Actin Filament Assembly,
Growth, Branching, Capping
and Disassembly are
Controlled by Actin Binding
Proteins
Actin polymerization is induced by Arp 2/3 protein complex at the surface of Listeria monocytogenes
Matthew D. Welch et al.
The pathogenic bacterium Listeria monocytogenes is capable of directed movement within the cytoplasm of infected
host cells. Propulsion is thought to be driven by actin polymerization at the bacterial cell surface and moving bacteria
leave in their wake a tail of actin filaments. Actin assembly by L. monocytogenes requires the bacterial surface protein
ActA and protein components present in host cell cytoplasm. We have purified an eight-polypeptide complex that
possesses the properties of the host-cell actin polymerization factor. The pure complex is sufficient to initiate ActAdependent actin polymerization at the surface of L. monocytogenes, and is required to mediate actin tail formation
and motility. Two subunits of this protein complex are actin-related proteins (ARPs) belonging to the Arp2 and Arp3
subfamilies. The Arp3 subunit localizes to the surface of stationary bacteria and the tails of motile bacteria in tissue
culture cells infected with L. monocytogenes; this is consistent with a role for the complex in promoting actin
assembly in vivo. The activity and subunit composition of the Arp2/3 complex suggests that it forms a template that
nucleates actin polymerization.
Actin Bundling
a-actinin and fimbrin cross-link actin filaments
Actin Bundling
a-actinin and fimbrin cross-link actin filaments
Diversity of Actin Bundling Proteins
a-actinin, fimbrin, and villin form parallel cables
-stiff connections between actin-binding domains
Spectrin and filamin form actin filament webs of gels
-flexible connections between actin-binding domains (filamanin)
-stiff bent connection (spectrin)
Microvilli (mv) are specialized actin-based structures
Increases surface area for
Absorption by20-fold
Villin and fimbrin
cross link
20-30 actin
filaments in mv
Villin introduced
into fibroblasts
Induces microvilli
formation
Filamin makes 3D
Crosslinks
Actin binds to Other Proteins That Influence its Function
CHI- complex haploinsufficiency and actin
haploinsufficiency has been gaining in
appreciation as an important influence on human disease
human individuals may have as many as 45 CHI gene pairs
Actin Filament Crosslinking
Spectrin, glycophorin, and band 3
Correspond to >60% of membrane
Proteins in RBCs
Spectrin is the most abundant, 25%
250000 copies/cell
Principle component of the cytoskeleton
If dissociated, the red blood cell
fragments into vesicles
Spectrin forms heterodimers
And tetramers, and is part of a
Junctional complex
Ankyrin joins spectrin to the PM by
The TM protein band 3
Look how small actin filaments are here!
Why? This weblike structure allows cells to deform in capillaries
Spectrin mutations cause round and fragile RBCs and anemia
PTEN and polarity:
phosphodiesterase and an inhibitor of
the phospho-AKT pathway by
removing the 3' phosphate group of PI
(3,4,5)P3.
Actin Is Required to Form Focal Adhesions
Formation of Intercellular Junctions during Wound Healing
Mammary epithelial cell monolayer was grown to confluence and
mechanically wounded. Cells at the free edge lose their intercellular
junctions, form lamellipodia and migrate to fill the empty space.
At the last stage of healing, cells from opposite sides of the wound have
made contact. Actin (red; rhodamine phalloidin) and occludin (blue, antioccludin antibody) appear at the newly forming intercellular contacts (arrow).
Nuclei were stained green (Syto) to help identify cells.
Reorganizing the cytoskeleton is critical for wound healing
Triple label showing microtubules (red), F-actin (green) and
myosin-2 (blue) around wound made in a Xenopus oocyte. The
microtubules are organized into a radial array that encloses the
ring of F-actin and myosin-2B.
Technology:
Novel antisense approach provides a rapid spatially and
temporally controllable knockdown of specific connexin
proteins. A single topical application of Cx43 antisense gel
results in a dramatic increase in the rate of wound closure.
Molecular Motors Allow motion
Actin depolymerizing factor (ADF) Cofilin binds to Actin
It twists the filament, making it easier for subunits at the
Minus end of the filament to dissassemble
Importantly, the stimulus-responsive function of
ADF/cofilin is regulated by phosphorylation of a single
serine residue. In response to stimuli, ADF is
dephosphorylated. The stimuli, such as growth factors,
chemotactic peptides, or agents increasing the levels of
[Ca2+]i and cAMP, promote the reorganization of the
actin cytoskeleton. In quiescent cells, ADF/cofilin appears
diffusely distributed in the cytoplasm, the activated
(dephosphorylated) protein translocates to regions of the
cells where actin filaments are highly dynamic like the
leading edge of ruffled membranes, the cleavage furrow
of dividing cells, or the neuronal growth clone.
Dephosphorylation correlates with increased motility and
extension of cellular processes (Carlier et al., 1999).
ADF/cofilin increases the turnover of actin filaments
which powers actin motility.