Lecture 09: Lecture 09: The Cytoskeleton (I -
Transcript of Lecture 09: Lecture 09: The Cytoskeleton (I -
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BME 42-620 Engineering Molecular Cell Biology
Lecture 09:Lecture 09:
The Cytoskeleton (I): Actin
C ( ) &The Cytoskeleton (II): Microtubule & Intermediate Filament
1BME42-620 Lecture 09, September 27, 2011
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Outline
• Overview of cytoskeletal filamentsy
• Actin and its associated proteins
• Microtubule and its associated proteins; Centrosome
• Intermediate filament and its associated proteins
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Outline
• Overview of cytoskeletal filamentsy
• Actin and its associated proteins
• Microtubule and its associated proteins; Centrosome
• Intermediate filament and its associated proteins
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The Cytoskeleton is Highly Dynamic and Regulated
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Overview of Cytoskeletal Filaments
Shape Diameter Subunits Polarized
i bl iactin cable ~6 nm actinmonomer
yes
microtubule tube ~25nm tubulin heterodimer
yes
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intermediate filament
rope ~10nm Various dimers
no
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Catalyzed and Regulated Self-Assembly of Macromolecules (I)
• Binding specificity between monomers is determined by multiple weakbonds on complementary surfaces.
• Demo: poliovirus capsid self assembly
Olson, A. J., Hu, Y. H. E., & Keinan, E. (2007). Chemical mimicry of viral capsid self-assembly. Proceedings of the National Academy of Sciences, 104(52), 20731-20736.
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Catalyzed and Regulated Self-Assembly of Macromolecules (II)
• Cellular macromolecules often form through self-assembly of smallsubunits.
• This process requires energy and is often catalyzed and regulated.
• Advantages:
- Reduced possibility of errorP ibilit f i- Possibility for repair
- Subunit can be recycled- Assembly provides multiple entry points for regulation
- Regulation of subunit synthesis and degradation- Regulation of subunit synthesis and degradation- Regulation of nucleation- Regulation by changing environmental conditions- Regulation by modifying subunits
Regulation using accessory proteins
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- Regulation using accessory proteins
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Example: Self-Assembly of a Tobacco Mosaic Virus
• The virus is made of 2130 protein subunits of 158 amino acids each.
• It also has a single-stranded RNA of 6390 nucleotids.
• Translation error 1 out of 3000 amino acids (0 99967)Translation error 1 out of 3000 amino acids (0.99967)Probability of making a correct TMV = 0.99967158=0.949
• If instead the TMV is assembled from one large sequence of amino g qacid, probability of making a correct TMV = 0.99967336540=1.87X10-49
• Self-assembly is one of the central principles in biologySelf assembly is one of the central principles in biology.
• The assembly information is encoded in weak but highly specific noncovalent interactions
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noncovalent interactions.Pollard & Earnshaw, Cell Biology, 2/e, 2008, Chapter 5,
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ATP (adenosine triphosphate) (I)( p p ) ( )• ATP is the most widely used energy carrier in cells.
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ATP (adenosine triphosphate) (II)( p p ) ( )• Synthesis of biological polymers is driven by ATP hydrolysis.
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Outline
• Overview of cytoskeletal filamentsy
• Actin and its associated proteins
• Microtubule and its associated proteins; Centrosome
• Intermediate filament and its associated proteins
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Organization of Actin with a Cell
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Actin Structure and Function• Each actin subunit is a
globular monomerglobular monomer.
• One ATP binding site per monomer.
• FunctionsFunctions- Cell migration- Cell shape- Used as tracks for myosin Used as t ac s o yosfor short distance transport
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Pollard & Cooper, Science,326-1208, 2009
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Basics Terms of Chemical Reaction Kinetics
• A reversible bimolecular binding reaction
A + B AB
• Rate of association = k+[A][B]
• Rate of disassociation = k-[AB]
• At equilibrium k+[A][B] = k-[AB]
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Actin Nucleation and Nucleotide Hydrolysis
• Actin polymerizes and depolymerizes substantially faster t th l d (b b d d) that the plus end (barbed end) than
at the minus end (pointed end).
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Actin Associated Proteins (I)
• More than 100 associated proteins identified so far.
• Functions- Monomer binding- Nucleation- Filament capping- Filament severing
Filament side binding and supporting- Filament side-binding and supporting- Filament crosslinking
- Signaling adapterg g p
• Functional overlap and collaboration between actin-binding proteins
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binding proteins
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Actin Associated Proteins (II)
• Monomer binding proteins- profilin: to bind actin monomer and accelerate elongationprofilin: to bind actin monomer and accelerate elongation- thymosin: to bind and lock actin monomer - ADF/cofilin: to bind and destabilize ADP-actin filaments
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Actin Associated Proteins (III)
• Actin nucleation- Formins: to initiate unbranched actin filamentsFormins: to initiate unbranched actin filaments- Arp2/3: to bind the side of actin and initiate branching
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Actin Associated Proteins (IV)
• Actin capping protein- Blocks subunit association and disassociationBlocks subunit association and disassociation
• Actin severing protein
• Three families of proteins perform both functions
- Gelsolin- Fragmin-severin- ADF/cofilin
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Actin Associated Proteins (V)
• Actin side-binding proteinstropomyosin nebulin caldesmontropomyosin, nebulin, caldesmon
• Actin crosslinking proteing p- -actinin- filamin- spectrin- ERM
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Actin Adapter Protein
• Adaptor proteins such as WASP (a branching mediating factor) & VASP ( l i ti di tiVASP (a polymerization mediating factor) server as connectors between signaling pathways and actin assemblyactin assembly.
