Lecture #12 - Motors Flashcards

Question

Duty ratio

Answer 1

Example – IF work 8 hours in a 24 hour day --> duty ratio is 8/24 (1/3) on and 2/3 off In Myosin example - How much time myosin is bound to actin in whole ATPase cycle - Low duty ratio = time bound to actin is small portion of the entire cycle time Walking = high duty ratio (always need foot on track ; Form a new contact with actin before releasing the first head) Running (Rowers) = Low duty ratio (have times where both feet are off track) - Generate force on actin and quickly release from it - Example - Skelatal mucles (have times where the motor is not connected to actin)

Answer 2

Myosin mechanochemistry cycle can be tuned in different isoforms to produce different properties Example - Skelatal Muscle myosin 2 = low duty ratio enzyme Vs. Other myosins have a higher duty ratio (higher than 50%)

Answer 3

Skeletal muscle myosin and non-muscle myosin are big assemblies with many heads Want to be able to contract muscles fast = Want the motor domains to contact the actin fast - When one head binds and pulls actin it has to move out of the way fast so that the other motor domains can bind --> MEANS sklatal myosin 2 has a low duty ratio (Like running)

Answer 4

High duty ratio = always have a foot clamped on the track High duty ratio = makes motors processicve - Myosins can be processive or non-processive (distiction is based on duty ratio)

Answer 5

Velocity of myosin movement = dependss on the distance (of motor steps) + the time spent in the strongly bound state V = Distance (d)/time spend in strongly bound state (t)

Answer 6

Myosin 2 = low duty ratio ; moves towards the barbed end (plus end) - Called barbed end because when there is no ATP in cell the motors stay in the post stroke phase (motors can't release from actin) and barbed end looks like how the motors are pointed

Answer 7

Work as dimers (NOT big assemblies) and hold onto vesicles to bring vesciles across actin Have high duty ratio (walkers) - always keep motor on actin or else the the motor/vesicle it is bound to will fall off and diffuse away - Coordinates between the heads so it can pull and not lose grip on the actin - High duty = spend more than 50% of ATPase cycle time bond to actin)

Answer 8

Mechanical stress can lock the motor onto actin --> increases the lifetime of the motor on the actin When have stress - When motor bound to ADP/Pi/bound to actin --> phosphate leaves but gets stuck in the isomeric state (transition state) --> then overcomes stress get to go to the post stroke state --> ADP comes off --> ATP binds - Isometric state = between Pi leaving and post stroke state Resistance locks the motor in a isometric state (in midpoint) - starts the power stroke BUT can't go to post stroke state because of the tsress resisting the transition to the post stoke state

Answer 9

In the transition state/isometric the motor can’t let go of ADP = motor can’t let go of the actin filament = the motor sits on actin and carries the cargo for longer = Myosin is waiting for the other motor domains on adjacent filaments to come in and bind Stuck motor is communicating to other motors to help by changing the track confirmation - Can communicate because actin is allosteric = when in isometric state get confirmation change along the actin that makes it a more desirble binding site for more motors to come in

Answer 10

Overall – use optical trap - Can see how the motor interacts with actin Process – tags beads onto end of actin and bead on myosin (myosin is anchored to slide) --> shine lasers onto the beads --> track where the beads are - If processive motor then can anchor the actin filament down and couple the motor to bead = only have 1 bead you have to track (motor will walk across the track) Found that myosin is a Processive motor turning actin down

Answer 11

Based on optical traps – See myosin 5 steps processive and myosin 5 takes 36nm steps and has a stalling force of 22.5 pN Chart – lines are fluctautions center of laser - Shows there is small thermal fluctauation at the begining (small peaks in the flat line at the start) --> then have a big jump (Jump correlates with the bead jump - Jump = increase in distance Pattern – Bead jumps (increase in distance) then plateu --> then jump

Answer 12

At plaetue the motor is dwelling and waiting --> then motor steps again --> then wait --> step --> wait For myosin 5 --> Distance between each plateau = 36nm - 36nm = length to get to pseudorepeat in actin - Myosin 5 takes 36nm processive steps happen UNLES there is resistance (myosin 5 = processive motor) NOTE - Pseudorepeat = next point where have step for motor to plant (has the same binding sites at each pseudorepeat)

