In this chapter of our web text, we will examine the architecture of the Actin Microfilament Cytoskeleton.
Microfilaments
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
minus
Microfilament
(thin filament)
structure
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
Actin can be purified from tissue homogenates by cycles of assembly and disassembly
G-actin
monomer
ATP
--- 7-8nm
25nm
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.
plus
assemble in the presence of ATP, magnesium and salts (K+ or Na+)
disassemble in low ionic strength
+2
G-actin (42-43kD; 375 aas)
(minus)
Animation
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.
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 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
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
Video
Polarity of assembly
(in vitro assay)
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
+ 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 +2
family - 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
Actin Binding
Proteins (ABPs)
modulate
architectures
“. . . the actin cytoskeleton is a complex
three-dimensional molecular jigsaw
puzzle” - Tom Pollard
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.
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
profilin
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 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
nucleation phase to form a trimeric seed (addition of F-actin fragments eliminates nucleation lag)
WASp
SCAR
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
ATP
translation
Formin
CCT
PIP2
WASp
VASp
Arp2/3
Dynamic
F- actinADP
microfilament
G-actinATP
ADP
(exchange)
ATP
Profilin
Stabilizing
ABPs
Thymosin
G-actinADP
VASp
Severin
Gelsolin
Stable
F-actin
microfilament
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.
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
+2
components are supplied by disassembly in the “retracting “tail”, triggered by local Ca entry across the plasma membrane which in turn triggers gelsolinmediated microfilament severing. Cell surface receptors play an important role in regulation of calcium channels, permitting the cell to make directed
progress across a surface.
Actin subunit
Video from http://www.abac.edu/SM/kmccrae/BIOL2050/Ch1-13/Animations/Animations.html cached 060208
Gelsolin (partial structure)
in presence of Ca++
Gelsolin structure from http://www.princeton.edu/~actin/pubs.htm cached 060208
Video
Amoeboid
motility uses
Gelsolin
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
Actin dynamics at
the leading edge
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.
GFP::actin in a
tissue culture cell
2 time-lapse video sequences
Source: http://cellix.imba.oeaw.ac.at/Videotour/video_tour_5.html , ccached 060205
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.
Signal transduction links
G-proteins
Cdc42
PIP2 hydrolysis by Phospholipase C "releases" gelsolin, cofilin, profilin activites (thereby turning over arrays)
Downstream Kinase/Phosphatase cycles also affect activities (e.g. gelsolin)
+2
WASp
Arp2/3
WASp*P
PIP
Rac
PIPkinase
Rho
MLCK
PIP2
Myosin
Filipodia
Localized Initiation
Lamellipodia
Frontal Initiation
Increased
Turnover
Stress fibers
Form and Tension
Formin
14
+2
+2
Ca levels, increased by IP3, also play a role (e.g.
activate gelsolin, Myo II), whereas
other ABPs (e.g. CapZ) are Ca insensitive; local Ca changes can
+2
+2
result in steering of gel-sol transitions (low Ca actin polymerization; higher Ca depolymerization)