• WASP: Wiskott-Aldrich syndrome t iprotein
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Actin Regulation
• GTPase: Molecule switch; Family of enzymes that areFamily of enzymes that are activated by GTP binding and inactivated by GTP hydrolysis and phosphate dissociation.
• GTPases:GTPases: cdc42: its activation triggers actinpolymerization and bundling at filopodia.
Rho: its activation promotes actinbundling.
Rac: its activation promotes
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Rac: its activation promotes polymerization at the cell periphery.
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GTP (guanosine triphosphate)
• GTP-binding proteins (GTPases) are important signaling molecules.
• When GTP is hydrolyzed into GDP, the GTP binding domain undergoes a g gconformation change that inactivates the GTPase.
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Rac on Actin Organization
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Summary: actin
• Relatively soft (quantification to follow in later lectures).y (q )
• Often form bundles; mechanical strength comes mostly from bundling and crosslinking.
• Mostly function to withstand tension rather than• Mostly function to withstand tension rather than compression.
• Relatively stable and easy to work with (biochemically).
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Summary: actin associated proteins
• Different associated proteins serve a broad range of p gfunctions.
• Proteins with multiple functional domains can have multiple functions.
• Some but not all of them are essential.
• Most of the proteins have functional overlap.
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Outline
• Overview of cytoskeletal filamentsy
• Actin and its associated proteins
• Microtubule and its associated proteins; Centrosome
• Intermediate filament and its associated proteins
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Microtubule Structure
• Polarized- plus (-tubulin) end- minus (-tubulin) end
• Outer diameter: 25 nm
• Typically 13 protofilaments; yp y pSome have 11, 15, 16, etc
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Microtubule Organization
• During interphase
- Radial pattern organized around the centrosome
• During metaphase
Bi l hit t- Bipolar architecture
30Fig. 34-1
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Microtubule Dynamic Instability
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Microtubule Dynamic Instability
T. Wittmann et al, J. Cell Biol., 161:845, 2003.
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Microtubule Dynamic Instability Parameters
Kinoshita et al, Science, 294:1340, 2001
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Microtubule Associated Proteins (I)
• Stabilizing MAPs
Tau Binds side and stabilizes microtubules
MAP2, MAP4 Binds side and stabilizes microtubules
• Destabilizing MAPs
Op18/stathmin Binds tubulin dimers & destabilizes MTs
Katanin AAA ATPase that severs microtubules
XKCM/MCAK Kinesin-related; destabilizes plus ends
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Electron Microscopy of Microtubules Decorated with MAPs
Fig. 34-11
Frozen deep-etched, shadowed microtubules with (upper) and without (lower) tau
Fixed, embedded sections of microtubules with (upper) and without (lower) MAP2without (lower) tau without (lower) MAP2
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Microtubule Associated Proteins (II)
Plus end track protein
CLIP170 Links membranes to growing plus endsCLIP170 Links membranes to growing plus ends
EB1 Links APC to MT plus ends
f
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Dynamics and mechanics of the microtubule plus end. Howard J, Hyman AA. Nature. 2003 422(6933):753-8.
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Summary: Microtubule
• Relatively rigid (quantification in following lectures).y g (q g )
• Often form bundles; mechanical strength comes mostly from bundling and crosslinking.
• Can withstand compression• Can withstand compression.
• Can denature easily; Difficult to work with (biochemically).y; ( y)
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Centrosome
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Outline
• Overview of cytoskeletal filamentsy
• Actin and its associated proteins
• Microtubule and its associated proteins; Centrosome
• Intermediate filament and its associated proteins
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Intermediate Filament
• So named because of its diameter in striated muscles (diameter ~10nm).
• Its core structure is an helical coiled coil• Its core structure is an -helical coiled coil.
• N- and C-terminal domains vary considerably in size.
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Intermediate Filament Classification
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Intermediate Filament• Biochemically most stable among the three filaments
• Assembly disassembly regulated by phosphorylation
- Phosphorylation: reaction in which a phosphate group is covalently coupledPhosphorylation: reaction in which a phosphate group is covalently coupled to another molecule.
- Kinase: an enzyme that catalyzes the addition of phosphate groups
- Phosphatase: an enzyme that catalyzes the removal of phosphate groupsPhosphatase: an enzyme that catalyzes the removal of phosphate groups
• Phosphorylation may stabilize or destabilize intermediate filaments.
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Intermediate Filament Associated ProteinsIntermediate Filament Associated Proteins
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Plectin Connects IFs to Actin & MicrotubulesPlectin Connects IFs to Actin & Microtubules
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Intermediate FilamentsIntermediate Filaments• Flexible but stable.
• Primary assumed function is to prevent excessive stretching.
I l h l i k b t i t di t• In general, much less is known about intermediate filaments
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An Example: Microtubule, Intermediate Filament & Organelles in a Frog Axon g
Nobutaka HirokawaN. Hirokawa, J. Cell Biol. 94:129, 1982
Nobutaka HirokawaUniversity of Tokyo
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Bar: 0.1m
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Required Reading
• Chapters 2 & 16p
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Questions ?
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