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 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

Microfilament (thin filament) structure minus Microfilament (thin filament) structure G-actin monomer ATP --- 7-8nm 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 040208 Like tubulin, actin 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 25nm plus

G-actin (42-43kD; 375 aas) (minus) Animation (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+2ATP-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 was discussed for tubulin assembly: dynamic instability and treadmilling. 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

Intrafilament contacts 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. 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) 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 Red arrow = minus end Green arrow = initial plus end Bulls-eye = contact point on surface + -

+ S1 Decoration The polarity of microfilaments is reflected in the asymmetry of each actin monomer. Thus, microfilament motor molecules (the myosins) (and other actin binding proteins) will bind to the filament in a directional way; this gives myosins an inherent polarity as they engage and step along the microfilament. Microfilament polarity can be revealed in the EM by Myosin S1 fragment decoration (proteolytic fragments of the actin motor myosin that binds to monomers in a directional way). The resulting pattern looks like arrowheads that point to the minus end of the polymer. The plus end is called the barbed end for its appearance. This particular example is likely an S1 decorated seed that was subsequently used for assembly (the minus end extension should be shorter than the plus end, although both run off the edge of this field). Negative stained EM from http://www.scripps.edu/~stoffler/publ/PDF/JSB_97.pdf , cached 040208 -

S1 decoration reveals microfilament polarities in situ Red arrows (and black arrowheads on original images) indicate the polarities of individual microfilaments (as indicated by S1 decoration), as they occurred within the cytoplasm of detergent-extracted cells. The red arrows point in the apparent direct of the decoration “points”, e.g. toward the minus end of each decorated filament In general cytoplasm (a), microfilaments often run in a variety of directions. In stress fibers (b) microfilaments are bundled laterally but without regard to polarity. In contrast, in the leading edge of a lamellipodium (c) the microfilaments are uniformly oriented with their plus (barbed) ends toward the membrane. http://www.borisylab.northwestern.edu/pages/supplemental/em/emfig4.html , cached 060209 a. Cytoplasm (mixed polarities) b. Stress fibers (bundled mixed polarities) c. Lamellipodia (uniform polarity, points in)

Phalloidin The Angel of Death Mushroom Amanita phalloides Actin binding drugs: Phalloidins bind to microfilaments and stabilizes them (showing that dynamic filaments are vital!). Phalloidin, chemically modified by the addition of a fluorescent tag, can be used for specific cytochemical localization of F-actin (but not G-actin. Jasplakinolide is similar in its action. Cytochalasins bind to the plus end of microfilaments, blocking assembly. Polymers disassemble from the minus end. Latrunculin binds G actin monomers and prevents them from polymerizing Swinholdine is another drug that actually severs filaments http://en.wikipedia.org/wiki/Amanita_phalloides, cached 0702123 http://habanero.ibme.wustl.edu/motility.php cached 070213 http://en.wikipedia.org/wiki/Phalloidin , cached 070213

Cells labeled with Phalloidin (F-actin) and DNase I (G-actin) Fluorescently-labeled phalloidins can be used as structural probes for assembled filamentous actin (f- actin). Interestingly, DNase I is a protein evolutionarily modeled on the “actin fold” (not as apparent from this structural comparison) and has an affinity for unassembled G-actin subunits; it will block assembly in vitro (the possible physiological relevance of this observation is not known). Intriguingly, G-actin appears to present within the nuclear compartment (at 43kDa is it just below the exclusion limit of the pore complexes), where it may play a role in gene expression! In any case, Fluorescent DNase I can be used as a probe for G-actin, as shown in these dual labeled cells, that is complementary to f-actin localization with phalloidin.Note that the Dnase I binds to part of the G-actin monomer that is “hidden” within the polymer. http://www.microscopyu.com/galleries/fluorescence/cells/ptk2/ptk2exlarge5.html , cached 070219 PTK2 cells labeled for G-actin (red), F-actin (phalloidin probe, green) and DNA (DAPI, blue) http://www.bms.ed.ac.uk/research/others/smaciver/DNase1.jpg , cached 070212

Actin Binding Proteins (ABPs) modulate architectures >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, PIP2 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 060204 Capping 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. “. . . the actin cytoskeleton is a complex three-dimensional molecular jigsaw puzzle” - Tom Pollard

Profilin is involved in ATP recharge 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 structural introgression 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 040208 A&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 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

Amoeboid motility uses Gelsolin Actin subunit Gelsolin (partial structure) in presence of Ca++ Video Amoeboid motility uses 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 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 060208 Gelsolin structure from http://www.princeton.edu/~actin/pubs.htm , cached 060208

F-actin motilities Composite time lapse segments Video timelapse sequences showing microfilament dependent motilities. Clockwise from upper left: mouse fibroblasts moving into an artificial wound (total time 3 hr); mouse melanoma cell (20 min); trout epidermal keratocyte (4 min!); chick fibroblasts (2 hr) http://cellix.imba.oeaw.ac.at/Videotour/Movies/fig3.mov , cached 040220 The microfilament cytoskeleton helps shape cells, structuring and organizing the cortical cytoplasm. This network also supports overlying membranes via cross-links to integral membrane proteins, control vesicular access and establishing membrane domains. Composite time lapse segments

