Lecture #12 - Motors Flashcards
Affect of Cytosklatal network on Molecules in cell
Cytoskeleton of the cell creates a sieve in cell (create a gel throughout the cytoplasm)
Can measure a diffusion coefficient of a moelcule
Chart - Log of the diffsuion coefficient against the log of the size of the particle
- IF have a purely aqueous environment –> Log of diffusion coefficient Vs. Log of the size of the particle gives a linear relationship
- NOT aqeous envirnment (cell is not purley aqous) –> get the same linear relatinoship for a little while BUT once you have a larger molecules get inflection point –> line goes down even faster
Diffusion in cell
In cell have a 3 fold drop in diffusion ability
Molecules expeirnece 3X high viscosiy in the cell
When diffusion ability drops means the molecules will move less
- Example - Organelles can’t move because the cytoplasm is packed with the cytoskalatl meshwork
Why do cells need the cytoskalaton
Cells need cytoskalaton for mechanical resileince and provide the shape conrtol to form all of the sgapes that are needed to create tissues/organs to be able to live
- No cytoskalaton = the cell would be a fluid droplet without the necesary structures
Problem the cytoskalaton poses
Precence of cytoskalaton makes many cellular tasks exceedingly slow or impossible because can’t move things (Teological problem for cell)
Example how do you move a vesicle to the membrane if the cytoskeleton is blocking the vesicle from moving
Solution – Evolve motor proteins that use the same cytoskeletal networks to activley move things around
What does it mean for something to be avtive
ACTIVE = means that you put in energy abive thermal energy (using ATP)
Structure of motor proteins
Motor proteins have 2 parts:
1. Catalytic force generating domain –> motor domain that converts ATP into mechanical work (binds to ATP)
- Tail Domain – confers the specificity of the crago cargo or higher order complexes that the motor binds to
Humans have 100 different motor proteins –> Motor proteins include – Kinesin + myosin (works on actin) + Dynein (works on microtubules)
Motor domain of a protein
Dimeric structure –> has 2 motor domains
Motor domain is coupled to a alpha helical coil coil tail
IMAGE - the two grey = two motor domains and each are connected to the alpha helical strcture
Dynein Structure Vs. Myosin Structure
Dynein - Has Triple ATPase domain AND Microtubulue binding site AND big complex of proteins that allow the Dynein to be incorporated into the various structures across the cell
Myosin - Has catalytic domain + actin binding interface + coverter domain
- Catalytic domain = connected to the lever arm (Lever arm = allows the motor to move by pivoting with respect to the tail of myosin –> therefore moves the actin filament)
Kinesin and Myosin Core
Kinesin and myosin motor domain has the same Core (also shared by Ras family)
- Core has alpha helix flanking Beta sheet + walker P loop where ATP binds
- RAS = GTPase with simialr structure
ALL use GEF (GDP exchange factor) an GAP (GTPase activating protein that stimulates hydrplyssi)
How do motor proteins work (OVERALL general concept)
Overall – take energy from ATP/energy released through hydrolysis and converting the energy to mechanical energy/work
Being able to convert energy to mechanical energy = governed by conformational changes in the molecule
In vitro motility Assays
Use - Shows that the myosin motor domain is sufficient for moving actin along its axis
Purify myosin from tissue –> isolate motor domains –> pin myosin on slide –> add actin (stain with rhodamine)
- Image – see myosin head pinned on slide and actin filament
Results:
- No Mg/ATP = nothing happens
- Add Mg/ATP = Actin filaments glide across the surface
First proof that the motor domain was the motor that powered the motion of filaments –> AFTER this could use the system to look at kinetic cycle and force generation
Histore of Assays
1920s- took muscle from animal –> put the muscle in calorimeter –> Stimulate the muscle to contract –> measure the heat coming from the muscle
- Add weight on the muscle and put in calirimeter–> msucle can’t contract even through stimulated it) –> is no heat coming from the muscle
- IF remove the weight –> muscle can contract when stimulated –> get heat
50s/60s - took muscle –> add X-ray or elctron beam –> see different defraction maps based on if the msucle was contracted or relaxed
- FOUND how conraction/relaxation works based on strcuture properties
1980s - could do in vitro motility assays
Myosin Cycle (Overall)
Myosin uses the energy from ATP hydrolysis to generate force to displace actin filament (ATPase Cycle)
- Start – Motor domain of Myosin is bound to ATP
- Myosin Motor domain intrinscially hydrolyzes ATP –> myosin is now bound to to ADP/Pi (hydrilysis caused confirmation change to the pre-stroke state)
- In pre-stroke state myosin is ready to bind to actin
- Myosin motor domain stores the chemical energy by KEEPING the phosphate (STILL IN PRESTROKE STATE)
- Myosin has a diffusional encounter with the actin filament (has weak interaction with actin filament)
