filament filopodia.

Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the protruding
edge of motile cells. Cortactin binds to Arp2/3 and is an essential molecule for dendritic
nucleation, additionally, it can stabilize the newly formed branching points.

Cell motility:
Phagocytosis:
Phagocytosis is the internalizaton of external particles into the cell and uses the protrusion of
the lamellipodia to enguf the particulate. Various signalling pathways can activate
phagocytosis such as tyrosine kinases and/or serine/threonine kinase C, this causes
phosphorlation at the receptor sites of phagocytosis.

Comet tail motility:

Actin comet tails are structures made up of polymerized actin sub units found in motile
bacteria such as Listeria . These comet tails uses the forces generated from actin
polymerization to propel them at speeds between 3 and 87 micrometers/ minute.[2]
 Contractions of the actin cytoskeleton:
The actin cyto skeleton generates a contractions via the process of myosin and actin filaments
sliding across one another.
Myosin is a superfamily of motor proteins which converts ATP into mechanical energy which
generates movement in the tissues. Class II myosin specifically are responsible for
contractions .
Myosin II polymerises into a bipolar filament which is held together with alpha helical and
coil-coiled interactions. These bipolar filamanets pull the actin filaments together which
causes contraction.
Actins are organised into bundles as this allows the contractile system to operate more efficiently.
Actin filaments are attached to a load which causes myosin driven contractions, the actins are
attached tot e load with their barbed ends and the myosin filaments then move towards these
barbed ends pulling the actin and so the load too.
We can also observe the principle of actin/myosin contractile systems in nonmuscle cells.
These actin myosin systems are less organised than their muscle counter-parts, being
organised into imperfectly aligned actin/myosin systems. However as these systems have less
organised it allows them to be more variable and dynamic.

Contractile forces in motile cells:

In migrating cells contractile forces can tear the lamellipodia from the cell membrane which
causes the cell edge to retract or the lamellipodia may be reinforced which will be used to
advance the protrusion. In the migrating cell typically it is the leading edge which is
reinforced and the rear of the cell which is ruptured. This bidirectionallity of
reinforcement/tearing reinforces the direction of cell motility.
Isomeric tension in actin myosin bundles is of great importance as it is responsible for
maintaining the cell structure and the physical properties of the cell surface.

Conclusion:
In conclusion actin structures such as the filopodia and lamellipodia are responsible for the
majority of cell motility in eukaryotic cells. The plasma membrane protrusions which form as
a result of the actin cytoskeleton is highly regulated process regulated by man differing
factors.

References:

(1) Schaks M. et al. (2019) Actin dynamics in cell migration. Essays in Biochemistry 63, 483-495
(1) Svitkina, T. (2018) The actin cytoskeleton and actin-based motility. Cold Spring Harb.
Perspect. Biol. 2018;10: a018267
(2)Robbins JR, Barth AI, Marquis H, de Hostos EL, Nelson WJ, and Theriot JA. Listeria
monocytogenes exploits normal host cell processes to spread from cell to cell. J. Cell Biol. 1999;
146(6):1333-50. [PMID: 10491395]
(4)Bernstein, B. W., & Bamburg, J. R. (2010). ADF/Cofilin: A Functional Node in Cell Biology.
Trends in cell biology, 20(4), 187
(5) Yarar D, To W, Abo A, Welch MD. The Wiskott-Aldrich syndrome protein directs
actin-based motility by stimulating actin nucleation with the Arp2/3 complex. Curr
Biol. 1999