The Cytoskeleton: Introduction and Actin

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The CytoskeletonIntroduction and Actin

• Alissa Weaver
• Department of Cancer Biology

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Introduction to the Cytoskeleton

• a network of fibrous elements, consisting
  primarily of microtubules, actin microfilaments
  and intermediate filaments found in the cytoplasm
  of eukaryotic cells.
• The cytoskeleton provides structural support for
  the cell and produces physical forces to allow
  movements
• directed movement of organelles, chromosomes, and
  the cell itself, muscle contraction, cytokinesis.

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The cytoskeletal components are divided up by the
size of the components

• Microtubules (24 nm in diameter) and are formed
  by
• polymerization of a,b-tubulin monomers and
  exhibit
• structural and functional polarity. They are
  important
• components of cilia, flagella, the mitotic
  spindle, and
• other cellular structures.

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Intermediate fibers (10 nm in diameter) formed
by polymerization of several classes of
cell-specific subunit proteins including
keratins, lamins, and vimentin. They constitute
the major structural proteins of skin and hair
form the scaffold that holds Z disks and
myofibrils in place in muscle and generally
function as important structural components of
many animal cells and tissues
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Microfilaments (7 nm in diameter) are formed
by polymerization of monomeric globular (G)
actin also called actin filaments.
Microfilaments play an important role in muscle
contraction, cytokinesis, cell movement, and
cell-cell and cell-matrix adhesion.
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MBC 16-1
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Two basic mechanisms for generating movement
One mechanism involves a class of enzymes
called motor proteins, which use energy from ATP
to walk or slide along a microfilament or a
microtubule.
Some motor proteins carry membrane-bound
organelles and vesicles along the cytoskeletal
fiber tracks (e.g. dyneins and kinesins on MTs)
while other motor proteins cause the fibers to
slide past each other (e.g. myosins and muscle
contraction).
The second mechanism involves the polymerization
and depolymerization of microfilaments and
microtubules
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The actin cytoskeleton
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The actin cytoskeleton is important for many
cellular functions

• Cell movement
• Lamellipodia
• Stress fibres
• Filopodia
• Cell-cell adhesion
• Formation
• Strengthening and maintaining
• Cytokinesis
• Formation of contractile ring
• Endocytosis/membrane trafficking
• Resistance to osmotic stress
• Functions in specialized cells
• Muscle contraction
• Cochlear hair cells
• Microvilli in intestine

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Actin
Actin is the most abundant intracellular protein
(5-10).
A protein consisting of approximately 375 residues
Actin exists as a globular monomer called
G-actin and as a filamentous polymer called
F-actin, which is a linear chain of G-actin
subunits
Each actin molecule contains a divalent cation
(Ca2 or Mg2) complexed with either ATP or ADP
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Addition of ions Mg2, K, or Na to a
solution of G-actin will induce the
polymerization of G-actin into F-actin filaments.
Assembly of G-actin into F-actin is accompanied
by the hydrolysis of ATP to ADP and Pi
All subunits in a filament point toward the same
filament end (i.e., they have the same polarity).

Actin is arranged into bundles and networks in
the cell achieved by actin binding proteins
Actin is bound to the plasma membrane by
membrane-microfilament binding proteins
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Nucleation of Actin Filaments

• rate limiting step for filament formation
• tightly controlled in cells
• essential for the creation of actin-based
  structures
• lamellipodia
• filopodia
• allows rapid changes in cytoskeletal structure
  and cell morphology
• Arp2/3 complex (branched), formins (unbranched)

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Actin filaments are polar and assembled from
ATP-actin

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Actin-binding proteins regulate filament assembly
and organization
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Major signals for regulation of cytoskeletal
assembly

• Phospholipids PIP2 PIP3
• Nucleotide state of actin ATP vs. ADP
• Rho GTPases
• Tyrosine kinase signaling

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Expression of constitutively active Rho GTPases
induces dramatic changes in the actin cytoskeleton
From A. Hall, Science 279509-514, 1998
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But we still dont understand how key signals
connect to actin rearrangements No effect of
Rac1/2Mtl KD in Drosophila S2 cells. Small
effect of Nck KD
From Rogers et al., JCB, 1621079-1088, 2003
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Actin Cytoskeleton and Cellular Migration
From Horwitz and Parsons, Science, 2861102-1103,
1999

• Cell shape changes and force generation
• Actomyosin contraction pulls the cell forward
• Actin polymerization drives plasma membrane
  protrusion

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Lamellipodial Extension directs Cell Motility
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Branched actin network in the lamellipodia
Svitkina et al. 1997. J. Cell Biol. 139, 397.
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Actin Assembly at the Leading EdgeFree Barbed
Ends
Rhodamine actin added to a permeabilized cell
reveals free barbed ends on actin filaments.
FITC-phalloidin stains all actin filaments
Chan et al., 1998
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Arp2/3 complexBranched actin
nucleationessential forcell motility and
invasionListeria motilityimportant
forendocytosis/membrane traffickingcell-cell
adhesion
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The Arp2/3 Complex binds to the side of actin
filaments and nucleates new filaments
Mammalian Activators WASps Cortactin
Volkmann et al., 2001
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Key Actin Regulatory Molecules in the
Lamellipodium

• Arp2/3 complex nucleator
• WASps and cortactin activate/regulate Arp2/3
• Profilin delivers actin monomer to free barbed
  ends
• Capping protein caps filaments
• Cofilin severs and depolymerizes filaments
• Result is dynamic assembly and turnover of the
  force producing machinery in seconds.