Answer 13

Myosin 5 and Myosin 6 display abaerant steping beahvior or they stall under high loads --> THIS allows force to be measured If the motor is trying to pull: - No resstent = myosin 5/6 motor takes 36 nm steps - With resistance = Myosin will take smaller steps (IF the resistnce is higher enough THEN can take a step back and try again) Example – motor steps normally then has resistance where it takes longer for the motor to get into next confirmation (has longer plateu) NOW myosin takes smaller steps because trying to fight against the resistence Right curve in graph = resistence is so high that the motor takes a step back and try again

Answer 14

Myosin 6 – walks in the minus end direction (opposite of myosin 5) - Motors can use the same track to go in different directions (Motors going in 2 directions allows the system to be responsive to road blocks) Myosin 6 steps = shorter 20nm steps (normal) - IF increases reisstnce = then Myosin 6 will toggle back and forth Vesicle uses myosin 5 and 6 - if one motor gets stuck --> the motor will toggle (wiggle back and forth) --> other motor will bind and move the vesicle backwards --> then the first motor re-engases and will move the vesicle back out

Answer 15

Atomic force microscopy feels changes in topology to make a map of the surface High frequencey atomic force micrscopy = used to image the surface of actin filament Found - loop sticking off of actin (loop is myosin 5) - When add ATP and Mg --> myosin 5 steps as walks across actin (see it lifts up and folds over again when takes step) HERE they could see the system for the first time

Answer 16

We consme nutrients to get glucose --> metabolize that glucose to amke ATP --> use ATP as the major energy source to do work Example - the cytoskalatol motors convert the energy to work - Energy to work conversion = burning ATP to get work 40% of ATP is consumed by actin/myosin systems in cell

Answer 17

Work (W) = Force (F) X Distance (D) How much energy is stored in the gamma phosphate of ATP (end phosphate being cut off of ATP) - ANSWER – 100 pNXnm Pn X nm = force X distance (measure of work)

Answer 18

When motor is stalled = get stuck in isometric state Example of stall force - Stall force = 3.5pN --> means need 3.5 pN of force to trap the motor in the isometric state Stall force = meausred using dual beam optical trap

Answer 19

Stall force = 3.5 pN Step size = 10 nm (how far moving on actin) (W) --> 3.5 X 10 = 35 pN X nm Efficieney = ~40% --> 40% of the 100 goes into mechansical work - Remaining 60% of energy goes into heat (process of using Myosin 2 to contract muscle releases heat) NOTE - no heat in calirimtery experiment because the motors were stalled in isometric state (can’t power through so not releasing ATP so not getting 60% of energy from ATP in form of heat VS. No weight then the muscle contacts and creates heat

Answer 20

Stall force = >2.5 pN Step size = 36 nm (1 psuedorepeat of actin) Work = >90 pNXnm Use approximates because did experiment in phosphte buffer so there is more phosphate so there was more effective energy in ATP because when hydrlyze the ATP you are pushing agaisnt a larger phophate gradinet (get higher numbers than should)

Answer 21

Stall force = 2.5 pN Step size = 20 nm Work = 45 pN X nm Efficiencey = 50%

Answer 22

Molecules are incredibly efficient at taking the chemical energy in ATP and converting it into mechanical energy ALL myosin motors = more efficinet than any car

Answer 23

Myosin superfamily = have a common motor domain with diverse tail structures - Tail structure (helical coil coil structre) – confers a multitude of functions because after the coil is the a cargo binding site where it will interact with the cargo - Myosin motor domain = often dimeric Example 2 – Myosin 5 and 6 has a long coil coil - After the coil it has a cargo binding site where it will interact with the vesicle

