Rao Cytoskeleton 1 and 2

Here again, we labeled lots of pictures. Double check with someone present to complete your notes.
Images and videos are not on test. Want to project key cell functions driven by cytoskeleton.
Filopodia are fingerlike projections. Sheetlike projections are lamellipodia. Focal adhesions are points of attachment to substrate.
During migration, cell shape changes.
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Stages of mitosis
Mitosis has a very coordinated mechanism. Any small mistake is lethal to cell.
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Important functions of cells related to cytoskeleton are listed. These are energy dependent mechanisms.
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3 types cytoskeleton. All polymeric proteins.
1. monomer is actin.
2. microtubule is bigger.
Cilia are bundles of microtubules.
Intermediate filaments appear to be entirely structural.
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Learning objectives.
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Actin: Difference between the amino acid sequences of alpha and beta is only 3-4 amino acids, but they have different functions. Monomeric is globular or G actin. Polymeric is F or filamentous actin.
Structure- G actin binds ATP. Without the ATP, the G actin gets denatured. ATP is necessary to maintain conformation. ATPase domain is only active when polymerized. Take G actin and add salt in vitro, get F actin. Polymerization and depolarization are important.
ATP hydrolysis is not necessary for polymerization. ADP G actin will still polymerize.
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If you fix filaments to a slide, and look in EM, with negative stain (stain is beside strand, but filament itself is not stained), you will see what is on this slide on the left.
F actin- one end is different from other (polarity) Added subunits are always added in same orientation. Myosin is an actin motor protein. Head group binds to actin. Used in myosin decoration experiment to see + and – end. See series of arrowhead-shaped structures.
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Bundle- filaments in cable or parallel arrangement.
Network- less organized-criss-cross arrangement.
See 2-D network in lamellipodium. If you take activated platelet and dissolve cell except for cytoskeleton, and take SEM, see what is on right. Organization of actin filaments provides shape of cell.
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Crosslinking proteins stabilize networks. Short proteins stabilize bundles.
Long proteins play role in crosslinking networks to stabilize them.
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Cortical network binds to plasma membrane proteins. Spectrins and junctionial complexes are organized in hub and spoke organization. Hub has small strand F actin bound to tropomyosin (stabilizes).Tropomodulin caps to prevent destabilization. Many spectrin can bind to one actin- many spokes.
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Unstable actins constantly undergo polymerization and depolymerization. Used for cell shape and movement. You can measure polymerization using mass of filaments. See lag phase, elongation, steady state. Nuclei are polymers of 3-4 subunits. They are very unstable. Add few more- takes off and becomes stable. Understand nucleation, elongation, steady state. If you add nuclei, or small fragments of actin filament, you see takeoff to elongation phase. Polymerization takes place at a critical concentration of G actin. No F actin forms until you reach the critical concentration . Then at that concentration, keep elongating. Elongation cccurs at both ends. At steady state, you are at critical concentration and units coming off and going on are in equilibrium. Below critical concentration, depolymerization occurs.
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Polymerization is different at + and – end. Polymerization is greater at + end. There is structural and functional polarity.
Why polarize at + end? Critical concentration is different at – and + end. If you cap one end, and study the critical concentration at other, it is different. Higher for minus end- when concentration G actin goes down, see polymerization only at + end.
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Measured association and dissociatiion constant for ATP-G actin and ADP-G actin. ATP is hydrolysed in polymerization. Once inorganic phosphate is off, the character of filament changes. Look up aricle for homework.Inorganic phosphate remains for 6 min. for some other purpose
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Toxins can modify dynamics.
Cytochalasin D causes depolymerization at negative end.
Phalloidin stabllizes F actin so it cannot depolarize. Lethal to cell.
Jasplakolinode also stabilizes F actin. Phalloidin used to determine whether depolarization is needed for cell function. This molecule is small and easy to label with fuorescent probe so it binds to filaments and you can see organization.
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Different in vivo and vitro:
In vivo, salt is high and concentration G actin is 5000 times critical. Expect it all in F actin, but not the case. About 40% is polymerized. Actin binding proteins control polymerization and depolymerization.
Thymosin binds cleft region to prevent binding of G actin at + end.
Profilin promotes polymerization.Binds at hinge region. Regulated by PIP2 hydrolysis.Has PH domain. When cell stimulated, profilin is released by PLCgamma, can bind to induce polymerization. Continued tomorrow.
Rao Cytoskeleton II

