Motors Flashcards
cytoskeletal network leads to seiving and how big a molecular, protein complex or organelle is impacts how they move
-dense meshwork that creates a gel throughout the cytoplasm
-measure diffusion coefficient of a molecule and it has inverse relationship where you take the log of the diffusion coefficient
-if you plot it against the log of the size of the particle, it’s very linear in purely aquaeous environment but in the cell, there’s a 3 fold drop in diffusion ability
-molecules are moving less if you’re a tiny ion and if you’re a large molecule, the movement is even less since you’re in densely packed cytoplasm
teleological relationships of the cytoskeleton
-without the cytoskeleton, cells would be liquid droplets
-needed ability to create shapes for cells, tissues, and organs
-presence of cytoskeleton makes many cellular tasks very slow because you can’t move anything
-evolved motor proteins that use the same cytoskeletal meshwork to actively move things around –> putting energy that’s above thermal energy- ATP
motor proteins
-dimeric structure typically but there are myosin ones that have just one motor domain and a tether Ex. stereocilia
-2 motor domains coupled to coiled-coil alpha helical coiled-coil tail
-they can be more diverse like in Dynein, a mega protein with triple ATPase domain with huge complex of proteins that enable it to be incorporated into various structures
-2 parts: catalytic force generating domain (converts energy from ATP into mechanical work) and large tail domain that confers the specific cargo or higher order complexes
-myosins work on actin, kinesins and dynein wokr on microtubules
kinesin and myosin share a conserved core domain that is also found in the Ras-family GTPases
-core that has similar structure between the two families
-alpha helices and beta sheets and walker P loop where the ATP docks
-small GTPases bind GTP and hydrolyze it to GTP + Pi and release Pi
-GEFs and GAPs that help stimulate hydrolysis- same cycle at play with kinesins and myosins except when the myosin binds the actin, helps trigger release of Pi and ADP- release process is coupled to conformational changes that help the motor undergo the conformational change and swing the arm
-once in post stroke configuration, it can let go of the actin and ATP will rebind and start cycle again
-kinesins do similar thing but with precise positioning of where the molecule is relative to the nucleotide state is slightly shifted
-taking energy from ATP and hydrolyzing it and converting mechanical energy into mechanical work is governed by conformational changes in molecules
in vitro motility assays show that the myosin motor domain is sufficient for moving actin along its long axis
-isolate a bunny or chicken muscle and stick in calorimeter then you could stimulate it to contract and measure heat from that
-stuck muscle in there with weight so it couldn’t contract then there was no heat production
-if you released the weight, it would contract and heat would come out
-chemical energy from ATP
-you could also stick it in an electron beam or X-ray beams then shine the beams through it and see how you would get diffraction maps based on whether muscle is contracting
-people started realizing that this thing would come across and bind –> you get different diffraction models depending on if muscles are contracted or relaxed
-deduced how it might work based on structural properties
myosin II slides actin filaments in vitro
-you can purify myocin from animal and isolate it in a tube and add in Mg and ATP –> led to the in vitro motility assay where you could isolate just the motor domains, pin them down on a glass slide, add in actin, label actin with color molecule, and nothing would happen until you added the magnesium and ATP –> actin filaments started gliding across the slide
-motor domains power the motion of filaments and you can go in to determine the kinetic cycle
myosin uses energy from ATP hydrolysis to generate force to displace an actin filament
-motor domain is bound to APT and in the ATP state it cannot bind actin filament but it has intrinsic ATPase rate where it will hydrolyze ATP to ADP + Pi –> releases chemical energy but wants to store it- myosin hydrolyzes ATP into ADP + Pi and doesn’t want to let it go yet
-always an intrinsic rate that will progress but mostly you hold it and has diffusional encounter with actin filament and actin serves as exchange factor for motor
- interacts weakly with actin
-motor locks on tightly as the Pi comes off
-tight bonding is highly coupled to one another and once it’s bound tightly then gets actin moving in post stroke state
-lives in ADP state and once ADP comes off then the myosin motors locked tightly to actin filament in post-stroke configuration until ATP comes and binds again to release motor from actin filament
-whole cycle time is governed by how long it takes the Pi to come off
-motors can bind ATP –> hydrolyze it and hang out waiting until they fire the rubber band
-once it enters actin and does weak to strong transition to kick off Pi then the process of moving is sub millisecond
-once the molecule gets into post stroke configuration and waiting for ADP to come off, the process takes 2-5 milliseconds depending on the specific paralog (myosin IIs)
-motor flexes and diffusional encounter where the Pi comes off and powers through for ADP come off and ATP rebinds to restart the process with ADP + Pi state
example of red meat
animal has passed and is not making ATP anymore so the meat get stiff and you want to hang beef in the cold room for it to age –> actin myosin interactions are now locked in so waiting for enzymes like trypsin to come in and clip the motors off for muscle to relax
myosin mechanochemistry cycle can be tuned in different isoforms to produce different properties
-muscle and non-muscle myosin IIs are typically low duty ratio enzymes
-big assemblies with many heads and you want to contract your arms -when one head binds and pulls, it has to get out of the way so others can join –> myosin called low duty b/c time spent tightly bound to actin is small in proportion to ATPase cycle time
-some myosin motor proteins have high duty ratio and spend large amount of time bound to actin
three diffeent myosins, three different sets of properties
-myosin II- typically low duty ratio- like to move towards the barbed end (fast growing)- when motor binds and pulls through and you don’t let any ATP be around, the actin filaments have motors that look barbed in post stroke state
