CSF Quiz 2

The Movement Of Kinesin

"Head as foot walking" -8 nm/step (up to 1μm total) -1 ATP/step -Walking motion: One head/"foot" detaches and binds a new B- tubulin; Trailing head/"foot" detaches, walks over the other head/"foot" and binds another new B- tubulin; Microtubule stays stationary -tail - carries cargo -WALKS HAND OVER HAND, NOT INCHWORM

Microfilament Assembly In Vitro

*Many similarities to MT assembly -Reversible assembly: critical concentration -Polarity of assembly; Plus/Minus ends and treadmilling -Basic units is ATP-bound, hydrolyzed once in filaments -Kinetics: Lag (nucleation) phase Rapid (elongation) phase Plateau phase: gain=lost

Intermediate Filaments: Contrast with MT and MF

-10-12 nm in diameter -Less organized appearance, but more stable and less soluble -No polarity: antiparallel tetramer subunits -Protein subunits fibrous -Protein sequences are tissue specific- Medical diagnostic use -No motor proteins, mainly structural

Microfilaments

-2 intertwined chains of Fi-actin - 7nm - G-action - +,- ends -Muscle contractions -Cell locomotion -Cytoplasmic streaming -Cytokinesis -Maintenance of animal cell shape -intracellular transport/trafficking

Intermediate Filaments

-8 Protofilaments joined end to end with staggered overlaps -8-12 nm -Proteins: Class V Nuclear Lamins -Structural support -Maintenance of animal cell shape -Formation of nuclear lamina and scaffoliding -Strengthening of nerve cell axons (neurofilament protein) -Keeping muscle fibers in register (desmin)

Microvilli

-Actin bundles in microvilli are the best-studied examples of ordered actin structures -Microvilli are prominent features of intestinal mucosal cells; they increase the surface area of the cells -The core of a microvillus consists of a tight bundle of microfilaments with the ends pointed toward the tip -The MFs are connected to the plasma membrane by crosslinks made of myosin I and calmodulin -The MFs in the bundle are tightly bound together by crosslinking proteins fimbrin and villin

Structure of a Sarcomere cont: THIN FILAMENT

-Actin filaments attached to each Z-disk by + end -Decorated by Tropomyosin and Troponin -capped at the (+)end by Cap Z and at the minus end by Tropomodulin (actin filament capping proteins) -long nebulin molecule lies along actin filament - length of nebulin = length of thin filament

Skeletal muscle regulation of Ca2+ concentration cont: TURNING OFF

-Ca2+ channels close -Ca2+ ATPases re-establish low Ca2+ concentration (<10-4 mM) in muscle cell cytosol -Ca2+ is lost from TN - TM moves to inhibitory 'off' position on thin filament

Other Actin (MF) -based cellular structures

-Cells that adhere tightly to the underlying substratum have organized bundles called stress fibers -Rapidly moving cells have the cell cortex, just beneath the plasma membrane, with actin crosslinked into a gel or loose lattice -Cells that crawl have lamellipodia and filopodia at their leading edge, allowing them to move along a surface

MTOC's in vivo

-Centrosome: two centrioles and associated pericentriolar materials -Centrioles: 9 sets of MT triplets; Oriented perpendicular to each other

Motility at the cellular level

-Cilia/flagella-based movement -Amoeboid movement -Tumor metastasis -Chemotaxis

Nuclear Lamins

-Class V -Lamin A: 70 kDa -Lamin B: 67 kDa -Lamin C: 60 kDa -Tissue: ALL CELL TYPES -Function: Forms a nuclear scaffold to give shape to nucleus

Inhibitor techniques to study cytoskeleton

-microtubule formation: Colchicine, Nocodazole, -Microtubule disassembly: Taxol -microfilament formation: Cytochalasin D

Interaction Between Motor Proteins And MT/MF Produce Motion

-Cytoplasmic dynein: motion towards MINUS END of microtubule -Kinesins: motion towards PLUS END of microtubule -Axonemal dynein: activation of sliding in flagellar microtubule

Function of cytoskeleton

-Cytoplasmic: C'some movements Structure and support Intracellular transport Contractibility and motility Spatial organization -Axonemal: cell motility

Treadmilling of Microtubules

-Different Cc at the two ends of the MT assembly (Lower Cc at the plus end) -The free tubulin concentration can thus be higher than Cc at the plus end but lower than Cc at the minus end -When that happens, polymerization will take place at the plus end while depolymerization takes place at the minus end -Tubulin dimers added to the plus end are lost after getting to the minus end "Blue" tubulin dimers go in at plus side, made their way down to minus end as more dimers are added, eventually are lost after getting to minus end.

