Protein Laboratory, University of Copenhagen

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Actin and Myosin Assembly
Vladimir Berezin
Protein Laboratory Institute of Molecular
Pathology University of Copenhagen
Protein Laboratory, University of Copenhagen
http//www.plab.ku.dk/berezin/index.htm Links
Actin and Myosin Assembly
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- Actin cytoskeleton
- Dynamics of actin assembly
- Myosin the actin motor protein
- Actin and myosin in nonmuscle cells
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- Cell locomotion
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Cytoskeleton
An internal network of 3 types of cytosol fibers
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Microfilaments
7-9 nm
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Role of Microfilaments
? Control of cell shape
? Cell Motility
? Intracellular Trafficking
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Actin Structure in a fibroblast
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Actin

• 10 (muscle cells) and 1-5 (nonmuscle cells)
  of the total protein

• 0.5 mM in the cytosol (platelets) and 5 mM in
  microvilli

• 375 AA residues long (42 kD), encoded by
  large, highly conserved
• gene family (humans have 6 genes)

• a-actins (4-6 AA residues differ)
• ß-actins
• ?-actin

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• Actin-related proteins (50 similarity with
  actin) are not part of
• the actin cytoskeleton

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Monomeric G-Actin and F-Actin Filaments
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Bundles and networks of F-actin in a
spreading platelet after treatment with detergent
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Actin Binding Proteins

• Actin Cross-Linking Proteins (Fascin, Villin,
  Fimbrin,
• a-Actinin, Spectrin, Dystrophin, Filamin)

Structure

• Membrane-Microfilament Cross-Linking Proteins
  (Ponticulin,
• Ankyrin, Dystrophin)

• Actin Capping Proteins (CapZ, Tropomodulin)

• G-Actin Sequestering Proteins (Thymosin ß4,
• Profilin)

Dynamics
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• Actin Severing Proteins (Gelsolin)

• Actin Dynamizing Proteins (profilin, cofilin)

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Actin Cross-linking Proteins
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Membrane-Microfilament Cross-linking
Proteins (platelets)
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Membrane-Microfilament Cross-linking
Proteins (erythrocytes)
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Actin bundles form the core of microvilli and
filopodia, finger- like projections of the plasma
membrane
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The Dynamics of Actin Assembly
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The Dynamics of Actin Assembly
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Cc (0.1µM) lt Cc- (0.8µM)
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Control of the Dynamics of Actin Assembly
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Control of the Dynamics of Actin
Assembly (severing and capping)
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Control of the Dynamics of Actin
Assembly (severing and capping)
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Control of the Dynamics of Actin
Assembly (sequestering and dynamyzing)
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Movements Driven by Actin Polymerization
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Myosin is a Molecular Motor for Actin Filaments

• Myosins are actin stimulated ATPases
• Conventional myosin (type II) forms bipolar
  filaments and is found in both muscle and
  non-muscle cells
• Unconventional myosins (type I III-VII) have a
  single head and are found in non-muscle cells
• Type I myosin binds to the plasma membrane

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Myosin Structure
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Myosin Structure
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In vitro Motility Assay
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3D-Structure of a Myosin II S1 Fragment
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ATP hydrolysis drives myosin along an actin
filament
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The Sliding-Filament Model of Muscle Contraction
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Actin and Myosin in Nonmuscle Cells
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The role of Myosin V in membrane transport from
the Golgi to the cell perophery (astrocytes)
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Actin and Myosin in Nonmuscle Cells
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Actin and Myosin in Nonmuscle Cells
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Actin and Myosin in Nonmuscle Cells
Stress fibers form only in cells tightly
attached to the substratum They terminate in the
basal surface of the cell at focal adhesion
plaques.
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Stress fibers in a cultured fibroblast revealed
by fluorescent anti-actin antibodies
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Cell Locomotion
Model for fibroblast motility
Protrusion of lemellipodium (actin mediated)
Adhesion of lemellipodium and formation of focal
adhesion plaque
Movement of cell over the attachment site
(stress fiber mediated)
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Retraction of the posterior part of the cell
(stress fiber mediated)
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Cell Locomotion
2 mechanisms for generating force at the
leading edge of a motile cell
Polymerization Extension by actin filament
elongation Molecular motor Extension by myosin
movement of actin filaments
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Signal Transduction Pathways and Cell Locomotion
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Conclusions
F-actin is a helical filamentous polymer of
globular G-actin subunits. Actin filaments
are organized into bundles and networks by a
variety of bivalent cross-linking proteins.
Cortical actin networks are attached to the cell
membrane by bivalent membrane-microfilament bindin
g proteins.
The actin cytoskeleton is dynamic, with filaments
able to grow and shrink rapidly. Actin filaments
grow considerably faster at their () end than at
their (-) end. The assembly, length, and
stability of actin filaments are controlled by
specialized actin-binding proteins. These
proteins are in turn regulated by various
mechanisms.
Protein Laboratory, University of Copenhagen
Myosins are motor proteins that interact with
actin filaments and couple hydrolysis of ATP to
conformational changes that result in the
movement of myosin and an actin filament relative
to each other. Cell locomotion occurs through a
common mechanism involving actin polymerization
and myosin I-generated movement at the leading
edge, assembly of adhesion structures, and
cortical contraction mediated by myosin II.