CYTOSKELETON (I)CYTOSKELETON (I)Actin filamentsActin filaments

Readings and ObjectivesReadings and Objectives

• ReadingReading– Russell : Chapter 1 (not complete information)– Cooper: Chapter 12

• ObjectivesObjectives• Actin and actin filament dynamism• Actin bundles and network• Actin and myosin: role in contractile assemblies• Role in cellular movement

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Introduction: CytoskeletonIntroduction: Cytoskeleton• A network of protein filaments and tubules, extends from the

nucleus to the plasma membrane• Structural framework

– cell shape, localize organelles, general organization of the cytoplasm

• Movement– Cell movement, internal transport of organelles, muscle

contraction• Dynamic structures, continually reorganizedComposed of three main types of protein filaments:

• Actin filaments (7 nm)• Intermediate filaments (8-11 nm)• Microtubules ( 25 nm)

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Structure and Organization of Actin FilamentsStructure and Organization of Actin Filaments• Actin filaments (microfilaments):

Polymer of Actin, flexible fibers 7 nm in diameter, several µm in length

• Actin first isolated from muscle cells in 1942

• Abundant in all types of eukaryotic cells

• Mammals have 6 actin genes: 4 are expressed in muscle cells and 2 in nonmuscle cells

• Highly conserved• Prokaryotic ancestor is MreB

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Actin and Actin FilamentActin and Actin Filament• 3-D structure determined in 1990• Actin: globular (G-actin), 375 aa (43 kD), barbed and

pointed ends, binds head-tail to nucleate a trimer• Filamentous (F-actin): monomers added to both end• Filament is polar pointed end vs barbed end

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G-Actin and Actin FilamentG-Actin and Actin Filament• Polymerization is reversible• The rate at which monomers are added to filaments is

proportional to their concentration

• ATP bound actin binds to barbed end with high affinity• ADP-actin has low affinity to the pointed ends• when ATP hydrolyses to ADP• ADP-actin dissociates from filaments more readily than

ATP-actin • Therefore, the critical concentration of actin monomers

is higher for addition to the pointed end than to the barbed end of actin filaments

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Treadmilling: polarity of F-actin growth• At cellular actin concentrations• Barbed end of a filament grows 5–10 times faster than

the pointed end• ADP-actin dissociates from pointed end• Exchange of ATP for ADP added to barbed-end• Process is called

Treadmilling• Dynamic growth• Direction?

PointedBarbed

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Actin Binding Proteins• Actin Binding Proteins (ABP): modulate the Assembly

and disassembly of actin filaments• ABD/Actin interaction has diverse functionality• Contribute to the cellular role of actin filaments

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Actin Remodeling• Some actin-binding proteins bind along the length of actin

filaments, stabilizing them or cross-linking them to one another• Others stablize by capping the ends and preventing dissociation• Others promote dissociation, while others regulate the

exchange of ATP for ADP.

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Initiation of Actin Filament polymerization• Nucleation is the rate-limiting step• Formin and the Arp2/3 complex determine where

filaments are formed by facilitating nucleation• Formins nucleate long unbranched actin filaments

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Initiation of Actin Filament polymerization• actin filaments actively turn over and branch extensively• These filaments are nucleated by the Arp2/3 (Actin

Related Protein) complex, which binds actin/ATP near the barbed ends

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Actin Filament Depolymerization• The ADF/cofilin (Actin Depolymerizing Factor) family modifies

existing filaments• enhance the rate of dissociation of actin/ADP monomers from

the pointed end, and remain bound to the monomers, preventing their reincorporation

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Actin Filament Severing• ADF/cofilin can also bind to and sever actin filaments• Profilin reverses the ADF/Cofilin effect • Stimulate exchange of bound ADP for ATP and dissociating the

actin/ATP monomers from cofilin• Become available for reassembly in two filaments

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Summary: Assembly of an Actin Filament

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Actin filament higher order assemblies• Actin bundles—cross-linked

into closely packed parallel arrays

• Actin networks—cross-linked in arrays that form 3-D meshworks

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Actin Bundles• Parallel filaments cross linked

by actin-bundling proteins • Have two domains to bind

actin and align the filamentsTwo types of actin bundles:1. Non contractile

– filaments (14 nm apart) aligned in parallel, same polarity, barbed ends adjacent to the plasma membrane

• Fimbrin: a 68 kD proteins, cross links by its two actin binding domains (ABD)

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Actin bundles: Intenstinal MicrovilliActin bundles: Intenstinal Microvilli• Actin bundles take part in

avariety of cell surface protrusions– cell movement– phagocytosis– absorption of nutrients