Proteins That Regulate Polymerization

-If the concentration of ATP-bound G-actin is high, microfilaments will assemble until the G-actin is limiting -In the cell, a large amount of free G-actin is not available because it is bound by thymosin 4. -Whether MFs can grow also depends on whether their filament ends are capped; Capping proteins bind the ends of a filament to prevent further loss or addition of subunits; CapZ binds to plus ends to prevent addition of subunits there

Microscopy techniques to study cytoskeleton

-Immunofluorescence microscopy -Live cell imaging (Fluorescent monomers & Time-lapse videos) -EM

Microtubule-Stabilizing/Bundling Proteins

-MAPs, microtubule-associated proteins, bind at regular intervals along a microtubule wall, allowing for interaction with other cellular structures and filaments -A MAP called Tau causes MTs to form tight bundles in axons; Tauopathies: Alzheimer's, Palsy -MAP2 promotes the formation of looser bundles in dendrites

MTOC: Microtubule Organizing Centers

-MT's originate from here -serves as the nucleation and anchoring site for MTs in the cells -basal bodies and centrosomes -Basal body located from the base of cilia serves as a MTOC -The MT will "grow out" from the basal body because tubulin dimers are added to the basal body EM examination reveals that MTs grow much faster from one end (plus end) than the other (minus end)

Structural components of cytoskeleton

-Microtubules (MT) -microfilaments (actin filaments) -intermediate filaments (IF)

Movement At The Cellular Level: Cilia And Flagella

-Motile appendages of eukaryotic cells: specialized projections of cell cytoplasm - inside Plasma Membrane; basal body (MTOC) at base of structure; -Common structure base but different size and number; Cilia: short and numerous; Flagella: general long and limited to one or few per cell

Structure of a Sarcomere cont.: THICK filaments

-Myosin bipolar filaments in center of sarcomere bundled together by Myomesin -In skeletal and cardiac muscle cells - ~300 myosins/bipolar filament (thick filament)

Demonstration of microfilament polarity

-Myosin subfragment 1 (S1) can be incubated with microfilaments (MFs) -S1 fragments bind and decorate the actin MFs in a distinctive arrowhead pattern -The plus end of an MF is called the barbed end and the minus end is called the pointed end, because of this pattern

Cell Motility at a subcellular level

-Separation of chromosomes during cell division -Intracellular movement of vesicles and organelles

The Sliding-filament Model For Muscle Contraction

-Thick filaments (A band) and thin filaments remain constant length but slide with respect to each other, increasing overlap. -I band in each half sarcomere shortens -Z-lines are pulled together all along the length of myofibril -sliding driven by "walking" of myosin head toward the plus end of thin filament; The amount of force/tension the muscle can generate during contraction depends on the number of myosin heads than can make contact with the thin filament

Cycle Of Cross-bridge Formation And Muscle Contraction

-Transient cross-bridges formed by interaction between the myosin head and F-actin in the thin filament holds the thick and thin filaments together loosely 1) Pi release cause pivot of head on neck and tighter binding 2) release of ADP is accompanied by a powerstroke that pulls the thin filament towards the center of the Sarcomere 3) ATP binding dissociates head from actin filament 4) Hydrolysis of ATP into ADP+Pi cocks myosin head toward actin (+)end (spring-loading) -head rebinds to actin filament upstream (towards the + end) of original position -Energy: 1 ATP/powerstroke

The GTP Cap and Its Role in the Dynamic Instability of Microtubules

-When tubulin concentration is high, a GTP cap is formed at the tip of the MT; The speed of adding tubulin-GTP onto the MT exceeds that of the GTP hydrolysis in the wall of the MT -A MT tip with a GTP cap is stable and allows more tubulin dimers to be added. -When tubulin concentration is low, no GTP cap is formed at the tip of the MT; The speed of GTP hydrolysis in the wall of the MT exceeds that of the addition of tubulin-GTP onto the MT. -A MT tip without a GTP cap is unstable and allows rapid depolymerization

Dynein "Walking" On MT Generates Sliding Force

-dynein arms (on A tubules) walks toward (-)end of adjacent B tubule - pushes adjacent tubule toward (+)end direction. This process is ATP dependent. -dynein is '(-)end-directed motor' -this "sliding" is normally restricted by nexin links: Axoneme treated with protease that breaks nexin links Addition of ATP - dynein arms generate force on adjacent doublet - telescopes axoneme to 9x original length

Cytoplasmic Dynein/Dynactin Complex

-heavy chains, intermediate chains and light chains -Heavy chains bind to MT and engage in "walking" toward the minus end -Light chains interact with a Dynactin complex, which in turn, binds to membrane of the cargo

