Lectures 8-10 Notes

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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CHAPTER 16 THE CYTOSKELETON •

Cells need to move, be structurally robust, and adopt certain shapes to conduct their function. o Think of ƒ muscle fiber contraction in muscle cells ƒ a nerve cell that extends from your foot to your head ƒ a sperm cell trying to find its way ƒ a macrophage crawling throughout your body ƒ skin cells that protect your body

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Large “work projects” like moving chromosomes, vesicles, and protein complexes requires large structural machines. The cytoskeleton provides this.

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The Self-Assembly And Dynamic Structure Of Cytoskeletal Filaments •

There are three major types of cytoskeleton filaments o Actin filaments – important for cell shape and motility, dynamic o Microtubules (made up of tubulin) – Tracks for intracellular transport, dynamic o Intermediate filaments – provides mechanical strength to the cell. very stable

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Motor proteins move vesicles and other large complexes along actin filaments and microtubules. Microtubules and actin filaments are dynamic – constantly assembling and disassembling. Fig. 16-2 Proteins must regulate these dynamics.

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Each Type of Cytoskeletal Filament Is Constructed from Smaller Protein Subunits and Multiple Protofilaments Filament Actin filament Microtubules Intermediate filaments

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Subunit actin tubulin lamins vimentin keratins many others

Diameter The thinnest, 5-9 nm The thickest, 25 nm Intermediate, 10 nm

Protofilaments 2 13 24

Each subunit is a protein. Thousands (millions?) of subunits line up head-to-tail via noncovalent interactions.

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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Remember from BMB/Micrb 251 what these noncovalent interactions are?

Also side-to-side interactions make for a very stable structure. Fig. 16-3

Filaments Formed from Multiple Protofilaments Have Advantageous Properties Fig 16-3 • Single protofilaments are thermodynamically unstable • Multiple aligned protofilaments produce a stable structure because each monomeric subunit is stabilized by multiple bonds

Nucleation Is the Rate-limiting Step in the Formation of a Cytoskeletal Polymer Fig 16-5 The Tubulin and Actin Subunits Assemble Head-to-Tail, Creating Filaments that Are Polar Fig. 16-6 •

The repeating unit in microtubules is a heterodimer of α-tubulin and βtubulin. o o

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The two are structurally related. Arrangement is head-to-tail

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Both subunits bind GTP, but only one can hydrolyze its GTP to GDP GTP hydrolysis regulates microtubule stability. Microtubules are cylinders (hollow tubes), with the walls consisting of 13 protofilaments Structurally they are very stiff

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The repeating unit in actin filaments is a monomer of actin. Fig. 16-7 Each actin monomer binds ATP (not GTP). ATP hydrolysis controls the dynamics. Actin filaments are not hollow, but have a helical twist. Two protofilamemts are twisted around each other Compared to microtubules, actin filaments are quite flexible. Why?

Filament Treadmilling and Dynamic Instability Are Consequences of Nucleotide Hydrolysis by Tubulin and Actin • • • • • •

A growing microtubule involves addition of GTP-bound tubulin to one end. Within the microtubule, GTP is hydrolyzed to GDP. GDP-tubulin dissociates from the microtubule end more rapidly. o However, once GDP-tubulin is internal to the filament, it no longer dissociates. Net polymerization/depolymerization is a race between GTP hydrolysis at the end and addition of new GTP-tubulin subunits. This is called dynamic instability. Fig. 16-11b o Allows filaments to grow and contract. What factors contribute to net polymerization? Net depolymerization?

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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GTP bound tubulin at + end stabilizes microtubule +ends, GTP hydrolyses destabilizes ends

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Same with actin but with ATP. Also rather than assembling and disassembling from one end, actin assembles from one end and disassembles at the opposite end of the filament. ADP-actin dissociates faster than ATP-actin Filaments are directional Monomer on and off rates at the + and -ends of filaments are not the same Fig. 16-14 At the point were momomeric and polymeric actin (or tubulin) are in equilibrium, monomers get added to the + end while at the same time they are removed from the - end Called treadmilling. Fig 16-10, Treadmilling is observed with both microt and actin filaments Allows them to move, which allows cells to change shape and move So what is the purpose of ATP and GTP hydrolysis with respect to filament dynamics?