-myosin Vs and myosin VIs- not going to work in big assemblies but operate in single dimer and hold onto big vesicle and help it carry it across –> have to have motor clamped on track every single time and need coordination between heads
-walks with high duty ratios and spend >50% of total ATPase cycle time bound to actin filament and ensure that one head is bound
mechanical stress can lock the motor onto the actin, increasing lifetime of the motor on actin
-myosin bipolar thick filament and comes in contact with actin filament to start process then phosphate comes in (transition state or isometric state) then post-stroke with ADP comes off and can release when ATP binds
-if you put resistance on myosin heads, the motors are trying to get to post stroke and traps motor in mid point where starting power stroke but can’t get through b/c a lot of resistive stress- locks motor in isometric state or cooperative binding state
-doing this b/c it can’t let go of ADP and actin filament so sits there longer and gives time for other motor domains to come in and bind
two different geometries for observing single molecule myosin motor activity in the optical trap
-take actin filament and couple little beads onto ends and focus lasers on beads- you can precisely track where beads are
-you can anchor the myosins in different ways by coupling them to beads and you can monitor how motor is interacting with actin filaments
Myosin-V steps processively: an Ex. distance trace with feedback control step size of 36 nm and stalling force of 2-2.5 pN
-fluctuations of the centroid coupled to the bead then it jumps from bead continuously jumping but there are plateaus where it dwells and waits for nucleotide exchange to happen
-36 nm is pseudorepeat of actin filament with myosin binding site
Myosin V and Myosin VI display aberrant stepping behavior or stall under high loads: allows force to be measured
Myosin V:
-motor experiences some stress and trying to do its best to pull
-no resistance –> can step right across those 36 nm steps
-might take smaller steps if it encounters resistance and if resistance is large enough, might take step back and try again
Myosin VI:
-minus end directed motor (goes in opposite direction of actin filament)
-swinging lever arm in opposite direction
-motor takes 20 nm steps and increase load it starts to toggle
essence of living
- consume nutrients for glucose
- metabolize glucose to make ATP
- use ATP as energy source to do work- 40% consumed to drive Na-P exchanger since we need electrical potential between inside and outside cells and the other 40% is consumed by actin-myosin systems in cell and 20% fuels everything else
- cytoskeletal motors are unique Ex. where efficiency to energy to work conversion can be conceptualized easily
function myosin II is assembled into thick filaments
-hexamer- functional unit is a hexameric monomer and hexamer has 2 heavy chains followed by long alpha helical coiled coil
-lever arm coming off the motor that is alpha helical structure wrapped by 2 light chains (essential and regulatory)
-2 heavy chains, 2 essential light chains, and 2 regulatory light chains- 6 polypeptides and functional units
-without enough light chains, it leaves alpha helix exposed and proteases come and clip it
-hexameric monomers come in to form big bipolar filaments and they can differ in size/length depending on type of myosin II
total internal reflection microscopy allows frame-rate, continuous imaging of an exponentially decaying field at the glass-liquid interface
-image right at the surface of the cell with epifluorescence
-excitation beam hits mirror –> goes through objective and specimen and illuminates fluorophores within and outside of the focal plane
-slide down the mirror and comes through objective at different angle- light gets bent inward and hits surface of glass then gets reflected internally and excitation comes down –> evanescent field that probes inside cell
-epifluorescence in middle of the cell then you can see right at the membrane cover slip interface- see bipolar filaments jiggling in cortex of cell
actin polymer assemblies help cells crawl
-actin polymers are all throguhout the cell
-leading edge of cell has Arp2/3 mediated assembly to help filaments grow and brownian ratchet model helps propel membrane forward
-myosin filaments all throughout the cell and more heavily enriched at the rear of the cell- contract to help the cytoskeleton to flow, constrict, and squeeze cytoplasm forward
-adhesions at the surface squeezing outward and helps to pause cell so it can’t retract
myosin II activity is required for malignant glioma cells to penetrate brain tissue
-brain tissue slices and introducing fluorescently labeled glioma cell and you can see cell body pushing outward then fingers stop and cell pulls up and they send fingers up again until they hit plateau and body moves forward
-myosin inhibitor- cell tries to send out protrusion but cell body can’t activate and pull itself forward –> cell stays local and grows faster so the animals die just as quickly
-myosin has feedback on some of the growth pathways and have tumor suppressive characteristics
muscles composed of muscle cells or myofibers
-myofiber- term for individual muscle cell and within myofiber you’ll have individual myofibrils –> myofibrils composed of sarcomeres with Z line
-actin filaments are prescriptive in their length
-large myosin bipolar filaments that are more extensive than what we would see for nonmuscle myosin
organization of myosin II into thick filaments provides 100s of heads available for force production
-actin filaments are 1 micron in length since there’s a nebulin, a molecular ruler, micron long subunit that layers along the actin filament coiling around it helping to establish and maintain the length of the actin filament
-minus end capping proteins- the plus end will anchor into the Z disc binding to a protein called cap Z and alpha actinins will bind there
-titin- largest gene in the human genome- elastic spring- when you’re contracting you’re pulling Z lines close together but if you’re flexing, pulling Z lines apart
-Ca release to get the troponins to release so the tropomyosin can get to its ideal spot and open up the actin filament for myosin to bind and pull
-myosin motors in skeletal and contractile types need to bind ATP and hydrolyze it then sit and wait to be ready to flex arm
-brain sends signal –> Ca released –> troponin opens up –> tropomyosin moves –> actin is ready –> myosin can pull
what happens if you stretch it so far apart that these myosin heads can no longer make contact with myosin beads?