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Listeria and E. coli (IcsA) motility can be
reconstituted using a purified protein system
Loisel et al., Nature 401613-616, 1999
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But Listeria tails are not the same as
lamellipodia...

• Ena/VASP proteins have opposite effects on
  lamellipodial protrusion as on Listeria speed
• clearly important in both systems different
  effects
• Lamellipodial actin assembly is nucleated from
  the plasma membrane and represents a different
  geometry
• Different Arp2/3 regulators are involved
• Lamellipodia are induced in response to a variety
  of external signals and must retain a dynamic
  responsiveness to external cues

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WASps

• Activate Arp2/3 complex
• 5 members of family
• WAVE1-3 lamellipodia formation
• WASp Hematopoietic isoform
• N-WASp Ubiquitous
• Roles of N-WASp WASp less clear
• membrane trafficking
• polarity
• ?filopodia formation?
• invasion

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WASp family proteins activate Arp2/3 complex on
cellular membranes
From Pollard and Borisy, Cell, 112453-465
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WAVE-2 is essential for lamellipodial protrusion
Control
WAVE-2 knockdown
Nicole Bryce, Weaver lab
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Formins unbranched actin nucleationimportant
for formation of bundled actin filamentsstress
fibres, filopodia, actin cables contractile ring
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Formins are required for actin cable (but not
patch) formation in yeast
Evangelista et al., NCB, 432-41, 2002
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Direct observation of filament nucleation by
formin cdc12 FH1FH2
Kovar and Pollard, PNAS,10114725-14730, 2004
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Higgs et al., 2005, TIBS, 30342-353
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Formins as processive capping proteins
elongation by walking along the filament barbed
end
Higgs et al., 2005, TIBS, 30342-353
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Myosin Motor Protein Family Tree
MBOC, 2004
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Myosins
-Myosin proteins are organized into head, neck,
and tail domains, which carry out different
functions. -The head domain binds actin and has
ATPase activity. -The light chains, bound to the
neck domain, regulate the head domain. -The tail
domain dictates the specific role of each myosin
in the cell.
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Evidence for the motor activity of the myosin head
James Spudich, MBOC
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The coupling of ATP hydrolysis to movement of
myosin along an actin filament. In the absence of
bound nucleotide, a myosin head binds actin
tightly in a "rigor" state. When ATP binds (step
1 ), it opens the cleft in the head, disrupting
the actin-binding site and weakening the
interaction with actin. Freed of actin, the
myosin head hydrolyzes ATP (step 2 ), causing a
conformational change in the head that moves it
to a new position, closer to the () end of the
actin filament, where it rebinds to the filament.
As phosphate (Pi) dissociates from the
ATP-binding pocket (step 3 ), the myosin head
undergoes a second conformational change the
power stroke which restores myosin to its rigor
conformation.
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Specialized Actin Based Structures Muscle
MBOC, 2004
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Sliding Filament Model for Muscle Contraction
MBOC, 2004
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Regulation of myosin attachment by troponin and
tropomyosin
MBOC, 2004
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Regulation of Myosin contraction in nonmuscle
cells
MBOC, 2004
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Specialized Actin Based Structures Microvilli
MBOC, 2004
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Contractile ring for cytokinesis
MBOC, 2004
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Commonly Used Techniques to Study the Actin
Cytoskeleton Pyrene actin polymerization with
pure proteins
MBOC, 2004
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Commonly Used Techniques to Study the Actin
Cytoskeleton Phalloidin Staining
Nicole Bryce, Weaver lab
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Commonly Used Techniques to Study the Actin
Cytoskeleton Quantitative Live Cell Imaging
GFP-Arp2/3 in fibrosarcoma cells Nicole Bryce,
Weaver Lab
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Commonly Used Techniques to Study the Actin
Cytoskeleton Quantitative Live Cell Imaging
GFP-paxillin, Emily Clark, Weaver Lab Donna
Webb, Biology Dept
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Conclusions

• Actin filaments provide the force for cell
  movement via polymerization (lamellipodia) or
  contraction (stress fibres)
• Actin binding proteins organize and assemble
  filaments into a variety of structures for
  specialized cell functions
• 2 known ways to nucleate filaments branched
  (Arp2/3) and unbranched (formins)
• Myosins provide contractile forces in both muscle
  and nonmuscle cells
• Major area of research is understanding how
  signals coordinate cytoskeletal reorganization