Dissecting the Complexity of Cell Structure 2: Microfilaments

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Dissecting the Complexity of Cell Structure 2
Microfilaments

• How actin-based polymers contribute to cellular
  structure, behavior, and motility

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An overview of the actin cytoskeleton in a
cultured epithelial cell, using antibodies and
immuno- fluorescence. Actin is at the cell
cortex and also organized into fibers that extend
through the cell.
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Formation of microfilaments (MFs) from the
protein actin
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Actin polymerization is controlled, in part, by
an initiating complex that includes actin-like
proteins
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The Arp2/3 complex not only initiates
MF polymerization, it can bind to the walls
of existing MFs, promoting the formation
of branches, which turn that region of the
cell into a gel (as opposed to a sol).
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Actin at the edge of a cultured cell, looking at
all actin (top) or only recently added actin
(bottom). Actin polymerizes at the cell
periphery
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Direct evidence for the addition of actin
monomers to the membrane-end of MFs
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Actin polymerization is also regulated by small
proteins that bind to soluble actin and modify
its behavior in solution. Here is profilin,
which catalyzes the exchange of ADP for ATP in
the actin cleft, increasing the pool of
polymer- ization-competent monomer
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There are also proteins that retard MF growth
While profilin enhances MF growth by
increasing the concentration of
ATP-actin, Thymosin reduces it by making
a complex that will not polymerize
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Diagramatic representation of the pathways The
help to regulate MF formation
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So how does a complex process like this actually
work?

• Amoeboid motility does not require MTs it
  proceeds normally in the presence of MT poisons
• It is immediately poisoned, however, by drugs
  that block actin polymerization, like
  Cytochalesins D and E or Latrunculin A
• It fails in several mutant strains that lack key
  actin assembly proteins (though it is amazingly
  robust to mutation)
• Infer, to understand amoeboid movement and
  related kinds of cell motion we need to
  understand the control of actin polymerization
  and organization

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Proteins that bind to the walls of MFs can
bind them together in different geometries
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Four proteins that bind the sides of MFs
and bundle them into different geometries
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Diagrams of examples of MF bundling
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TEM image and diagram of MF Bundling in a
microvillus
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Diagram of MF web formed when cross-linking is
done by the long, fibrous protein, Filamin
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When filamin is under-expressed, due to mutation,
cell morphology and motility are abnormal.
Cells depleted for Cell
expressingfilamin by LOF mutation normal
filamin levels
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Like MTs, MFs can bind some associated proteins
that alter the properties of the polymer.
Cofilin binds to F-actin and distorts the
polymer, making it less stable.
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Gelsolin also bind the MF wall, distorting is and
inducing breaks that shorten the average fiber.
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Gelsolin and proteins like it can break up
MF either in vitro (as shown here) or in vivo,
leading to rapid reshaping of the actin
cytoskeleton
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Breaks in MFs mean both more and shorter MFs
therefore more ends (for a given amount of
polymer). Thus, both growth and shrinkage of
polymer can be faster after MF severing.
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Capping proteins, like Cap-Z can silence one MF
end for further subunit addition in this case it
is the fast-growing (plus) end that is turned
off.
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All these processes can be regulated to make For
a quite complex behavior of the MFs in
vivo. Blood platelets (thrombocytes) looking as
they do in circulating blood (left), after
attachment to a surface (center) and during a
platelet reaction, where they attach to a
substratum and contract. All this is MF
assembly-dependent.
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But What Initiates MF Polymerization at the PM?
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Projection morphology and other actin-dependent
shape changes suggest that controlled
polymerization can also give rise to
unidirectional fibers, the filopodia

• Microvilli are of this form
• Dynamic projections from not only platelets but
  also neuronal growth cones are of this form
• Even projections from sperm and egg, involved in
  fertilization, are of this form
• There must be a membrane-associated actin
  initiator that does not involve branching

