Microfilaments cytoskeleton

Figure 16-10. Migratory keratocytes from a fish epidermis. (A) Light micrographs of a keratocyte in culture,
taken about 15 sec apart. This cell is moving at about 15 µm/sec. (B) Keratocyte seen by scanning electron
microscopy, showing its broad, flat lamellipodium and small cell body, including the nucleus, carried up above
the substratum at the rear. (C) Distribution of cytoskeletal filaments in this cell. Actin filaments (red) fill the large
lamellipodium and are responsible for the cell's rapid movement. Microtubules (green) and intermediate
filaments (blue) are restricted to the regions close to the nucleus. (A and B, courtesy of Juliet Lee.)

Figure 22-1. Actin structures in a fibroblast. (a)
Scanning electron micrograph of a cultured
fibroblast. At the front of the cell, filopodia,
lamellipodia, and ruffles project from the cell
membrane. At the rear of the cell, the tail is firmly
attached to the surface. The arrow indicates the
direction of movement. (b) Fluorescence micrograph
of a fan-shaped fibroblast, stained with rhodamine
phalloidin. Visible are numerous actin bundles in the
lamellipodia and stress fibers in the cell body. [Part
(a) courtesy of J. Heath; part (b) courtesy of B.
Hollifield.]

Figure 22-9 The three phases of G-actin polymerization in vitro. (a) During the initial nucleation phase, ATP G-actin monomers (pink) slowly form stable
complexes of actin (purple). These nuclei are more rapidly elongated in the second phase by addition of subunits to both ends of the filament. In the third
phase, the ends of actin filaments are in a steady state with monomeric ATP G-actin. After their incorporation into a filament, subunits slowly hydrolyze
ATP and become stable ADP F-actin (white). Note that the ATP-binding clefts (black triangles) of all the subunits are oriented in the same direction in F-
actin. (b) Time course of the in vitro polymerization reaction (pink curve) reveals the initial lag period. If some actin filament fragments are added at the
start of the reaction to act as nuclei, elongation proceeds immediately without any lag period (purple curve).

Figure 22-5. Actin cross-linking proteins bridging pairs of actin filaments. (a)
When cross-linked by fascin, a relatively short protein, actin filaments form a
bundle. (b) Long cross-linking proteins such as filamin are flexible and thus can
cross-link pairs of filaments lying at various angles.

Figure 22-6. Cross-linkage of actin filament
networks to the plasma membrane in platelets,
muscle cells, and epithelial cells. (a) In platelets a
three-dimensional network of actin filaments is
attached to the integral membrane glycoprotein
complex Gp1b-IX by filamin. Gp1b-IX also binds
to proteins in a blood clot outside the platelet.
Platelets also possess a two-dimensional cortical
network of actin and spectrin similar to that
underlying the erythrocyte membrane. (b) In muscle
cells dystrophin attaches actin filaments to an
integral membrane glycoprotein complex. This
complex binds to laminin and agrin in the
extracellular matrix (ECM). (c) In epithelial cells,
the ERM protein, ezrin, and EBP50 crosslink an
actin filament to the cystic fibrosis transmembrane
conductance receptor. After activation, ezrin
unfolds and oligomerizes to form head-to-tail
dimers. The head domain binds EBP50, while the
tail domain binds actin.

Figure 18-34. The circumferential
belt is located near the apical surface
of epithelial cells. In epithelial tissue,
a belt of actin and myosin filaments
rings the inner surface of the cell
adjacent to the adherens junctions,
where cell-cell contacts are
maintained. The circumferential belt
is attached by linker proteins to cell-
adhesion molecules in the plasma
membrane (Chapter 22).

Figure 22-38. Experimental demonstration that
myosin II is required for cytokinesis. The activity of
myosin II was inhibited either by deleting its gene or
by microinjecting anti-myosin II antibodies into a
cell. A cell that lacked myosin II was able to replicate
its DNA and nucleus, but it failed to divide; this
defect caused the cell to become large and
multinucleate. In comparison, an untreated cell
during the same period continued to divide and
formed a multicellular ball of cells in which each cell

contained a single nucleus.