Actin dynamics at the leading edge GFP::actin in a tissue culture cell 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 2 time-lapse video sequences

Axonal growth cones Video GFP-actin dynamics Video Microtubules are excluded from the periphery The color IMF image shows the relationship of the microtubule and microfilament cytoskeletons in a axonal growth cone. The rapid and dynamic treadmilling of actin in the ruffling edge (GFP-actin sequence) creates an exclusion zone that prevents microtubules, vesicles and other materials from entering (as seen in a fluorescent tubulin injected cell at right). This is one way in which cells can structure their cytoplasm. http://www.fmi.ch/members/andrew.matus/video.actin.dynamics.htm , cached 040208 http://www.yale.edu/forschlab/ , cached 060205 http://www.bio.umass.edu/biology/wadsworth/movies/Fishkeratocytes/ , cached 060204 Cell microinjected with fluorescent tubulin to label microtubules

Treadmilling within the lamellipodium In this sequence, the treadmilling of microfilaments in the lamellipodium is demonstrated using speckle microscopy. http://cellix.imba.oeaw.ac.at/Videotour/Movies/fig10.mov , cached 040220 Video was produced by Clare Waterman-Storer. Video

Keratocyte motility Video Fish keratocytes are highly motile cells that can be isolated from the scales of fish that are normally involved in wound repair. This zoomed sequence involved live cell microscopy seamlessly transitioned into electron microscopy, showing the thick brush of branched microfilaments in the leading edge of the crawling cell. http://www.borisylab.northwestern.edu/pages/movies.html , cached 060205

Branching out on the family tree: the Arp2/3 complex Arp2/3 immunolocalization Branching out on the family tree: the Arp2/3 complex Arp2/3 is a 7 peptide complex involving two actin-related protein subunits that forms a nucleation seed at a membrane or on an existing actin microfilament to create a branch. The addition end of the Arps is structurally similar to the cleft end of G-actin monomers; the Arp2/3 + a G-actin subunit basically “looks” like the end of a microfilament which then starts to grow. Arp2/3 remains at the minus end as an anchor site, whereas n-WASp remains at the membrane recruiting G-actin N-WASp (Wiskott-Aldrich Syndrome protein - defective in an immunological B-/T- cell disorder)/SCARp (supressor of G-protein coupled cAMP receptor)/VASp (Vasodilatory protein)/Wave initiate assembly at membrane surfaces by recruiting the Arp2/3 complex and G-actin subunits. Coronin acts as a negative regulator of assembly by binding the Arp2/3 complex and inhibiting nucleation Model from http://www.cgl.ucsf.edu/chimera/ImageGallery/entries/actin/actin.html , cached 060204 , Arp2/3 comples in blue Colorized EM from http://www.borisylab.northwestern.edu/pages/supplemental/arp.html , cached ca. 2002 Diagram from http://bilbo.bio.purdue.edu/%7Eamcgough/ActinProjects.html , cached 040208 plus 70o minus Nucleation Promoting Coronin Factors (NPFs) WASp/SCAR/Wave

The Actin Family Tree The actin family tree consists of both “true” actins and divergent actin-related protein (Arps) There are about 10 Actin-related proteins (ARPs) highly divergent (45% identity); they play a role in filament initiation (Arp2/3 mimics a nucleation " seed”) Source: http://jcs.biologists.org/cgi/content/full/115/13/2619/FIG1 cached 080115

Formin those long filaments - + Formins are actin nucleating complexes that act as dimers (each “lassoed” to the “post” of the other) to cap the end of a growing fliament in such as way as to both stabilize the plus end and literally “step” a new monomer into place. This model explains how the complex can hold on to a growing polymer end. The linker is just long enough to accommodate insertion of a G-actin monomer as it reversible transitions from a helix to a disordered loop. Formins are responsible for promoting the growth of long microfilaments, and represent a nucleation pathway distinct from the Arp2,3 pathway. They regulated by the small G-protein Rho. Formins remain associated with the plus ends of microfilaments, promoting continued assembly and protecting the + end from capping factors. This creates long unbranched arrays (often bundled by crosslinkers into parallel arrays as in microvilli). Image from http://focus.hms.harvard.edu/2004/March19_2004/biological_chemistry.html , cached 070212 G-actin monomer

Assembly Cycles WASp SCAR translation Formin Arp2/3 VASp Dynamic ATP translation Formin Arp2/3 VASp Dynamic F- actinADP microfilament CCT G-actinATP ADP (exchange) ATP PIP2 Profilin Stabilizing ABPs WASp 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. Thymosin Stable F-actin microfilament G-actinADP Severin Gelsolin