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Formins are now recognized as molecules that can
initiate MF polymerization at the PM and allow
continued addition of actin at the PM while an
association with the membrane is maintained

• Formins contain conserved domains, the
  Formin-homology (FH) domains. FH1 and 2 define a
  comparatively short polypeptide that can serve as
  an efficient nucleator of MF polymerization
• Formin-initiated polymerization adds actin
  subunits AT the PM, pushing the already formed MF
  away
• This allows PM-controlled events to regulate
  aspects of MF organization, much as MTOCs control
  MT formation

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Movements of PM-associated cortical actin can
affect the position and organization of other
components of cytoplasm, like MTs
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Conversely, the behavior of MTs can affect
behavior of the actin cortex
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Actin stress fibers are initiated by and
attached to the cell cortex.Cell margin seen
with The same region seen withoptics
that show proximity fluorescence optics and
anbetween the cells surface antibody that
lights up actin
and the underlying substratum
These are focal adhestions that mark site of
cell attachment
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Another view of stress fibers (green) and focal
adhesions, stained with antibodies to vinculin,
a component of stress fiber adhesion sites
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There are specific trans-membrane proteins and
adaptor proteins that connect MFs with the
extracellular milieu
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To promote tissue strength, cadherins are
specifically but non-covalently bound to the MFs
of the cytoskeleton. The linking proteins
include catenins, which play a role in signaling
between tissues.
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Cadherins bind the MF cytoskeleton of one cell to
that of its neighbors, forming a mechanical
unit. This coupling contributes to the
mechanical integrity of a tissue.
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In some cells, cadherins and actin MFs form
ordered arrays that can work like a contractile
ring and control the cells diameter.
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Integrins are membrane proteins that bind cells
to the Extra-Cellular Matrix (ECM). The
integrins make bonds between the actin
cytoskeleton and the fibers of the ECM, such as
collagen and fibronectin.
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Transmembrane proteins link the actin-dependent
cytoplasmic motility that pulls on MTs with
extracellular material, such as beads that can
attach to the cells surface
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Thus, we can recognize a mechanical continuum
that runs from cytoplasmic MFs, controlled in
part by MTs, through the plasma membrane to the
molecules of the extracellular matrix (ECM).

• The questions defined by this idea include the
  identity and behavior of the linker molecules
  (cadherins, integrins, and others) and the
  processes and molecules that control the behavior
  of the fibers and their states of polymerization
  and linkage

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Signal transduction cascades that activate small
G-proteins regulate MFs in a wide range of cells
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Reminder of the mechanism for signaling by small
G-proteins, like Rac, Rho, and CDC-42 GTP-bound
state is active and turns on down-stream
proteins. GAPs and GEFs control the relative
concentrations of GTP- and GDP-bound forms of the
protein
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In budding yeast there is a signaling cascade
that links a pheremone to regulation of the actin
cytoskeleton
Discovery of such control pathways has motivated
a Search for homologous paths in other cell types
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There are gt8 down-stream targets for each of the
small G-proteins involved in regulation of the
actin cytoskeleton

• Rho-GTP leads to the activation of cytoplasmic
  myosin, which in turn promotes actomyosin-dependen
  t structures, like stress-fibers
• Rac-GTP activates WASp, which in turn activates
  the Arp2/3 complex, and it also promotes
  uncapping of MFs by removing CapZ or Gelsolin,
  inducing extensive, branched -end MF growth
• CDC42-GTP also activates the WASp protein but
  Formin is also activated, promoting MF assembly
  into membrane-associated bundles

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A plausible mechanism for the organization of
actin in the cells cortex and for its role in
extension of a lamellipod
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Signal transduction cascades that activate small
G-proteins have now been found in a wide range of
cells
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Functional Rac is important for
mebrane-associated actins response to MT
invasion of the cell cortex
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Compare cortex behavior with a cell expressing
wild-type Rac during MT regrowth