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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) Microfilaments http://www.biology.purdue.edu/research/groups/motility/gallery/pages/3neurons.htm cached 070213 Showing 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. Microtubules Microfilaments Text and image sources are included using the notes function of this file F-actin (filamentous) microfilaments were originally called thin filaments for their consistent 7-8nm diameter. Each consists of a double start helical polymer minus of G-actin (globular) monomers. Each monomer is asymmetric, with its deep ATP binding pocket oriented toward the minus end of the microfilament. 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 040208 (thin filament) Actin can be purified from tissue homogenates by cycles of assembly and disassembly structure G-actin assemble in the presence of ATP, magnesium and salts (K+ or Na+) monomer disassemble in low ionic strength ATP --- 7-8nm 25nm plus +2 G-actin is a 42-43 kDa globular protein (~375 amino acids) that consists of several domains hinged around an Mg 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. G-actin (42-43kD; 375 aas) 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 (minus) 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 Animation 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 060205 Actins 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 tube Prokaryotic 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 (plus) Changes upon hydrolysis Each 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. Intrafilament contacts 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 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. Video Total interference reflection microscopy uses evanescent wave excitation to image events close to a surface. 300x time compression. http://www.pnas.org/ Polarity of content/vol0/issue2004/images/data/0405902101/DC1/05902Movie3.mov cached 041013 assembly (in vitro assay) + G-actin, ATP Red arrow = minus end Green arrow = initial plus end + Bulls-eye = contact point on surface - >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 +2family - ezrin, radixin, moesin), severing and depolymerization (villin, gelsolin) of microfilaments. ABPs can be regulated by phosphorylation, PIP and Ca binding 2 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 060204 Capping proteins are highly abundant in cells and dynamically unstable filaments are rapidly capped and stabilized. This means that most microfilament Actin Binding arrays consist of lots of short filaments meshed by ABPs. Proteins (ABPs) modulate architectures “. the actin cytoskeleton is a complex three-dimensional molecular jigsaw puzzle” - Tom Pollard 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 Profilin is involved in ATP recharge 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 profilin Cofilin (white) is an actin binding protein (ABP) that literally “shoehorns” into the filament, untwisting the two-start helix and weakening inter-monomer Cofilin weakens 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 040208 subunit interactions A&B from http://www.bio.umass.edu/vidali/web/cell_motil/sept_28_long3.htm cached 080115 by structural introgression Alpha-actinin dimers bridge microfilaments to form regularly spaced parallel arrays and bundles EM image from http://www.sb.fsu.edu/~taylor/current_members/dtaylor.html , cached 080115 Bundling model from http://www-ssrl.slac.stanford.edu/research/highlights_archive/actinin.html cached 080115 Alpha-actinin crosslinks microfilaments Assembly kinetics Assembly Cycles WASp nucleation phase to form a trimeric seed (addition of F-actin fragments eliminates nucleation lag) SCAR elongation phase plus-end growth (as polymer accumulates, monomer level decreases) ATP equilibrium phase is a balance point at the critical concentration for templated-assembly, on rate = off rate translation Formin Arp2/3 VASp treadmilling can occur as long as G-actin is recharged with ATP Dynamic CCT G-actinATP F- actinADP 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 microfilament signaling cascades. ADP PIP2 (exchange) Profilin ATP Stabilizing ABPs WASp Thymosin VASp Stable G-actinADP F-actin Severin microfilament Gelsolin 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
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