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Treadmilling and dynamic instability use energy but serve an essential purpose! Other polymeric proteins also use nucleotide hydrolysis to couple a conformational change to cell movements (i.e. dynamin, a protein involved in endocytosis of vesicles) Tubulin and actin have been highly conserved during eukaryotic evolution

Intermediate Filaments Impart Mechanical Stability to Animal Cells Fig 16-18 • Keratins o Finger nails, hair, scales, and skin are made up of keratins. o There are many different kinds of keratin intermediate filaments. • Nuclear lamina o Lamins A, B, and C o Make up the inter lining and structure of the nucleus = nuclear lamina. • Neurofilaments o NF-H, NF-M, NF-L (NF-L forms heterodimers with L or H) o Give structural stability to neuronal axons. • GFAP: astrocytes • Desmin: Muscle cells • -> Many are cell type specifically expressed

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

Intermediate Filament Structure Depends on The Lateral Bundling and Twisting of Coiled Coils Fig. 16-16 • • • • • • •

Intermediate filaments impart structural strength to cells. Things that tend to undergo mechanical stress tend to be anchored to the intermediate filaments. Unlike actin and tubulin, intermediate filaments have monomeric subunits that are elongated and alpha-helical. Monomers dimerize via coiled coils. Dimers come together to form tetramers, and so on to form long ‘cables’. There is no head-to-tail arrangement, no dynamic instability, no treadmilling, no nucleotide binding. Assembly/disassembly is regulated by protein phosphorylation.

Filament Polymerization Is Sensitive To Plant And Fungal Toxins That Alter The Assembly/Disassembly Of The Cytoskeleton • • • • •

Fungal phalloidins stabilize actin filaments o fluorescent derivatives for labeling of microfilaments Fig. 16-50 Taxol from the yew tree stabilizes microtubules. o Used as a mitotic inhibitor to treat cancer. Colchicine from a crocus cause tubulin depolymerization o -> cell cycle arrest of cells in metaphase of mitosis, What is this useful for? Latrunculin (from a sea sponge) stabilizes actin monomers, thereby inducing actin depolymerization o Used to study the function of actin dynamics in research Acrylamide disassembles neurofilaments. o Neurotoxic! Watch out – it is used in the lab to make polyacrylamide gels

Summary • Microtubules are made up of globular tubulin subunits and form long structural cylinders. • GTP hydrolysis controls rates of net assembly and disassembly. • Microtubules function in intracellular transport. • Actin filaments are made up of globular actin subunits. • ATP hydrolysis controls the dynamics of assembly and disassembly. • Actin filaments provide cell shape and is the treadmill for cell movement. • Neurofilaments provide mechanical strength to the cell. • Subunit structures of neurofilaments are unlike actin and tubulin. • Drugs can affect cytoskeleton assembly/disassembly.

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MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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How Cells Regulate Their Cytoskeletal Filaments Microtubules Emanate from the Centrosome in Animal Cells Fig. 18-18, • Centrosomes and microtubule organizing center (MTOC) are essentially the same thing in animals • Plants lack centrosomes but have numerous locations corresponding to MTOCs Microtubules Are Nucleated by a Protein Complex Containing γTubulin • • •

Microtubule assembly is initiated at the MTOC Fig. 18-18 Fig. 16-22a A number of proteins are part of MTOC. One in particular is γ-tubulin. γ-tubulin is related to α- and β-tubulin, but serves only to anchor one end of the growing microtubule filament.

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γ-tubulin is part of a ring complex at the MT (-)end containing accessory proteins and involved in MT nucleation embedded in the centrosome is a pair of centrioles. Fig 16-24 (16-31) The centrioles are orientated at right angles to each other. Centrioles organize the centrosome matrix. During cell division, they duplicate, move to opposite sides of the cell, and help pull apart duplicated chromosomes at mitosis. What are centrioles made up of? o Modified microtubules and other proteins.

