Microfilaments

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Microtubules Microfilaments

In this chapter of our web text, we will examine the architecture of the Actin Microfilament Cytoskeleton.Microfilaments are polymers of actin subunits, and can comprise 1-10% of total cell protein (0.1-0.5uM)http://www.biology.purdue.edu/research/groups/motility/gallery/pages/3neurons.htm cached 070213Showing three neuronal cells in culture, stained for microtubules (green) and microfilaments (red) emphasizing the role of the actin cytoskeleton in the extending processes of each cell.

Microfilament (thin filament)

structure

25nm

--- 7-8nm

G-actinmonomer

ATP

minus

plus

F-actin (filamentous) microfilaments were originally called thin filaments for their consistent 7-8nm diameter. Each consists of a double start helical polymer of G-actin (globular) monomers. Each monomer is asymmetric, with its deep ATP binding pocket oriented toward the minus end of the microfilament.Images from http://www.bi.umist.ac.uk/users/mjfjam/1CAT/l007.htm , cached 040208 and http://www.scripps.edu/~stoffler/publ/PDF/JSB_97.pdf cached 040208Actin can be purified from tissue homogenates by cycles of assembly and disassembly

assemble in the presence of ATP, magnesium and salts (K+ or Na+)disassemble in low ionic strength

G-actin (42-43kD; 375 aas)(minus)

(plus) Changes upon hydrolysis

Animation

G-actin is a 42-43 kDa globular protein (~375 amino acids) that consists of several domains hinged around an Mg+2

ATP-binding site that bridges a deep cleft (oriented toward the minus end in the microfilament). It is useful to consider it as a clamshell-like form that will close tightly when ATP bridges the two halves, and become floppy when hydrolysis breaks the bridge into ADP and Pi bound halves.

ATP bound in this site makes contacts across the cleft, stabilizing the monomer in an assembly competent form. Addition into the polymer covers this cleft and stabilizes the G-actin shape in the “closed” form. Subsequent hydrolysis internally weakens the monomeric shape but the microfilament does not disassemble because of of these intrafilament stabilizing contacts. However, depolymerization can begin from the ends of the polymer and subunit loss occurs rapidly.The dependency of structure on nucleotide status generates certain “behaviors” as we will discuss for tubulin assembly: tread milling (simultaneous addition at the plus end and loss at the minus end, driving subunit flux through the polymer) and (to a lesser extent) dynamic instability. Proteins that interact with the ends of the polymer will influence these processes dramatically.Hydrolysis converts a flexible loop in subdomain 2 into an alpha helix (forming Dnase I binding site) as seen from the side (view rotated relative to the left structure) in this dynamic simulation (right). This view shows how an actin binding protein might interact with an interface that would differ in structure depending upon the nucleotide status of the monomer. http://www.bbri.org/faculty/dominguez/Structure-gallery-frame1.htm , cached 060205

http://www.bbri.org/faculty/dominguez/Movies/Actin-ATPtoADP_Movie.gif cached 060205Actins are a highly represented by a highly conserved group of isoforms (6 in humans,17 in Dictyostelium, many in plants). Universal actins (sometimes called non-muscle but expressed in all cell types including muscle) differ in about 25 aas (93% identity) include beta-actin (1 in mammals) found in lamellipodia and gamma actins (2 in mammals) found in stress fibers. Muscle alpha-actins differ in only 4-6 aas (98+% identical); expressed only in muscle (3 in mammals - unique forms for striated, cardiac and smooth). Curiously, alpha-actins do not coassemble with other types in vivo, even though in same cytoplasm. They will co-assemble in the test tubeProkaryotic proteins with structural and biochemical similarities to eukaryotic actins have been identified. MreB forms cables that direct cell wall peptidoglycan synthesis and ParM is involved in the partitioning of plasmids