- Weak intercation will trigger the release release of the energy (release of phosphate) –> NOW motor locks tightly on actin
- Tight binding is coupled to release of Pi (happens condornatley)
- Release of the energy (phosphate) causes a confirmational change of the lever (drives the myosin power stroke) –> NOW myosin is in the post stroke state bound to actin (bound to ADP)
- Myosin stays in ADP bound state for a while a the ADP will leaves BUT the myosin motor is still on actin tightly in the post stroke configuration
- ATP binds to mysoin –> releases release myosin form actin (restart the cycle)
Myosin when bound to ATP
In ATP state myosin CAN NOT bind to actin
What happens when ATP is hydrolyzed to ADP/Pi
When hydrolyze ATP to ADP/Pi myosin has cofirmation chnage to pre-stroke state
Hydrolysis releases chemical energy BUT myosin wants to store that chemical energy = stores the energy by holding onto the phosphate
- Waits time until releases the energy (releases the phosphate)
- Analogy (like rubber band storying chemical energy as elastic energy when you stretch it)
- HERE - motor bound to ADP is bound to actin –> phosphate is still in = in the isomeric state (transition state)
When myosin stores the chemical energy it does NOT want to randomly let go of the energy (wants to let productley let go of the energy)
Nueocetide state when Myosin has random diffusion and weakly interacts with Actin
When motor bound to ADP is bound to actin –> phosphate is still in = in the isomeric state (transition state)
GEF in myosin ATPase cycle
Actin = acts as the GEF (exchnage factor) for the motor
When the motor encounters actin it will interact weakly with the actin filemnt –> the weak intercation will trigger the release of the energy (release of the phosphate)
Change in lever in Myosin Cycle
Release of the phosphate causes a confirmational change of the lever –> the lever arm swinngs = causes the actin filament to moved with respect to myosin (actin moves inward 10nm)
- Release of phosphate drivers the myosin power stroke
The lever arm swinging is force/work that generate the step (because makes the actin filament move)
Why does myosin stay in the ADP state bound to actin after the lever swing
Myosin stays on the ADP state for a while so that when the ADP does eventually come off a new myosin is already tightly bound on actin in the post stroke configuration
NOW ATP binds and releases the motor form the actin filament ONLY when have a new myosin tightly bound to actin
When ADP is released –> Post stroke nucleotide binding site on motor domain opens = allows ADP to be relased
Myosin Rigor state
Nucleotide free state (when release ADP AFTER power stroke) = rigor state
If have high ATP concteration then ATP binds fast (have short Rigor state)
What dominates the timing of the Myosin Cycle
Cycle is dominated by how long it takes for the phosphate to come off
Motors can bind ATP and hydrolyze ATP to ADP/Pi and then hang out waiting to have weak interacton with actin before they release the phosphate
Example:
1. Skelatal muscle – Myosin bound to ADP/Pi (no release of Pi) = lasts 97-100 miliseconds
- Having strong interaction/releasing the phosphate/going to post stroke = sub milisecond
- Molecule in post stroke waiting for ADP to leave = 2-5 millisocons
ATP binidng to myosin Vs. ADP leaving myosin
ADP is micromolar concetration Vs. ATP is milimolar concentration) –> MEANS ATP binding is a faster that ADP leaving
- If have high ATP concteration then ATP binds fast (have short Rigor state)
The amount of time that myosin spends tightly bound to actin depends on the kinetics of ADP release
Rigor mortis (beef)
When kill animal it won’t make ATP –> ADP levaes BUT ATP can’t bind to myosin so myosin stays bound to actin (stays in rigor state) –> meat gets stiff
Hang meat in a cold room to age to tenderize the meat
- When age meat in the cold room waiting for enzumes (proteases) to cleave the motors off of actin so the muslce will relax and tenderize
Rigor mortis = myosin 2 locks on the actin and makes the corspse rigid
What happens when actin experiences resistance
Example - Actin anchored by cortexilin resistents being dragaed = mysoin 2 motor gets locked mid stroke
Know actin is allosteric sp binding of myosin on 1 actin binding site causes a confirmational chnage that causes cooproative bidning on sites nearby (myosin dimers will bind to each other and their motor domains will bind to actin)
Motor stays bound to actin for short time (10 milliseconds) but the staggard action of nearby myosin filament maintains a grip on filament to keep pulling
- Promote myosin binding to help pull actin
Duty ratio
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)
Different isoforms of Myosin
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%)
Skelatal mucles assmeblies/duty ratio
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)
High duty ratio Myosin
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)
Velocity of myosin movement
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)
Myosin 2
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
Myosin 5 and 6
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)
Myosin cycle when have stress