Figure 22-17. Structure of various myosin molecules. (a) The three major myosin proteins are organized into head, neck, and tail domains, which carry out
different functions. The head domain binds actin and has ATPase activity. The light chains, bound to the neck domain, regulate the head domain. The tail
domain dictates the specific role of each myosin in the cell. Note that myosin II, the form that functions in muscle contraction, is a dimer with a long rigid
coiled-coil tail. (b) Proteolysis of myosin II reveals its domain structure. For example, most proteases cleave myosin II at the base of the neck domain to
generate a paired-head and neck fragment, called heavy meromyosin (HMM), and a rodlike tail fragment, called light meromyosin (LMM). Further
digestion of HMM with papain splits off the neck region (S2 fragment) and leads to separation of the two head domains into single myosin head fragments
(S1 fragments).

Figure 22-19. The sliding-filament assay. (a) Schematic diagram illustrates movement of actin filaments across myosin molecules attached to a coverslip.
After myosin molecules are adsorbed onto the surface of a glass coverslip, excess myosin is removed; the coverslip then is placed myosin-side down on a
glass slide to form a chamber through which solutions can flow. A solution of actin filaments, made visible by staining with rhodamine-labeled phalloidin, is
allowed to flow into the chamber, and individual filaments are observed under a fluorescence light microscope. (The coverslip in the diagram is shown
inverted from its orientation on the flow chamber to make it easier to see the positions of the molecules.) (b) Sliding movements of fluorescent actin filaments
generated by the myosin head can be quantified by video microscopy. These photographs show the positions of three actin filaments (numbered 1, 2, 3) at
30-second intervals. In the presence of ATP, the actin filaments move at a velocity that can vary widely depending on the myosin tested and the assay
conditions (ionic strength, temperature, ATP concentration, calcium concentration, etc.). [Part (b) courtesy of M. Footer and S. Kron.]

Figure 22-28. The titin-nebulin
filament system stabilizes the
alignment of thick and thin
filaments in skeletal muscle. (a) A
titin filament attaches at one end to
the Z disk and spans the distance to
the middle of the thick filament.
Thick filaments are thus connected
at both ends to Z disks through titin.
Nebulin is associated with a thin
filament from its (+) end at the Z
disk to its ( ) end. The large titin
and nebulin filaments remain
connected to thick and thin
filaments during muscle contraction
and generate a passive tension when
muscle is stretched. (b) To visualize
the titin filaments in a sarcomere,
muscle is treated with the actin-
severing protein gelsolin, which
removes the thin filaments. Without
a supporting thin filament, nebulin
condenses at the Z disk, leaving titin
still attached to the Z disk and thick
filament.

Figure 22-26. Schematic diagram showing location of capping
proteins that stabilize the ends of actin thin filaments. CapZ (green)
caps the (+), or barbed, ends of filaments at the Z disk, and
tropomodulin (yellow) caps the ( - ), or pointed, ends of thin
filaments. The presence of these two proteins at opposite ends of a
thin filament prevents actin subunits from dissociating during
muscle contraction.

Figure 22-27. The sliding-filament
model of contraction in striated
muscle. The arrangement of thick
myosin and thin actin filaments in
the relaxed state is shown in the
top diagram. In the presence of
ATP and Ca2+, the myosin heads
extending from the thick filaments
pivot, pulling the actin thin
filaments toward the center of the
sarcomere. Because the thin
filaments are anchored at the Z
disks (purple), this movement
shortens the sarcomere length in

the contracted state (bottom).

Figure 22-22. The coupling of ATP hydrolysis to
movement of myosin along an actin filament. In the
absence of bound nucleotide, a myosin head binds actin
tightly in a "rigor" state. When ATP binds (step 1 ), it
opens the cleft in the head, disrupting the actin-binding
site and weakening the interaction with actin. Freed of
actin, the myosin head hydrolyzes ATP (step 2 ), causing a
conformational change in the head that moves it to a new
position, closer to the (+) end of the actin filament, where
it rebinds to the filament. As phosphate (Pi) dissociates
from the ATP-binding pocket (step 3 ), the myosin head
undergoes a second conformational change the power
stroke which restores myosin to its rigor conformation.
Because myosin is bound to actin, this conformational
change exerts a force that causes myosin to move the actin
filament. The diagram shows the cycle for a myosin II
head that is part of a thick filament, but other myosins
attached to a membrane are thought to operate according
to the same mechanism. [Adapted from I. Rayment and H.
M. Holden, 1994, Trends Biochem. Sci. 19:129.]