Answer 24

Myosin 2 motor domain uses chemical energy from ATP hydrolsyis into mechanical work - ATP hydrolysis drives confirmation change to get mechanical work Funcational unit is a hexameric monomer- 2 heavy chains and 2 pairs of light chains - 2 pairs of light chains = 2 essnetial light chains and 2 regulatory light chains Each heavy chain has – motor domain + lever arm + long coil coil tail - Lever arm comes off of the motor and has 2 light chain binding sites (Binding sites are wrapped by 1 essential light chain and 1 regulatory light chain)

Answer 25

Not enough light chains then alpha helix in lever arm exposed and proteases will come and cleave it (THIS IS what happens when hand the meat hangging when meat becomes tenderized)

Answer 26

Coil-coil tail farthest from motor = assmebly domains Assembly: Two myosin monomers bind --> forms a parallel dimer Two dimers join to make a anti paraellel tetromer (more teteromers can bind to that tetromer) END = get bipolar thick filament (actin form of myosin 2) that does work

Answer 27

Use of TIRF - visualize Bipolar thick filament structures in living cells (see indivdiual thick filaments) - See what is at the membrane coverslip interfacte --> can image at the surface of the cell (see cell cortext) - Better reolution than epiflorusnece or confocal When the light hits the objective it gets bent inwards and hits the surface of the glass --> when hits the sirface of the glass the light is internally relefcted = creates an evencent feild that pentrates 125 nm into the cell - Cell cortecxt (with actin meshwork) = 100nm thick

Answer 28

Actin polymers assemble at the leading edge using Arp2/3 mediate assmbley (filament growth) - Expansive forces generated at the front of a cell by actin polymerization ALSO have the ratchet model to propel the membrane foward ALSO have myosin filaments throughout the cell cortext (more enriched at the back of the cell) --> myosin contracts to cause the cytoskelaton to contract to sqeeuze the cytoplasm fowrad ALSO have focal adhesions to the surface (Membrane moves outward as the filaments grow then have adhesion --> cell moves foward --> repeat) - Focal contacts help pause the cell so it can’t retract

Answer 29

Cell coordinates the pushing out (membrane moving foward) and back of cell squeezes forward to catch up

Answer 30

Myosin 2 activity is required for malignant glioma cells to penetrate through brain tissue (required for glioma invasion process) When add a mysoin inhibitor --> glioma tries to send protrusions BUT the cell body can't activate and pull itself fowards (Cell stays in place) Tried to leverage this for anti-cancer --> If add myosin inhibior it reduces the invasivness of cancer cells BUT the cells locally grow very fast - Myosin has a feedback on pro growth pathways = myosin can have tumor supressive roles (no myosin = get more cancer growth)

Answer 31

Haves have 3 non-muscle myosins Myocin 2C = upregulated in PDAC Image – shows Mysosin 2C in normal pancreatic duct (left) Vs. Mysocin 2C in PDAC (right)

Answer 32

Muscle fiber = 1 muscle cell = myofiber --> Muscle fiber (myofiber) has mypfibrils --> Myofibril is composied of sarcameres Sarcameres - Have Z line + actin filaments + extensive myosin bipolar filamets + have motor domains + have barrier zone (in middle) - Motor domains (sticking off of myosin) = arrayed so they are tiled with periodicity along length in both directions

Answer 33

Organization of myosin 2 into thick filaments produces 100s of heads available for force production Sarcamere in depth - has Actin + Titin + tropopmycin and troponins - Have a minus end caping proteins on actin + have plus end of actin bound to capZ (plus end anchors to the Z disc)

Answer 34

Actin 1 micron because of nebulin protein Nebrulin is a molecular ruler --> micron long SU that coiling around actin to confirm and maintain the length of actin

Answer 35

Titan = elastic spring How do you relax the muscle? - ANSWER = need to flex the other muscle When you contract a muscle you pull the Z lines close together ALSO when contract one muscle you flex the other muscle --> when flex the other muscle it stretches the Z lines apart - When flex the other muscle you can stretch the Z lines so far apart that the myosin heads can’t contact the actin filament (NOW would be stuck and would not be able to contract back) Solution - Have titin elastic string so when you let stop contracting it brings back the Z lines so the motors can contact actin to extend back again