Tubulin heterodimers (Tubulin subunit - alpha/beta dimer)

-highly conserved protein across all eukaryotic species-house-keeping proteins -the alpha -tubulin - GTP bound permanently the beta -subunit - has GTPase activity and hydrolyzes GTP after the dimer is incorporated into the MT

Microtubule assembly in vitro: initiation

-tubulin dimers associate end-to-end to form short protofilaments 1. Aggregation of dimers to form oligomers, known as nucleation 2. Joining of oligomers to form protofilaments 3. protofilaments associate side-by-side to form sheets 4. sheet closes to form tubular structure Tubulin dimers->ogliomers->protofilament->sheets of protofilament->closing microtubule

The Structure Of Kinesin 1

-two globular heads (blue feet, ATP binding and one coiled-helical tail -heads: MT binding domain ATPase site generate force on MTs through ATP hydrolysis -Light chain tail - binds cargo

Microtubule assembly in vitro: MT elongation

5. tubulin dimers (both GTP bound) associate and dissociate at each end tubulin dimer in wall of MT (b-subunit) hydrolyzes GTP to GDP+Pi

Microtubule-Based Motility

ATP dependent Motor proteins walk on MT track Motor proteins carry cargos (vesicles)

Regulation of Contraction in Striated Muscle

Actin-myosin alone -unregulated -myosin head crossbridges free to interact continuously with actin filaments -Muscle would contract continuously Tropomyosin -lies along both sides of actin filament -2 positions : "normal" state - blocks myosin head interaction with actin filament; "sliding-off state" - allows myosin head interaction with actin filament Troponin - 3 protein complex -TN-T: binds complex to Tropomyosin -TN-I: required to position TM in 'normal' state -TN-C: related to calmodulin binds Ca2+ with KD ~ 10-3 mM Ca2+ bound to TN - C -forces Tropomyosin into "sliding-off" state position on thin filament (removes inhibition) -allows force production

The Structure of Cilium/Flagellum: an Axoneme connected to a Basal body by a Transition zone

Anoneme: "9+2" Nine MT outer doublets; Central pair Transition zone: Nine MT doublets Basal Body: Nine sets of MT triplets; Same as a centriole

Actin-based motility in non-muscle cells

Cell crawling through Lamellipodia and filapodia -Lamellipodia: protrusion of a sheet of cytoplasm -Filapodia: protrusion of thin, pointed structure -Focal adhesion: local contact with surface to provide traction. -All contain actin filaments

Cross-section of an Axoneme

Central Pair (2) -13 protofilaments each Outer doublets (9) -doublet MTs -A tubule: 13 protofilament; -B tubule: 10 or 11 protofilament, shares with A tubule -dyneins attached to A tubule can interact with B tubule on adjacent doublet

Ca2+ Regulation of smooth muscle myosin II

Common: Ca2+ GOES UP , Myosin binds actin, muscle contraction Ca2+ GOES DOWN , Myosin can not bind, muscle relaxation Difference: Ca2+ come from outside of the cell Calmodulin and phosphorylation is involved No Troponin based regulation

MT sliding within the Axoneme causes cilia and flagella to bend

Connection (resistance to sliding) translates the force of doublet sliding into a bending action

What is the Cytoskeleton?

Definition: a network of interconnected filaments and tubules that extends throughout the cytosol, from the nucleus to the inner surface of the PM

Chemotaxis Is a Directional Movement in Response to a Graded Chemical Stimulus

Directional migration occurs through the formation of protrusions predominantly on one side of a cell. Diffusible molecules can act as cues for directional migration; when a cell moves in response to a chemical gradient, it is called chemotaxis Chemoattractants: cells move toward a higher concentration of the diffusible molecules Chemorepellants: cells move toward a lower concentration of the diffusible molecules Binding of the molecules to cell surface receptors (G protein-linked receptors) leads to corresponding cytoskeletal changes

Microtubule dynamics in cells

Dynamic Instability - individual MTs oscillate between phases of elongation and 'catastrophic' shortening; intrinsic property of cytoplasmic MTs in cells 1. Growth: GTP-tubulin added(length change goes up); 2. catastrophe: GTP hydrolyzed, MT depolymerizes rapidly(length change plummets); 3. Rescue: growth continues(length changes begins to go back up)

Summary: MT and Cilia/Flagellum

Growth at MT plus end contributes to the formation of cilia/flagellum Dynein-mediated MT sliding cause bending and motion of cilia and flagellum Kinesin and Dynein-mediated vesicle transport delivers/retrieves materials to/from the tip (plus end) of cilia/flagellum. This is called Intraflagellar Transport.