End of lecture 1
Depolymerization responds after inorganic phosphate is released. Generates ADP or ATP F actin- ADP more unstable. Important aspect of treadmills. Review simplifies many things. If start with nuceus of F actin, start with ADP-F actin. In reality in cell undergoing polymerization, there is possibility that piece at positive end is ATP-F actin. Kinetics would be totally different. No exam question, just thought question.
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Arp 2/3 demonstration- can visualize polymerization under fluorescence microscope if construct the fusion so that the proteins fluoresce upon polymerization.
Add ARP 2,3- get branching instead of just elongation under high salt.
Formins- bind to G actin and enhance nucleus formation. Stabilizes nucleation for bundles. FH is formin homology domain.
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Listeria cell uses actin network to stroll around cell.

Lecture II
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Pulling cell body forward requires additional force –more than explained by polymerization.
Myosins generate additional force along with actin filaments. Act like motor- bind and walk along actin filament using head chain with ATP hydrolysis for fuel. Can carry vesicles and organelles along.
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Learning objectives
Do not worry about cytoplasmic streaming. Know the rest.
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myosin structure. Myosin I is ubiquitous.
Myosin has intrinsic ATPase activity. Hydrolysis leads to conformational change leads to walking along actin filaments. Actin is track. Myosin is motor. Myosins have 3 parts:
Head or globular region has ATPase and actin binding site. ATPase is activated upon binding 6-8 fold.
Neck domain-regulatory subinuts and translocation
Tail domain-long not conserved. Determines specificity of function. Can bind membrane components and transport vesicles. In myosin II-muscle contraction by binding each other. Know function and how light chains affect function heavy chain.

2 heavy chains. Can regulate by Ca or phosphorylaiton
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Details of binding of myosin II to each other.
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Demo of walking along filament. Microfilament sliding assay. Bind tail of myosin to slide with head free. Use fluorescent F actin fragments. Visualize –you can see filament slide. Myosin only walks minus to plus. Speed depends on length of neck region.
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Mechanism of walking along filament
This is model proposed at moment. Conformational change is important. 3 changes per step.
1. ATP binds, 1st conformational change lifts head group from actin.
2. Hydrolysis- head pivots to bind 2 subunits later.
3. Release pi, induces conformational change and neck straightens .
4. Process repeats for walking along filaments.

Properties of tail domain determine its role in cell.
Neck conformational change is important. In reality, in the cell actin is stable point and the myosin moves, unlike in vitro system used to explain the process.
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review sarcomere function.
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How does Ca binding regulate muscle contraction?
Topomyosin binds to actin. Troponin c subunit regulates where tropomyosin is placed.
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smooth muscle contraction- primitive. Regulated by signal transduction. Light chains in neck region regulated by phosphorylation- myosin light chain kinase- increase Ca., activate light chain kinase.MLCK phosphorylates the light chain. Off- muscle is relaxed. Active- induce contraction. Dephosphorylate-bring back to relaxed state.
Links signal transduction to muscle contraction through actin-myosin dynamics
CaM is calmodulin
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epithelial cells with tight junctions. Has perijunctional actomyosin ring. Tight junction forms seal in extracellular region between cells. Regulated by actin dynamics. Disrupt tight junction by disrupting cytoskeleton. Proteins forming junction bind too cytoskeleton. Activate MLCK, regulate tight junction. Cortical actin network supports plasma membrane. Myosin also present and stiffens plasma membrane to provide tension for extra support. Plasma membrane stiff- see illusration on back.
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during cytokinesis- presence of myosin helps in formation of cleavage furrow. Presence myosin- formation new cells.
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cell migration 4 phases
1. Cell extends lamellipodium at leading edge. Involves polymerization of actin.
2. Adhesion- do not want to retract back.
3. Translocation cell body
4. Deadhesion at back of cell.
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lamellipodium extension
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Bacteria Hijack host cell actin cytoskeleton. One end of bacterium induces polymerization, see actin behind. Movement is compressive. Actin polymerizes at one end- force at back and side to squirt it forward. These bacteria can spread faster than others because of their ability to use the host cytoskeleton for travel..
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Bacterial toxins can change the actomyosin ring.