• Intenstinal microvilli:• Membrane projections,

increase absorption surface

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Actin bundles: Intenstinal MicrovilliActin bundles: Intenstinal Microvilli• closely packed parallel

bundles of 20 to 30 actin filaments

• Relatively permanent • The filaments are cross-linked

in part by fimbrin and villlin• The actin bundles are

attached to the plasma membrane by the calcium-binding protein calmodulin in association with myosin I

• At the base attach to actin cortex

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Actin bundles: membrane protrusionsActin bundles: membrane protrusions• Other surface protrusions are transient and form in

response to environmental stimuli• Pseudopodia- responsible for phagocytosis and the

movement of amoebas• Lamellipodia- broad, sheetlike extensions at the leading

edge of fibroblasts• Filopodia- thin projections of the plasma membrane in

migrating cells

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Actin Bundles: Contractile2. Contractile bundles: more

widely-spaced filaments (40 nm), cross-linked by α-actinin

• α-actinin: a 102 kD protein with single ABD and an α-helical spacer

• Interacts with actin as a dimer

• Increased spacing allows actin interaction with motor protein myosin II

• Important in muscle fiber contraction

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Higher order Actin assembly: Actin NetworkHigher order Actin assembly: Actin Network• Filamin (280 kD) form flexible

cross-links • Filamin dimer: flexible V-shaped

molecule– actin-binding domains at the end

of each arm– dimerization domain– Β-sheet spacer

• Binds actin orthogonally, form 3-D network beneath the plasma membrane

• network (cell cortex) determines cell shape, and cell movement

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Actin Network and cell cortexActin Network and cell cortex• Red blood cells as mode• lack other cytoskeletal components, so the cortical cytoskeleton is the

principal determinant of cell shape• Spectrin, major actin-binding cortex protein• tetramer of two polypeptide chains, α and β• ends of the spectrin tetramers bind actin filaments, resulting in the

spectrin-actin network

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Actin Network and Cell CortexActin Network and Cell Cortex• Ankyrin links the spectrin-actin network and the plasma

membrane• Protein 4.1 is another link that binds spectrin-actin

junctions and the transmembrane protein glycophorin

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Association of Actin filaments with Motor proteinsAssociation of Actin filaments with Motor proteins

• Brings higher level of functional complexity to cells– Cellular or organismal movement– Intracellular cargo transportation, cell division

Association with motor protein myosin• Myosin is a molecular motor: converts chemical

energy (ATP) to mechanical energy force and movement.

• Muscle contraction: model for understanding actin-myosin interactions and the motor activity of myosin molecules

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Actin-Myosin and Muscle Contraction Actin-Myosin and Muscle Contraction • muscle fibers, large cells

(50 µm in diameter and up to several centimeters in length)

• Cytoplasm consists of myofibrilsmyosin filaments and thin actin filaments

• sarcomeres, myofibril units of skeletal and cardiac muscle

• actin filaments attached at their barbed ends to the Z disc

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Sacromere: a structural and contractile unitSacromere: a structural and contractile unit• Titin is extremely large protein; extend from the M line

to the Z disc – keep myosin II filaments centered in the sarcomere – maintain the resting tension that allows a muscle to

snap back if overextended• Nebulin, associated with actin, regulate assembly of

actin filaments

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Sliding Filament Model• was proposed in 1954• Myosin slides on actin filament• Sarcomere shortens, bringing the Z discs closer• There is no change in the width of the A band, but the I

bands and H zone almost disappear

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Sliding Filament Model• Tropomyosin binds along

actin filaments, also bound to troponin

• No Ca2+, tropomyosin-troponin block binding of myosin to actin

• nerve impulses, stimulate release of Ca2+ from the sarcoplasmic reticulum

• Ca2+ binds troponin C, shifts the complex

• Allows myosin binding to actin

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Sliding Filament Model• Myosin II (the type in muscle), large protein with two heavy chains

and two pairs of light chains• heavy chains have a globular head region and a long α-helical tail • Tails twist around in a coiled-coil• globular heads bind actin• myosin moves the head

groups along the actin filament in the direction of the barbed end

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Sliding Filament Model• ATP hydrolysis is

required• Binding of ATP

dissociates myosin from actin

• ATP hydrolysis induces a conformational change that displaces the myosin head group

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Sliding Filament Model• myosin head binds to a

new position on the actin filament and Pi is released

• The “power stroke”: Myosin head returns to its original conformation, which drives actin filament sliding, and ADP is released

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Muscle contraction and the role of Actin thin filaments

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Actin and myosin in cell divison

• Cytokinesis—division of a cell following mitosis

• A contractile ring of actin and myosin II is assembled underneath the plasma membrane

• Contraction of the ring pinches the cell in two

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Actin and myosin: vesicular transport• Myosin I: much smaller

than myosin II,contains a globular head group, acts as a molecular motor

• Short tails bind to other structures

• Movement of myosin I along actin filament

• transport cargo, such as a vesicle

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