Actin-Based Cell Motility: The Myosins

Head domain (2 for myosin II): -actin-binding site (punching gloves) -ATPase site Neck region: -binds light chains (collar of punching glove) -4 for myosin II (calmodulin related) Different 'tail' domains: -specific cell functions -some forms dimerizes through coiled-coil domain interactions (e.g. myosin II).

Kinetics of microtubule assembly in vitro

In vitro assembly of MT is temperature dependent: polymerization (assembly) at 37C and depolymerization (disassembly) at 4C or 0C Critical concentration Cc of tubulin dimer: MT forms/grows when the dimer concentration is above Cc and MT shrinks when it is below Cc . Lag phase (nucleation): individual dimers ->olgiomers Elongation phase: protofilaments to growing microtubule Plaeau phase: microtubule with subunits coming on and off.

What did the dye show for the myosin hands

Instead of inchworm, it was hand over hand

How does calcium regulate muscle contraction? It causes a conformational change in myosin. It causes a conformational change in microfilaments. It causes a conformational change in tropomyosin. It enhances the rate of ATP hydrolysis by myosin.

It causes a conformational change in tropomyosin.

Microvilli versus Cilia

Microvilli are tiny protrusions on the villi of digestive system walls for instance, they increase the surface area to increase the efficiency of diffusion and such like Cilia is an organelle found in eukaryotic cells, they are sensory organelles which protrude out of sells (so a there are some similarities in that sense to Microvilli, but are much more flexible). They are found in the respiratory system, they catch dust and mucus in the lungs etc. Structurally they are the same as flagella, distinctions can only be made when you assess their length and function

Organization of Skeletal Muscle Tissue

Muscle - tissue -bundles of myofibers Myofiber - muscle 'cell' -fusion of many cells into multinucleated cell with large interior cytoplasmic space filled with myofibrils Myofibril -end-to-end linkage of sarcomeres Sarcomeres -contractile unit

Motility at the tissue level

Muscle contraction: the combined effect of many muscle cells moving simultaneously

Many proteins bind to microtubules and microfilaments inside the cell, which of the following is a major microtubule-binding protein: Troponin Myosin Tau Myomesin

Myosin

Ca2+ regulation of skeletal muscle contraction

Rise in Ca2+ concentration to ~10-3 mM -Ca2+ binds to TN-C -Tropomyosin slides out of the way -Contraction Decrease in Ca2+ concentration , <10-4 mM -Ca2+ removed from TN-C -Tropomyosin blocks Myosin binding -Relaxation

Skeletal muscle regulation of Ca2+ concentration

Sarcoplasmic reticulum (muscle smooth ER) -membranous system wrapping around myofibrils/sarcomeres T-tubules -invaginations of plasma membrane penetrate deep into myofibril cytosol -close proximity to SR -SR membrane Ca2+-ATPase pumps: pump Ca2+ from cytoplasm into SR lumen actively establish resting level of Ca2+ in cytosol (<10-4 mM) -voltage-gated Ca2+-release channel: Can be activated by membrane depolarization opens to release Ca2+ down concentration gradient

Types of microtubles

Singlet (O), Doublet (OO), and Triplet (OOO)

Structure of a Sarcomere

Skeletal and Cardiac muscle -Striated: alternating dark and light bands -A band and I band Sarcomere -One A band, two half I band on each side of A band -A band: thick or thick+thin -H zone: thick only -I band: thin only boundaries: -center of I band: Z line -a sarcomere runs the lengths between two Z lines

Which of the following sentences describes characteristics shared by all myosins? They are motor proteins that bind to GTP. They are motor proteins that are active in muscle cells. They are motor proteins that carry vesicles during intraflagellar transport. They are motor proteins that bind to and exert force on microfilaments.

They are motor proteins that bind to and exert force on microfilaments.

What do all motor proteins have in common? They can be used for muscle contraction. They can bind to intracellular vesicles. They convert chemical energy into motion. They convert actin networks into actin bundles, or axonemal microfilaments into cytoplasmic microtubules.

They convert chemical energy into motion.

MT serves as "Tracks" for vesicle transport inside the cells

Transport of secretory vesicles -Vesicles from ER delivered to Golgi (near centrosome); Dynein -Retrograde transport;Kinesin To the plasma membrane; kinesin

Skeletal muscle regulation of Ca2+ concentration cont: TURNING ON

nervous stimulation - muscle contraction -Nerve cell stimulation initiates voltage depolarization in myofibril -T-tubules transmit voltage depolarization that opens SR voltage-gated Ca2+ channels -Ca2+ concentration in myofibrils rises - Ca2+ binds to TN-C and allows actin-myosin force production