Figure 22-32. Three myosin-dependent mechanisms for regulating muscle contraction. (a) In
invertebrate muscle, binding of Ca2+ to the myosin regulatory light chain (LC) activates contraction. (b)
In vertebrate smooth muscle, phosphorylation of the myosin regulatory light chains on site X by Ca 2+-
dependent myosin LC kinase activates contraction. At Ca 2+concentrations <10 6 M, the myosin LC
kinase is inactive, and a myosin LC phosphatase, which is not dependent on Ca 2+ for activity,
dephosphorylates the myosin LC, causing muscle relaxation. (c) Activation of Rho kinase also leads to
phosphorylation of the myosin regulatory LC at ser 19.

Figure 18-42. Steps in keratinocyte movement. In a fast-moving
cell such as a fish epidermal cell, movement begins with
extension of one or more lamellipodia from the leading edge of
the cell (step 1); some lamellipodia adhere to the substratum via
focal adhesions (step 2). Then the bulk of the cytoplasm in the
cell body flows forward (step 3). The trailing edge of the cell
remains attached to the substratum until the tail eventually
detaches and retracts into the cell body (step 4). See the text for

more discussion.

igure 22-43. A model of the molecular events at the
leading edge of moving cells. The polymerization of actin
filaments at the (+) end, stimulated by profilin located at
the leading-edge membrane, pushes the membrane
outward. Other proteins like Vasp and Arp2/3 may
participate in directing assembly. Simultaneously, cofilin
induces the loss of subunits from the ( ) ends of
filaments. Arp2/3 and actin cross-linking proteins
stabilize the actin filaments into networks and bundles. In
addition, myosin I is thought to link actin filaments to the
leading-edge plasma membrane.

Figure 22-12 Model of the complementary roles of profilin and thymosin β4 in regulating
polymerization of G-actin. (a) At the cell membrane, profilin is bound to PIP 2, a membrane
lipid, while most of the G-actin is complexed with thymosin β4 and thus cannot polymerize. (b)
In response to an extracellular signal, such as chemotactic molecules that stimulate actin
assembly, profilin is released from the membrane by hydrolysis of PIP 2. The released profilin
displaces thymosin β4, forming profilin G-actin complexes that can assemble into filaments.
(c) The profilin-actin complexes interact with proline-rich proteins in the membrane, where
profilin adds actin monomers to the (+) end of actin filaments. Eventually, the incorporation of
monomers into filaments depletes the pools of profilin-actin and thymosin β4 actin
complexes. (d) ADP G-actin subunits that have dissociated from a filament are converted into
ATP G-actin by profilin, thus helping to replenish the cytoplasmic pool of ATP G-actin.

Figure 22-39. Cytoplasmic streaming in cylindrical giant
algae. (a) The center of a Nitella cell is filled with a single
large water-filled vacuole, which is surrounded by a layer
of moving cytoplasm (indicated by blue arrows). A
nonmoving layer of cortical cytoplasm filled with
chloroplasts lies just under the plasma membrane
(enlarged lower figure). On the inner side of this layer are
bundles of stationary actin filaments (red), all oriented
with the same polarity. A myosinlike motor protein (blue
dots) carries portions of the endoplasmic reticulum (ER)
along the actin filaments. The movement of the ER
network propels the entire viscous cytoplasm, including
organelles that are enmeshed in the ER network. (b) An
electron micrograph of the cortical cytoplasm shows a
large vesicle connected to an underlying bundle of actin
filaments. This vesicle, which is part of the endoplasmic
reticulum (ER) network, contacts the stationary actin
filaments and moves along them by a myosinlike motor.
[Part (b) from B. Kachar.]

Microfilaments cytoskeleton

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Microfilaments cytoskeleton