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
Myosin in Isometric/transition state
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
How to study single molecule myosin motor activity
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
Results of optical Trap (myosin 5)
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
Plateu in optical trap
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)
Myosin 5 and 6 stepping when have stress
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
Myosin 6 direction
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
Atomic force microscopy
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
What is the essence of living
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
Work calculation
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)
What happens when a motor is stalled
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
Myosin 2 Efficieney
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
Myosin 5 Efficieney
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)
Myosin 6 Efficieney
Stall force = 2.5 pN
Step size = 20 nm
Work = 45 pN X nm
Efficiencey = 50%
Efficiecey of all myosin motors
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
Myosin superfamily
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
Myosin 2 Monomer strcuture
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)
What happens when myosin 2 does not have enough light chains
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)
Myosin 2 - binding of monomers
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
TIRF
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
How cells crawl
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
How cells crawl END overall
Cell coordinates the pushing out (membrane moving foward) and back of cell squeezes forward to catch up
Myosin + glioma
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)
Non-muscle myosin in humans
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)
Organization of skelatal muscle
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
Organization of myosin 2 into thick filaments
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)
What maintais the length of actin
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
Titan
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
Tropopmycin and troponins
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
Why does myosin use ATP and how does the brain cause muscle to flex when think about flexing muscle
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
Drugs to inhibit Myosin
- 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)
- Block phosphate release–> Myosin can touch actin BUT only weakly binds because it can’t release phosphate
Drugs for cardiomyopathy
There are other drugs that target cardiac myosin that are used to treat cardiomyopathy
Kinesin Walking
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
What filament does Kinesin walk on
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
Kinesin Structure and overall walking
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
Ordered state of kinesin
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
Kinesin walking
Kinesin walks by coupling confirmationcal changes of the neck linker to ATP binding and hydrolysis (uses same ATPase cycle)
- One Kinesin motor domain binds to the microtubulues in a nucleotide free state
- 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
- Ordered region lays along the motor domain bound to the microtubulue –> propels the rear head foward
- 1st Bound motor hydrolyzes ATP to ADP/Pi –> once hydrolyze ADP the motor can let go
- ATP binds to the 2nd motor protein –> get the same disorder to ordered tranistion proleling the rear head foward
- Second motor has ATP hydrolysis and the kinesin will fall off the microtubule
Step size of Kinesin
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)
Efficiency of Kinesin
Stall force = 8pN
Step size = 8nm
Work = 64 pNXnm
Efficieney = 60%
Dynein (Overall)
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
Dynein complex
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)
Dynein Steps
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
Clutch activity of Dynein
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)
Flexibility that Dynein and Kinesin provide
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
Efficiecey of Dynein
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
Vesicle transport (overall)
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
What happens when vescile has roadblock
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
Switching protofilaments in low resistnce
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’
Use of intermediate filaments
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’
What happens once a vesicle is in the cortect
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
Use of myosin 6
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 - C
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
Summary
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)