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Actin Filament Nucleation occurs at the Plasma Membrane and depends on ARPs • • • • •

Actin filament nucleation occurs mainly at the plasma membrane. So the highest density of actin filaments is just under the cell surface where it determines cell shape, plasticity, and movement. Nucleation of actin is regulated by external signals in response to changing environments. Nucleation is catalyzed by ARPs (actin-related proteins). Fig 16-28ab o Analogous to the γ-tubulin ring complex ARP-dependent nucleation of microfilaments is facilitated by ARPs binding to actin filaments -> tree-like web of actin filaments Fig 16-28c

Actin Filament Elongation Is Regulated by Proteins That Bind to Actin Monomers Fig.16-30 •

About half of the actin in a cell is in filaments. The rest is free monomer.

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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Proteins like thymosin are inhibitory to monomer incorporation

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Profilin competes with thymosin binding, and enhances monomer incorporation into the “+” end of filaments It is activated by phosphorylation and binding to phospholipids This specifically promotes microfilament assembly at the membrane Directional polymerization results in cell movement. Figs. 16-90, -86, -92 Extracellular signaling molecules localized to specific cell surfaces (cell contacts) can lead to local profilin activation -> burst of actin filament assembly -> filopodia/lamellipodia extendion -> cell movement Similar processes (different proteins and different cellular location) happen with tubulin.

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Proteins That Bind Along the Sides of Filaments Can Either Stabilize or Destabilize Them • • • • • •

These proteins tend to bind throughout the filaments. Tropomyosin holds together actin bundles. Cofilin depolymerizes actin. Cofilin preferentially interacts with the ADP form of actin, which tends to be near the minus end, in the treadmilling process. ->Cofilin preferentially depolymerizes the older microfilaments or microfilament sections Proteins that bind MTs are called MAPs (microtubule-assoc. proteins). o

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Differential distribution of different MAPs o

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The MAP Tau is limited to axons (and soma), MAP2 is limited to soma and dendrites of neurons

Some MAPs stabilize MTs, including the formation of large MT bundles. Fig. 16-33 o

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Do not confuse with MAP kinase (mitogen activated protein kinase)

Exampe: Tau, hyperphosphorylated Tau leads to insoluble microfibrillary tangles in Alzheimer’s disease and other Tauopathies And how do microf tangles differ from β-amyloid plaques?

Other MAPs link microtubules with other cellular components.

Proteins That Interact with Filament Ends Can Dramatically Change Filament Dynamics •

Capping proteins ‘cap’ the plus end of actin filaments. o

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Subject to regulation by signaling molecules via PIP2

ARPs cap the minus end. Microtubules have their own set of capping proteins as well.

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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Cross-linking Proteins with Distinct Properties Organize Different Actin Assemblies • •

Actin filaments can be arranged in bundles or in a web-like network. Specific proteins are designed to crosslink actin filaments in different ways. o α-actinin makes loose bundles with room for myosin -> contractile bundles. Fig. 16-40 o fimbrin makes tight parallel bundles that prevent myosin Fig.1640 o Villin and fimbrin crosslink actin filaments in microvilli Fig. 16-41 o Filamin also makes networks. Fig. 16-42 ƒ

Important for making flat lamellipodia for crawling along surfaces.

Actin Filament Severing Proteins such as Gelsolin Regulate the Length and Kinetic Behavior of Actin Filaments Fig. 16-47 • Severing actin filaments with gelsolin Î greater number of actin filament ends Î increased rate of actin dynamics

Cytoskeletal Elements Can Attach to the Plasma Membrane •

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ERM (=Ezrin and related moesin and radixin) proteins attach actin filaments to the plasma membrane. Fig. 16-48 Actually, the ERM proteins attach to transmembrane glycoproteins such as CD44. Unlike the ‘permanent’ attachment that occurs in red blood cells and muscle cells, the ERM attachment is dynamic and regulated. Phosphorylation and PIP2 regulate this in response to external signals.