NPF: VASp (vasodilator stimulated phosphoprotein) F-actin And VASp Mouse melanoma cells transfected with a GFP::VASp fusion construct. VASp is a nucleation promoting factor that recruits Arp2/3 and profilin to establish sites of microfilament assembly. Note the broad, diffuse signal associated with the leading edge, as well as punctate foci at the ends of stress fibers. http://cellix.imba.oeaw.ac.at/Videotour/video_tour_5.html , cached 040220 http://www.molcell.rwth-aachen.de/fig-antonio/TCR.htm , cached 070212 http://www.uvm.edu/~akhowe/howefg4.jpg , cached 070213 NPF: VASp (vasodilator stimulated phosphoprotein) Video

Signal transduction links G-proteins WASp WASp*P PIPkinase MLCK Cdc42 Rac Rho Filipodia Localized Initiation Lamellipodia Frontal Initiation Increased Turnover Stress fibers Form and Tension Arp2/3 PIP PIP2 Myosin Formin 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)

Stress fibers distribute forces Stress fibers are parallel, bipolar bundles of actin microfilaments that connect to cell surface focal adhesion sites. Mini-myosin filaments promote bundling and can create tension, allowing cells to distribute stresses. We will examine myosins in greater detail in the following chapter. http://www.borisylab.northwestern.edu/pages/supplemental/emc/emc1b.html , cached 050205 http://www.physik.uni-bayreuth.de/lehre/biophysik.html , cached 050205 Arrows highlight microtubules adjacent to the stress fiber bundle

Getting in shape Activation of different G-proteins from the signal transduction cascade on the previous slide reveals the controlling effects of these key components. http://med.mc.ntu.edu.tw/~anatomy/smwang/lecture/2006actin-myosin.ppt , cached 070211 (looks like a text illustration but no reference was given)

Neutrophil motility Video This “classic” video sequence shows a human neutrophil chasing a bacterium in a fresh blood preparation. Rapid motility on the part of this phagocytic white blood cell is critical if it is to catch and destroy quickly moving parasites. Note that agility is just as important as speed, and surface receptors must trigger signalling cascades that in turn control microfilament polymerization to let the cell turn on a dime as it tracks its prey. http://expmed.bwh.harvard.edu/projects/motility/neutrophil.html , cached 040208

Microvilli Great Superlab photos taken at Haverford! Microvilli are polar bundles of parallel microfilaments 1-2 um length x 100nm diameter. Microvilli are a rich source of ABPs that link the filaments to each other (fimbrin, villin) and the membrane (calmodulin, 110kDa myosin I), and cap the filaments in the electron-dense tip. Left image is a longitudinal section; right mage is a cross-sectional view. Microvilli increase the surface area of an epithelial layer, as in these sections through the enterocyte lining of the small intestine.

Anchored in the terminal web The base of each microvillar bundle is anchored in the terminal web, a mesh of cytoskeletal elements in the apical cytoplasm, by fodrin cross-links. http://bio.winona.msus.edu/berg/IMAGES/cortex.jpg , cached 040208

Ca++ regulated adaptation MyoVIIa is involved in Ca++ regulated adaptation The stereociliar bundle is a molecular vibration meter. Sound entering the inner ear is focused in a frequency-dependent manner onto different regions along the length of the seashell-shaped spiral cochlea. Resonance causes movements of the stereociliary bundles of microvilli, triggering the opening of tethered ion channels to trigger nerve impulses that are sent to and processed by the brain. An actin microfilament bundle holds each stereocilium erect although how the stepped lengths are controlled with such precision and geometry remains a mystery. http://www.iurc.montp.inserm.fr/cric/audition/english/corti/hcells/transd/transd.htm , cached 040210 See also http://www.hhmi.org/news/images/story/coreydp.gif and http://www.nidcd.nih.gov/news/releases/05/1_26_05.asp for some more images! Stereocilia

Act’in Up Video Listeria monocytogenes moving in PtK2 cells Listeria and Shigella are human pathogens that are phagocytosed by intestinal epithelial cells but break out into cytoplasm before delivery to lysosomes. Within 15 minutes they start zipping around the host cell’s cytoplasm on microfilament tails at speeds of up to 1 um/sec. A single bacterial gene product (IcsA in Shigella and ActA in Listeria) is sufficient to give motility in a cell extract (E. coli expressing a single gene (Shigella IcsA) also will move in vitro. The protein is localized to one end of the bacterium where it recruits host cellular WASp and Arp2/Arp3. Short microfilaments form and are crosslinked and capped by host factors. As initiation continues, a tail of short filaments grows and the cell pushes off on I, generating forward motility driven by polymerization along. The pathogens can push from one cell to the next, spreading an infection without exposure to the host immune system. This nucleation/crosslinking/release model is not dissimilar to processes used by the cell to move the leading edge forward in lamellipodial motiilty. http://cmgm.stanford.edu/theriot/movies.htm#Hits , cached 060205 , 150x compression Listeria monocytogenes moving in PtK2 cells