Harvey Lodish • Arnold Berk • Paul Matsudaira •
Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell
Molecular Cell Biology
Fifth Edition
Chapter 19:
Cytoskeleton I: Microfilaments and
Intermediate Filaments
Copyright © 2004 by W. H. Freeman & Company
Cytoskeleton: a cytoplasmic system of fibers, is critical to cell motility
Macrophage cytoskeleton
Intermediate filaments
Distributions of cell adhesions (actin and vimentin)
The major components of eukaryotic cell architecture
and shape. The cytoskeleton is NOT a passive framework, but is a
dynamic and adaptable arrangement of filaments that constantly
undergoes rearrangement.
Three types of cytoskeletal fibers:
Microfilaments: actin filaments, thin filaments, 7-9 nm
intermediate filaments: about 10 nm diameter
microtubules: about 24nm diameter
Needs energy
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Depiction of the distribution of cytoskeletal filaments in eukaryotic
cells and bacterial cells
Main Functions of cytoskeleton
Epithelial
Maintains or changes cell shape
Chapter 5
Provides structural support
Contraction (muscle and nonmuscle)
bacterial
Organelle movement
Cytokinesis
FtsZ protein is homology of tubulin (MTs), may participates cell division, it can
polymerization protofilament
MreB is similar to actin.
Epithelial cell: actin near apical region, microtubule along long axis, intermediate
filaments cell periphery junction with neighborhood cells
Bacteria: MreB actin homolog constrict width, FtsZ cell division sites
Overview of the actin and intermediate filament
cytoskeletons and their function
1. Polymerization and
depolymerization of actin
filaments → membrane forward
2. Support cell structure
3. 1,2 connected to myosin, →
movement with (intracellular)
and contractions of cell
membrane
4. 3, 4 integrate cell and content
Cell movement
Movement of flagella or cilia
Binding site for some RNA and proteins
Involved in signaling in the cell
Cytoskeletal filaments are all constructed from
smaller protein subunits
Intermediate filaments: smaller
elongated and fibrous subunits
Actin and microtubule filaments:
compact and globular subunits
All form as helical assemblies
of subunits
Noncovalent interactions:
rapid assembly and disassembly
Intermediate filaments (IM)
Ch 5
2
Actin is ancient (古老), abundant, & highly conserved.
Actin cytoskeleton in a
moving cell
Most abundant intracellular protein in most eukaryotic cells; 1-5% of cellular
protein in nonmuscle cells. Cytosolic concentration of actin about 0.1-0.5
mM (local can be 5mM).
About 42kDa.
Single-celled organisms: 1 or 2 actin genes; multicellular organisms:
multiple genes, e.g. human has 6 actin genes: 4 α-actins in various
muscle cells, β- & γ-actins in nonmuscle cells (They differ at only 4 or 5
positions).
Actin sequences from amoebas & from mammals are 80% identical.
Humans have 6 genes
• α actins used in contraction
• β actins in stress fibers
• γ actins in leading edge of moving cells
G-actin monomers assemble into long, helical Factin polymers.
ATP to ADP and Pi
Actin: a globular monomer called G-actin, each molecule contain Mg2+ and 1
ATP (ADP); ATP-G-actin, ADP-G-actin, ATP-F-actin, ADP-F-actin
Filamentous polymer celled F-actin
G-actin
Polymerization (Mg2+, K+ Na+)
F-actin
De-polymerization
Ionic strength lowered
ATPase fold
F-actin has structural & functional polarity.
Myosin S1 heads bind to actin filaments in a polar, spiral (螺旋)) fashion, with
the barbed (倒鉤) ends pointing to the negative end of the actin filament.
Exposed to the
surrounding
solution
Mg2+
G-actin (4 sub-domain) formed F-actin
Twisted strands of bead produced thinner 7nm diameter and thicker 9nm.
3
F-actin has structural and functional polarity
Experimental demonstration of polarity of an actin filament by binding of myosin S1
head domains.
Myosins bind to actin with a slight tilt. When all subunits are bound by myosin, the filaments
appear decorated with arrowheads that all point towards one end of the filament. This end
is referred to as the “pointed end”.
CH-domain & other proteins (actin cross-linking proteins)
organize microfilaments into bundles & networks.
CH: calponin homology –domain superfamily
Fimbin
- monomer
- binds actin in a tight
parallel bundle (14 nm)
偽足
Spectrin
- tetramer
- binds actin in a loose
parallel arrangement
銳利
+
Filamin
- dimer
- crosslinks actin into a
network with actin
filaments at almost right
angles to each other
_
+
+
+
α-actinin
- dimer
- binds actin looser than
fimbrin
_
Actin bind to “binding protein “ → extension or formed network
Fimbrin, villin, & α-actinin crosslink actin filaments into bundles.
Filamin cross-links actin filaments into a threedimensional gel-like network.
Polymer actin
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A two-dimensional skeleton formed by spectrins & short actin
filaments helps to shape and stiffen (變硬) plasma membrane.
Cell functions for actin
Fig. 5-31, p.176
Actin polymerization in vitro, proceeds in three steps
Can be monitored by viscometry, sedimentation, fluorescence spectroscopy.
Critical concentration
Each filament is composed of 2 helically intertwined chains of G-actin monomers
To polymerize into filament
G-monomer must have an ATP bound
G-ATP hydrolyzes to ADP + Pi (faster at + end, slower at - end)
Become stable ADP-F-actin
This is reversible: F-actin depolymerizes into G-actin monomers.
exchange
Nucleation
- assembly of trimers
- lag phase can be reduced if polymerized
fragments added first
Elongation
- ATP-G-actin is hydrolyzed to stable ADP-Factin;
Steady State
- rate of subunit addition = rate of subunit loss
Concentration of G-actin determines filament formation
Critical concentration (Cc): concentration of G-actin in
equilibrium with actin filaments.