Answer 36

Have tropopmycin = make actin better filaments for myosin to bind to in muscle Tropomodulin complex = interacts with calcium to help make tropomycin move so either actin is not accessible to myosin or freely accessible

Answer 37

Why does Myosin have ATP --> Brain activates neurons = trigegrs calcium release = get troponins to release = trpomycin goes to ideal spot = opens actin filament = myocin can bind BUT will sit and wait to realse the phosphate = then can pull actin NOW in relaxed state Mysoin is ready with ADP/Pi state wiating for breain to tell it to conract --> myosin can release Pi for when you think about flexing arm - Once brain sends signals Calcium will go in and open torponin so tropopmycin moves = now actin is ready and myosin can pull

Answer 38

1. Promote tight binding (Omecamtiv Mecarbril) - Helps when the myosin samples the actin it promote the binding of myosin to actin (triggers tight binding) AND slows the release of myosin from actin (myosin stays bound to actin for longer) - Drug = used for cardiac myosin (treat heart failure) --> worked BUT need you be dosed specifically for each pateint so it is implactical - Preventing release of myosin = keeps myosin in the rigor state = BAD (lose ADP but the myosin stays bound to actin) 2. Block phosphate release --> Myosin can touch actin BUT only weakly binds because it can’t release phosphate

Answer 39

There are other drugs that target cardiac myosin that are used to treat cardiomyopathy

Answer 40

Kinesine = Highly processive (takes >100 8nm steps) per encounter with microtubule (moves crago a speed of 0.8 um/s) Some kinesins walk towards the plus end (conventional kinesin and Nod) or the minus end (Ncd) on the microtubule

Answer 41

Kinesin interacts with Microtubules --> Kinesin supports the movment of microtubulues - NOTE - Microtubules = longer and more flexible than actin Do an in vitro motility assay --> anchor Kinesin on the glass surface AND add Microtubulues --> kinesive drives motility of microtubules

Answer 42

2 heads held together by coil coil Kinesin = Binds to ATP to power work step Vs. Myosin uses phosphate release to produces work - BOTH produce work using confirmational change IN Kinesin - ATP binding causes a confirmation change in neck linker sequence to go from disordered to ordered state --> In order state– kinesin is thrust towards the plus end of the microtubule

Answer 43

Once in ordered state – kinesin = deufses along the microtubulue like browian ratchet using energy from ATP hydrolysis and using the swing and docking of the neck linker onto the motor domain

Answer 44

Kinesin walks by coupling confirmationcal changes of the neck linker to ATP binding and hydrolysis (uses same ATPase cycle) 1. One Kinesin motor domain binds to the microtubulues in a nucleotide free state 2. ATP binds to bound motor--> causes a flexible strand to go from a disodered to order orientation ; AND the second motor binds to microtubules - Becomes ordered when it lays alng the motor domain upon ATP binding 3. Ordered region lays along the motor domain bound to the microtubulue --> propels the rear head foward 4. 1st Bound motor hydrolyzes ATP to ADP/Pi --> once hydrolyze ADP the motor can let go 5. ATP binds to the 2nd motor protein --> get the same disorder to ordered tranistion proleling the rear head foward 6. Second motor has ATP hydrolysis and the kinesin will fall off the microtubule

Answer 45

Step size of kinesin = 1 Alpha/beta tubulin dimer of the microtubule Alpha/beta tubulin dimer = 8nm longer (Kinesin takes 8nm steps) Kinesins are small enough and take precise enough steps that they walk on1 protofilamnt on the microtubule (can't step off of the protofilament)

Answer 46

Stall force = 8pN Step size = 8nm Work = 64 pNXnm Efficieney = 60%

Answer 47

Dynein moves on microtubules (moves towards minus end) Has motor domain (AAA ATPase) + has Microtubule binding doman + 2nd alpha helix + complex with many components - has stilt alpha helic and alpha helix connecting to other components dynein = walks on 'stilts' - Stilt = signal antiparaellel coil coil alpha helix that connects the the ATPase to the microtubulus binding site (between binidng site and ATPase) - Antiparalelle coil coil helix = comes from 1 peptide