Special Bundles of Cytoskeletal Filaments Form Strong Attachments Across the Plasma Membrane: Focal Contacts, Adhesion Belts, and Desmosomes • • • • •

When cells are slithering across a surface they need to grab on to things. Cells do this through focal contacts. Integrins are transmembrane proteins that bind to the extracellular matrix. Vincullin binds to actin filaments at focal contacts (Fig 19-45 new book) Adhesion belts: cell-cell junctions at epithelial cell layers o Cadherins are Intracellularly attached to catenins, which attach to actin filaments. o Desmosomes are different cadherins family members that are attached intermediate filaments (i.e. keratins). (Fig 9-17 new book)

Different Extracellular Cues Signaling through Different Monomeric

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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G-Proteins Induce Formation Of Different Actin Filament Structures Fig. 16-50 • Rho -> stress fibers • Rac -> lamellopodia • Cdc42 -> filopodia Summary • Microtubules nucleate at centrosomes • Actin nucleates at the plasma membrane • Assembly and disassembly is regulated by binding of nucleotide triphosphates • Microtubule and actin filament can be lashed into strong bundles by crosslinking proteins. • Microtubule and actin filament assembly and disassembly is dynamic, directional and controlled by capping and other proteins • Extracellular signals can control actin filament assembly/disassembly by signaling through different monomeric G proteins • Actin filaments are anchored to the cell membrane by integral membrane proteins that serve to attach cells to the extracellular matrix and other cells.

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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MOLECULAR MOTORS Generic structure • Globular head domain attaches to a particular type of cytoskeleton filament • The tail attaches to a particular cargo o Mitochondria o Vesicle o chromosome o Other filament (muscle contraction) • ATP hydrolysis moves the motor protein relative to the filament.

Actin-based Motor Proteins Are Members of the Myosin Superfamily •

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Myosin is a protein responsible for force generation during muscle contraction. o 2 heavy chains + 2 x 2 light chains o myosin II heavy chain o extended alpha helical tail region that dimerizes o globular head with ATP activity containing domain o Bound to each heavy chain are 2 copies of myosin light chain o Myosin II bipolar thick filament (frog muscle) o Heavy chain tails form bundles that are symmetric. Fig. 16-52 o Bundles get together to form myosin filament. There are many different kinds of myosins each having different functions, but all have similar structural arrangement, particularly in the head region. Fig 16-54 o Myosin moves along the ‘+’ end of the actin filament upon ATP hydrolysis, one step at a time.

Microtubule-directed Motor Proteins: Kinesins and Dyneins • • • • • •

Kinesins = superfamily of >10 families of kinesin-related proteins Classical kinesins move cargo from the (-) towards the (+) end of microtubules Some unusual kinesins have the ATPase domain at the C rather than N terminus and move cargo in the opposite direction Some have lost motor activity all together and act to destabilize MTs -> called catastrophins structurally and evolutionarily related to myosin. Fig. 16-55, Fig. 16-57

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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Dyneins are motor proteins that move toward the ‘-‘ end of microtubules. They are unrelated to kinesin and are very fast. Axonemal dyneins are used to move cilia and flagella Which type of motor protein do you think moves vesicles from the soma to the distal end of neural axons? What about in the other direction?

Motor Proteins Generate Force by Coupling ATP Hydrolysis to Conformational Changes •

The myosin cycle is like paddling a canoe Fig. 16-58

step ATP binding site 1 empty 2 3

ATP ADP + Pi

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ADP empty

What happens to the myosin head Bound to actin filament, in a state of rigor (cause of rigor mortis in death) Detaches from actin filament (at position ‘n’) ‘Cocks’ forward (conformational change induced by ATP hydrolysis) Re-attaches to actin filament at n+1 position, causes release of Pi Loss of bound ADP, Myosin head shifts back to original conformation (power stroke)

-> Be able to identify what happens at each of these steps: • ATP binding • ATP hydrolysis • Release of Pi (inorganic phosphate) • Release of ADP Unbinding of which molecule eventually produces the energy needed for the power strke? •

The kinesin cycle is like walking with at least one foot always on the ground Fig. 16-59a step Head Head B What happens to the kinesin heads A Release of (A) from tubulin = ‘free’ 1 ADP empty 2

ADP

ATP

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ADP empty

ATP ADP

(B) bound to tubulin Binding of ATP to head (B) throws ‘free’ rear head (A) forward past ‘attached’ leading head (B) Both heads bound to tubulin Simultaneous release of ADP from foot (A) and hydrolysis of ATP on foot (B) brings dimer back to original stance with the positions of the two heads switched

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

-> Be able to identify what happens at each of these steps.