<Cc = no polymerization;
>Cc = filaments formed.
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Actin filaments grow faster at (+) end than at (-) end.
+ end elongates 5-10 times as fast as dose
the opposite (-)
Can measured by capping protein.
-: free site
Cc more lower → easy polymerization
Cc at (+) end is much
lower than Cc at (-) end
Treadmilling (滑動) of actin
filaments:
When critical concentration
between -+, →
preferentially to the + end.
In steady state phase, Gactin continue to be added
at +, and lost from the –
end → the length of
filament remain constant
→ can move
Treadmilling (踏車) type
mechanism for move.
Treadmilling - if both ends of the polymer are exposed, subunits undergo a net
assembly at the (+) end and a net disassembly at the (-) end with the polymer
remaining a constant length, even though here is a net flux of the subunits
through the polymer.
Cc: critical concentration
Toxin perturb (擾亂) the pool of actin monomers.
ADP
Monomers
È
oligomers (nucleus)
ATP
È
assembly
+ ATP
È
dynamic equilibrium (treadmilling) +
ADP
ATP
ATP
ADP
ADP ADP
.Pi
-
Cytochalasin D:
– A fungal alkaloid.
– Binds to (+) end of F-actin & blocking further addition of subunits.
Latrunculin:
– A toxin secreted by sponges.
– Binds to G-actin & inhibits it from adding to a filament.
When these two toxins are added to live cells, actin cytoskeleton
disappears and cell locomotion & cytokinesis are inhibited.
Phalloidin:蕈毒素
– Isolated from the "angel of death" mushroom.
– Binds between subunits in F-actin, locks adjacent subunits together
& prevents actin filaments from depolymerizing.
– Fluorescent-labeled phalloidin is commonly used to stain actin
filaments for microscopy.
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Actin polymerization in vivo is regulated by proteins
that bind G-actin.
Thymosin β4, an actin-sequestering (隔離) protein, keeps cellular G-actin
conc. (~200 mM) above its Cc (0.1 mM).
Profilin, another G-actin-binding protein, promotes actin filament assembly.
– As a nucleotide-exchange factor (ADP -> ATP).
– Assists actin addition to (+) end of filaments.
Competing roles of profilin & thymosin β4 in regulating actin
polymerization.
Thymosin β4
Binds ATP-G-actin (1:1)
Sterically blocks ATP binding site
Prevents polymerization
Sequesters ~70% of G-actin
Profilin
Binds ATP-G-actin (1:1)
buffered ~20% of free G-actin
Only actin binding protein that permits
exchange of ATP for ADP, thus it likely
promotes binding to actin filament.
Membrane phospholipid PIP2 binds profilin & inhibits its binding to G-actin.
(PIP2 is hydrolyzed in response to certain extracellular signals.)
Membrane-associated signaling proteins (Ena/VASP family) bind profilin &
localize profilin-actin complexes to the membrane.
1. Actin complex (with thymosin) dissociate
Profilin and thymosin β4 in
2. Actin add to the end of a filament
regulated polymerization of
3. ATP hydrolysis ADP
G-actin
4. ADP-G actin with a profilin and formed complex
5. ATP to ADP, ADP-G actin convert to ATP-G actin
6. Can dissociate → free ATP-G actin → add to the end of filament
7. Profilin dissociate →bind to thymosin β4
Profilin vs. signal transdution
Filament-binding proteins that control actin
polymerization.
Beside actin bind protein, second group of filament-bind proteins can control actin
polymerization
Filament-binding severing proteins create new actin ends
Change the structure” such sol to gel. Ameba
G-actin bound to thymosin is “unavailable” for polymerization
Capping: severing protein breaks a filaments at one site, it remains bound at the + end
of one of the resulting fragments where it prevents the addition or exhange of actin
subunits
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Severin (切割) proteins (gelsoin) promote turnover of actin
filaments by creating new (-) ends.
Cofilin (actin depolymerizing factor) binds, twists, and
depolymerizes actin filaments.
Binds preferentially to ADP-containing actin filaments, thus promote
turnover of actin filaments from (-) end.
Regulated by PIP2 binding & phosphorylation.
The activity of
gelsolin is regulated
by Ca+2 and PIP2.
European Journal of BiochemistryŹ268Ź(24),Ź6426-6434., 2001
ADF/cofilin intercalates between
subdomains 1 and 2 of two
longitudinally associated actin
monomers causing twisting of the
filament.
Tropomodulin: Actin-capping proteins stabilize F-actin.
-
•
The activity of gelsolin is inhibited by PIP2 binding & activated by increase
in cytosolic Ca2+ to ~10-6 M.
Cap Z: Actin “capping” proteins
+
bind to – ends of filament
Bind to + ends of actin filaments,
prevents the addition or loss of actin
subunit form the + end; it inhibited
by IP3 signal transduction
Both capped protein can stabilized
Cap Z is a plus end capping protein
Tropomodulin is a minus end capping protein
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Arp2/3 complex binds at 70o to the side of actin
filaments to nucleate a daughter filament.
The actin-related proteins: ARP 2/3
Actin-related protein (Arps) about 50 % similarity with actin.
Arp2/3 can stimulates actin assembly.
Arp2/3 combined mother and daughter filaments → created ends of filament elongate
and create the force to push the membrane forward
The Arp2/3; active state
Intracellular movements and changes in cell shape are driven by
actin polymerization
Actin polymerization can generate forces that move
certain intracellular bacteria & viruses.
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