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Lecture 7 - Actin
Names and functions of proteins and molecules reguarding Actin
39
Biology
Undergraduate 3
03/05/2013

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Term
Functions of the Cytoskeleton
Definition
Support of the cell - Intermediate filaments
Support of the membrane - Actin filaments
Targeted Delivery - Microtubules
Term
Monomers (G-Actin)- 'G' refers to 'globular'
Definition
Molecular weight ~ 43 kDa (4-5 nm in diameter)
Binds ATP or ADP
Innate ATPase activity
Term
Polymers (F-Actin) - 'F' refers to 'filamentous'
Definition
Diameter 7-9 nm
Two helical strands (periodicity of one strand ~ 72 nm)
Innate ATPase activity (enhanced within filaments)
Polarized filaments (due to ATP hydrolysis)
Term
Steady state of F-Actin (Critical Concentration)
Definition
rate of monomers coming on is equal to the rate of monomers coming off
Term
Pointed end of F-Actin
Definition
- end, monomer deletion
Term
Barbed end of F-Actin
Definition
+ end, monomer addition
Term
Treadmilling
Definition
added to positve side, falls of negative side.
Term
Cytochalasin
Definition
Filament Depolymerization - barbed-end blocking (caps barbed positive end of filament)
Term
Latrunculin A
Definition
Filament Depolymerization - monomer binding
Term
Phalloidin
Definition
Filament Stabilization - lateral binding - fluorescence-tagged, used for observation
Term
Jasplakinolide
Definition
Filament Stabilization - lateral binding (cell permanent)
Term
Viscometry
Definition
Biochemical method for measuring Actin polymerization
- formation of filamentous actin increases viscosity
Term
Sedimentation Assays
Definition
Biochemical method for measuring Actin polymerization
- F-Actin pellets at 100,000 xg for 30 minutes
Term
Fluorescence Video Microscopy
Definition
Biochemical method for measuring Actin polymerization
- optical measurements of filament growth
Term
Pyrene Fluorescence
Definition
Biochemical method for measuring Actin polymerization
- Actin-pyrene fluroescence intensity (intensity is increased in filamentous actin - 20 fold increase)
Term
Arp2/3 Complex
Definition
Nucleation of Actin Filament
- forms orthogonal/branched arrays of Actin filaments
- set of seven proteins
- mimics an "actin nucleus"
Term
Formin Family of Nucleating Proteins
Definition
Nucleation of Actin Filament
- forms bundles of parallel Actin filaments
Term
Tandem Monomer Binders
Definition
Nucleation of Actin Filaments
- binds, orients, and stabilizes Actin nuclei
Term
FH2 domain
Definition
Dimer of Formin - de novo nucleation and processive monomer addition
Term
Arp: actin-related protein
Definition
- high degree of homology with actin
- form identical folds as found in actin
Term
Properties of Arp2/3 complex
Definition
1. Lateral F-Actin binding
2. de novo Actin nucleation
3. Pointed-end Actin binding
Term
WASP proteins
Definition
activates Arp2/3 complex
- activated by cdc42 (small GTPase protein) and PIP2
Term
Profilin
Definition
Small Actin Binding Protein
- enhances barbed-end addition
- 12-15 kDa
- localized at membranes
- Binds to PIP2
- regulated by phosphorylation
- blocks pointed end addition
- promotes barbed end addition
- accelerates ADP-ATP exchange
- lowers Km at the barbed end
Term
Thymosin B4
Definition
Small Actin Binding Protein
- buffers available monomers
- ~ 5 kDa
- very abundant protein (up to 0.5 mM in some cell types
- enriched at cell periphery
- inhibits monomer addition
- inhibits nucleotide exchange
- inhibits nucleotids hydrolysis
- G-Actin buffer (regulated by mass action)
Term
Cofilin
Definition
Small Actin Binding Protein
- pointed-end severing
- 15-22 kDa
- Activates monomer binding, pointed end capping, filament binding (ADP-actin >> ATP-Actin), filament severing (twisting)
- net effect: - end depolymerization
- inhibited by: acidic/neutral pH, phosphorylation (ser-3), PIP2-(4,5), tropomyosin
Term
CapZ
Definition
Actin Capping Protein
- barbed end capping (+ end)
- moderates direction polymerization
Term
Tropomodulin
Definition
Actin Capping Protein
- pointed end capping (- end)
- caps and protects
Term
Tropomyosin
Definition
Lateral Actin Binding Protein
- stiffens filaments
- stabilizes tropomodulin-actin
- impedes cofilin binding
- covers ~ 6 actin monomers
- enhances myosin II binding
- sterically hinders numerous lateral actin interactions
Term
Fibrin, villin
Definition
Actin Bundling Proteins
- two actin binding domains
- "dense" filament bundling (~12 nm)
Term
Dimer (alpha-actin)
Definition
Actin Bundling Protein
- single actin binding domains
- antiparallel coiled coil tail domain interactions
- "loose" filament bundling (~35-60 nm; stress fibers)
Term
Membrane Blebbing
Definition
occurs with disruption of the membrane-cytoskelton linkage
Term
Ankyrin
Definition
Binds Na+ channels to spectrin (fodrin) which is bound to F-Actin
Term
Spectrin
Definition
- tetramer from head-to-head heterodimers
- tetramer extends 200 nm
- chain formed of 106 aa repeats
- multiple membrane protein interactions
- actin association via protein 4.1 at tail
Term
Filamin (ABP-280)
Definition
- Actin binding (CH) domain
- six Ig-like repeat domains
- self-association via 6th repeat
- multiple membrane protein interactions
- crosslinks orthogonal actin arrays
- concurrently binds F-Actin and various integral membrane proteins
Term
Filamin mutant melanocyte
Definition
Filamin (ABP-280) deficient cells
- continuous blebbing
- diminished locomotion
- viable
Term
Spectrin Knock-Down
Definition
Constituitive membrane Blebbing
Term
Duchenne Muscular Dystrophy
Definition
mutation in the dystrophin gene
- x-linked recessive mutations
- 1 in 3,500 males
- 100s of distinct mutations identified
- most result in loss of protein
- progressive muscle weakness and atrophy
- sarcolemmal membrane and fiber damage
- ~12 y.o = wheelchair bound
- ~18 y.o = death by pulmonary failure
- subset have cardiomyopathy
Term
Dystrophin
Definition
- Gene: 2.6 x 10^6 base pairs (largest in human genome)
- 427 kDa protein
- expressed in skeletal and cardiac muscle
- some expression in neurons
- binds F-Actin
- binds B-dystroglycan
Term
Function of dystrophin
Definition
- provides a mechanical link between the actin
- provides a structural support against the mechanical stress from repeated contraction and relaxation
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