Answer 48

Dyenin = mega complex with many components Having many components allows Dynein to have different types of dyneins - Example - Have popultion of dynein on vesicles Vs. population of dynein in cortext Vs. population of dynein at chromosome kinetichore (sits at kinetichor to help keeps the chromosome so it can follow the microtibules as microtubules do dynamic instability)

Answer 49

To find step size = do optical trap High load (high resistance) - 8nm steps (consistent with length of alpha/beta tubulin dimer) - Can only get step by step at high load (very precise) Low low (no resistence) - 32nm steps - IF increased the resistence then Dynein goes back to taking shorter steps - Because CAN take bigger step it can go from one protofilament to the next protofilament

Answer 50

Having 2 different step sizes for 2 different loads = clutch activity - shifting gears is done with Dynein motor Example – car on a freeway would go in high gear (32nm steps) to move fast BUT if the car is pulling a large load then would downshift to a lower gear so can have more power (8nm steps) Can go fast with low resitnece (bigger steps) or can go to smaller steps in higher resistence (shift to a lower gear)

Answer 51

Kinesin and Dynein motors build more adaptability for moving a vesicles or chrosmomes More adaptability because have motors that can step forward (kinesine) or move backwards (Dynein) IF there is a road block and you can’t get the vesicle across THEN can use Dynein to move vescile backwards and can step to an adjacent protofilament before moving fowards again

Answer 52

Low stall force --> stall force = 0.3 pN ; step size is 32nM --> work is 9.6 pNXnm Higher stall force --> Stall force = 1.1 ; Step size = 8 nm ; Work = 8.8 pNXnm IN BOTH cases the efficieny is ~10% (same efficince at high and low resistence) - Deals with high resistance by taking smaller steps - Energetics don't chnage BUT molecules evoloved to have flexibility to make shorter steps

Answer 53

Vesicle transport moving through meshwork of cytoskalaton occurs through tug of war between multiple motor types assembled on a common vescile Have many motor types on 1 vescle --> Can have myosin 5 or 6 or kinsin or Dynein Start – vescile is ‘on the freeway’ riding on the Microtubules and will use kinesin - Use microtubule because long distance travel

Answer 54

What ifhave a roadblock when vesicle moves using Kinesin on the microtubule (Ex. Intermediate flament blocking vescile movment) To deal with road block --> might wnat to back up the vescile (tranistion to using dynein) --> Dynein can go to a different protofilament --> might use Intermeidate filemnts for a bit --> when on the microtubule again (after using intermediate filaments) the motor with vescile can go to the cortext of cell

Answer 55

When vesicle backs away from road block have low rsistnce and NOW dynein can move to a different protofilament (like going to a side road to bypass a traffick jam) Dynein can go to a different protofilamnt using Intermeidate filaments to diffuse into a new ‘lane’

Answer 56

Lots of motors can interact with intermediate filaments BUT can’t do direction motion because Intermeidate filamemnts are non-polar STILL can use intermdieate filamenys to diffuse into a new ‘lane’

Answer 57

Once at the cortect of the cell --> vescle want to exit the ‘freeway’ (get off the microtubule) --> NOW vescile will travel on myosin 5 and go onto actin to get the vescile to the membrane --> vesile can fuse with membrane - At cortext have a lots of actin filament and fewer microtubules IF have a roadblock in the cortext then use myosin 6 (instead of myosin 5) to back up and then find a better path and go foward again

Answer 58

Myosin 6 helps in endocytosis to move the vescile away from the mmebrane going through actin until Dynein can move the vescile on micritubule freely (move traffic inward)

Answer 59

Answer - C

Answer 60

Answer - B --> promotes strong binding Ablity for motor to enter strongly bound state but have risk that the motors get stuck and make cardiac failure worse

Answer 61

Actin push against surface of cell ; Microtubules pull Actin and microtubules are tracks (polar) Myosin and Kinesin carry cargo and move filament boxes = shows that the words apply to Kinesin and Dynein = work on MT Drugs target Microtubules, myosins, and kinesin Can target paralogs that are not as ubiquitoous (wont affect all of the cells)