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MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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Motor Proteins Mediate the Intracellular Transport of Membraneenclosed Organelles • • •

Microtubules emanate from the cell center (minus end) to the periphery (+ end). Kinesin moves organelles toward the plus end (periphery). o Endoplasmic reticulum (ER) splays out toward the periphery due to Kinesin Dynein moves things toward the (-) end (i.e. cell center). Fig. 16-63 o Axonal MTs have their (+) end directed towards the axon tip -> dyneins mediate retrograde transport of vesicles in axons, kinesins are responsible for anterograde axonal transport o Attachment of dyneins to cargo involves accessory proteins such as the actin nucleation protein ARP, as well as the submembrane cytoskeleton factors ankyrin and spectrin (Fig 1031)

Motor Protein Function Is Regulated

• Rapid change in myosinV-dependent localization of pigment granules in melanocytes is responsible for rapid change in coloration in several species of fish (Fig 16-65) • Myosin II activity is regulated by myosin phosphorylation: o Phosphorylation by the myosin light chain kinase MLCK switches myosin V from inactive to active state (Fig 16-67)

Muscle Contraction Depends on the Sliding of Myosin II and Actin Filaments • Muscle fibre consists of many myofibrils • Myofibrils = cylindrical structures of 1-2 uM diameter that span entire length of a syncytial muscle cell • Consist of long repeated chains of contractile elements called sarcomeres = contractile units of a muscle fiber. Fig 16-68 • Z disk – actin – M line – actin – Z disk • -> Striated appearance Fig. 16-69, 16-72 • composed of actin/myosin filaments and diverse accessory proteins Fig 16-72 –Titin (25,000 amino acids!) connects the two Z discs. Largest know protein; acts as a highly elastic bungee cord, adjusting to the length of the sarcomere during contraction –Nebulin (also huge!) = molecular ruler for actin filament. Consists almost entirely of 35 aa actin binding domains that determine the length of the actin filament –Tropomodulin = actin filament capping protein

MICRB/BMB 252 lecture 8-10 notes (prepared by B. Luscher)

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Actin filaments encircle myosin filament Fig. 16-70 Muscle fibrils are contractile. During muscle contraction the myosin filaments slide past the actin filaments Fig 16-70

Muscle Contraction Is Initiated by a Sudden Rise in Cytosolic Ca2+ Concentration • • • • • •

What happens when you decide to contract a muscle? Depolarization mediated by V-gated Na+ channels spreads from motor neuron endplate to T- tubules Fig. 16-73a T-tubules wrap around the myofibrils. Depolarization in T-tubule membrane is relayed from V-gated ion channels in tubule membrane to a Ca2+ sensitive Ca2+ channel in membrane of underlying sarcoplasmic reticulum ->Ca2+ rushed out of SR into the cytosol and initiates the contraction of the myofibrils Fig. 16-73C 16-77 in new book What stops the signals? Increase in Ca2+ is transient because it is rapidly pumped back into SR by a ATP-dependent Ca2+ pump known as Ca2+ ATPase

Muscle Contraction Is Initiated By Sudden Rise In Cytosolic Ca2+ • • •

At rest, tropomyosin blocks myosin binding site on actin filament Upon initiation of contraction Ca2+ binds to the troponin complex causing it to dissociate from tropomyosin. Fig. 16-74a,b Once tropomyosin is out of the way the myosin binding site on actin is accessible to myosin -> contraction begins

Summary Nerve impulse Æ motor neuron endplate, depolarization of target muscle endplate membrane Æ spread of depolarization to T-tubules Æ Ca2+-gated ion channels Æ Ca2+ release from SR Æ Ca2+ –| (Troponin Æ Tropomyosin –| myosin/actin ATP hydrolysis). •