Intrafilament contactsEach monomeric unit makes many contacts with adjacent subunits (here the contact residues are colored uniquely for each interface); these weak interactions sum to hold the polymer together. As was the case with tubulin, the nucleotide status of the monomer influences its shape and, consequently, the strength of these interactions. ATP bound monomer is predisposed to assembly; hydrolysis to the ADP form after assembly then weakens these contacts (although not destroying the polymer), predisposing it to disassembly.http://www.ks.uiuc.edu/Research/cell_motility/actin/lorenz.gif cached 040206

Polarity of assembly

(in vitro assay)

Red arrow = minus endGreen arrow = initial plus end

Bulls-eye = contact point on surface

Video

+-

+ G-actin, ATP

As with microtubules, there is a basic asymmetry in the polymer with one end (plus) favored for assembly and the other end (minus) less favored for assembly. In addition, G-actin monomers must be charged with ATP in order to assemble and hydrolysis after polymerization predisposes the subunits for disassembly. This opens the door for polymer behaviors like dynamic instability and treadmilling, as previously introduced for microtubule polymers. Total interference reflection microscopy uses evanescent wave excitation to image events close to a surface. 300x time compression. http://www.pnas.org/content/vol0/issue2004/images/data/0405902101/DC1/05902Movie3.mov cached 041013

Actin Binding Proteins (ABPs)

modulate architectures

“. . . the actin cytoskeleton is a complex three-dimensional molecular jigsaw puzzle” - Tom Pollard

>60 kinds of actin binding proteins control ATP-recharge of G-actin (profilin) and nucleation, extension, capping (vinculin), stabilization, crosslinking (fimbrin, spectrin, filamin, fodrin, fimbrin, villin), anchoring (110K myosin I, alpha-actinin, dystrophin; ERM family - ezrin, radixin, moesin), severing and depolymerization (villin, gelsolin) of microfilaments. ABPs can be regulated by phosphorylation, PIP

2 and Ca

+2 binding

Many of these ABPs share homologous actin binding domains. For example, the calponin-homology domain is a 24 kDa domain that occurs once per monomer in dimer-forming alpha-actinin and spectrin, and in pairs in the cross-linking fimbrin and filamin ABPs.http://www.bio.brandeis.edu/goodelab/research.html cached 060204Capping proteins are highly abundant in cells and dynamically unstable filaments are rapidly capped and stabilized. This means that most microfilament arrays consist of lots of short filaments meshed by ABPs.

Profilin is involved in ATP recharge

profilin

Profilin is an abundant cytoplasmic 15kDa protein originally discovered in profilamentous bodies (i.e. the sperm acrosome). It binds to G-actin 1:1, covering the polymerization site. Unbinding occurs upon pH rise and/or PIP2 binding (an intermediate in the phosphatidyl-inositol signalling pathway). Profilin is also recruited by Nucleation Promoting Factors like VASp and WASp to increase locally the concentration of ATP-bound actin near assembly sites.

Profilin promotes nucleotide exchange 1000-fold to "recharge" actin (note it “gooses” the G-actin to close the nucleotide binding cleft, promoting ADP->ATP exchange.Thymosin is a another abundant cytoplasmic 5kDa protein that also binds actin monomers, but does so at the opposite end of the monomer from profilin,blocking the nucleotide binding cleft. This interferes with assembly, allowing cells to modulate the pool of unassembled subunits (permitting buffering of the critical concentration for assembly independent of polymer mass).Profilin image http://bio.winona.msus.edu/berg/ILLUST/actin3D.gif cached 040208

Cofilin weakens subunit interactions

by structuralintrogression

Cofilin (white) is an actin binding protein (ABP) that literally “shoehorns” into the filament, untwisting the two-start helix and weakening inter-monomer contacts. Binding of cofilin is regulated by dephosphorylation and pH and increases microfilament turnover by 20-30 fold.http://biomachina.org/research/projects/actin/ cached 040208A&B from http://www.bio.umass.edu/vidali/web/cell_motil/sept_28_long3.htm cached 080115

Alpha-actinin crosslinks

microfilaments

Alpha-actinin dimers bridge microfilaments to form regularly spaced parallel arrays and bundlesEM image from http://www.sb.fsu.edu/~taylor/current_members/dtaylor.html , cached 080115Bundling model from http://www-ssrl.slac.stanford.edu/research/highlights_archive/actinin.html cached 080115

Assembly Cycles

translation

G-actinATPDynamicF- actinADP microfilament

StableF-actinmicrofilament

G-actinADP

Thymosin

ProfilinADP

(exchange)ATP

ATP

CCT

Arp2/3

WASpSCAR

StabilizingABPs

SeverinGelsolin

PIP2

WASpVASp

Formin VASp

Assembly kinetics

nucleation phase to form a trimeric seed (addition of F-actin fragments eliminates nucleation lag)elongation phase plus-end growth (as polymer accumulates, monomer level decreases)equilibrium phase is a balance point at the critical concentration for templated-assembly, on rate = off rate

treadmilling can occur as long as G-actin is recharged with ATP

Assembly/Disassembly of actin (white) is regulated at many points by ABPs (pink), which in turn are controlled by input from nucleation factors and cell signaling cascades.

Amoeboid motility uses

Gelsolin

Actin subunit

Gelsolin (partial structure) in presence of Ca++

Video

Amoeba show a beautiful form of bulk cytoplasmic flow. This mode of locomotion is driven by local sol-gel transitions regulated by calcium levels. Calcium levels are low in the leading pseudopod (“False-foot”) and microfilaments assemble and are crosslinked by ABPs, notably filamin. These components are supplied by disassembly in the “retracting “tail”, triggered by local Ca

+2 entry across the plasma membrane which in turn triggers gelsolin-

mediated microfilament severing. Cell surface receptors play an important role in regulation of calcium channels, permitting the cell to make directed progress across a surface.Video from http://www.abac.edu/SM/kmccrae/BIOL2050/Ch1-13/Animations/Animations.html cached 060208Gelsolin structure from http://www.princeton.edu/~actin/pubs.htm cached 060208

Actin dynamics at the leading edge

GFP::actin in a tissue culture cell

2 time-lapse video sequences

Right: GFP::Actin transfected rat embryo fibroblast. Note the distinct, dynamic ruffling edge as well as the more static stress fiber bundles in this relatively slow moving cell. Source: http://www.fmi.ch/members/andrew.matus/video.actin.dynamics.htm , cached 040208

Left: Another GFP::Actin expressing cell locomoting more quickly across the field of view. This cell has both a broad lamellipodium and thin, spike-like filipodia at the leading edge, and a long retraction tail.Source: http://cellix.imba.oeaw.ac.at/Videotour/video_tour_5.html , ccached 060205

Signal transduction links

Cdc42

Rac

Rho

WASp

WASp*P

PIPkinase

MLCK

Arp2/3

PIP

PIP2

Myosin

Formin

FilipodiaLocalized Initiation

LamellipodiaFrontal Initiation

Increased Turnover

Stress fibersForm and Tension

G-proteins

Several small G-proteins (downstream of tyrosine kinase surface receptors) modulate the microfilaments cytoskeleton via a variety of intermediates. Notably, they also interact in a cascading fashion to coordinate the assembly of different architectures.

PIP2 hydrolysis by Phospholipase C "releases" gelsolin, cofilin, profilin activites (thereby turning over arrays)Downstream Kinase/Phosphatase cycles also affect activities (e.g. gelsolin)Ca

+2 levels, increased by IP3, also play a role (e.g. activate gelsolin, Myo II), whereas other ABPs (e.g. CapZ) are Ca

+2 insensitive; local Ca

+2 changes can

result in steering of gel-sol transitions (low Ca+2

actin polymerization; higher Ca+2

depolymerization)

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