elastic spring brings it back so the motors can make contact in this muscle and extend it back again
kinesin supports movement of microtubules in vitro
-longer than actin filaments and straighter since run risk of breaking
-kinesin drive motility
processive two-headed kinesin walks by coupling conformational changes of the neck linker to ATP binding and hydrolysis
-kinesin binds in nucleotide free state –> ATP will bind and you have flexible strand that goes from disordered to ordered transition
-becomes ordered when it lays along the micromotor domain upon ATP binding and when it lays along that, it’s propelling rear head forward (in ADP state)
-ATP has to rebind one and the other is hydrolyzed to ADP + Pi then it can let go
-ATP rebinds and causes disordered to ordered transition again and moves the rear head forward
processive two-headed kinesin walks by coupling conformational changes of the neck linker to ATP binding and hydrolysis
-both kinesins start with ADP states and for one head, ATP comes on and kinesin sticks to microtubule then other head sticks onto the microtubule then the other head has ATP hydrolyzed to ADP + Pi
-second head has ATP bind to it while the other heasd that has ADP + Pi has Pi come off and process continues
-8 nm step size of kinesin and spans one alpha beta tubulin dimer
-walk along single protofilament of microtubule
dynein is a large minus-end directed microtubule motor with a motor domain related to AAA ATPase
-microtubule binding sites at the very tips like walking on stilts
across b/c you have single alpha helix that’s anti-parallel to b/c it’s coming from the same polypeptide
-alpha helix comes out to form loop then folds back in to create the alpha helical coiled coil
-big mega complex with diverse components to bind and interact with this
-dynein in the cortex, vesicles, kinetochore on chromosome, minus end directed protein and by sitting on the kinetochore it helps keep the chromosome so it can toggle and follow the microtubule during dynamic instability
dynein takes longer (up to 32 nm) steps under low load and shorter (8 nm) steps at higher loads
-high load- 8 nm- consistent with exact length of alpha beta tubulin dimer- motor can only go step by step
-low load- it can take big long steps with 32 nm steps but if you increase resistance, goes back to short steps
-can deviate and step off of one protofilament to next adjacent protofilament
-if you’ve got a vesicle or chromosome, kinetochores have kinesin family proteins there and you’re building adaptability with ability to step forward precisely with kinesin
-encounter roadblock- use dynein to back it up a little and if there’s no resistance, we can even step off and move off to adjacent protofilament before re-engaging and moving forward
real vesicle transport occurs through tug-of-war between multiple motor types assembled on a common vesicle
-kinesins, dyneins, myosin V, and myosin VI will sometimes be on all the same molecule
-when riding on microtubules, uses kinesin then intermediate filament lying across then backs up with dynein and you can do a lot of toggling and perhaps switch protofilaments
-get out to cortex and microtubules not abundant but more actin filaments
-myosin V comes on and tries to get the vesicle up to membrane to get it to exocytose
-when vesicles endocytose, myosin V helps vesicles move away from the membrane and get it to the actin filaments until the dynein can take it away –> move it inward
what is one way a drug could make a muscle contract more forcefully?
ADP release
cells are like houses scaled all the way down
-must withstand external forces, divide, and its contents are held within the cell membrane and cortex
-cytoplasm has contractile meshwork of proteins
-actin filaments are semi-flexible polymers assembled from globular actin (G actin) and distributed throughout the cytoplasm of cortex
overview video
-global cross linkers help push the poles apart expanding the cell
-cleavage furrow forms when the cell divides –> cortexilin joins and anchors the actin filaments
-third type translates chemical energy into mechanical work, helping the cell contract at the furrow- myosin II
-cytoplasm has reserve of myosin II in its inactive form in hexameric monomer- single unit that consists of 6 parts (2 heavy chains, 2 essential light chains, 2 regulatory light chains)
-each heavy chain has motor domain, lever arm, and long coiled coil tail
-2 light chains binding sites along the lever arm are wrapped around by essential light chain and regulatory light chain
-assembly domain is all the way at the end of the coiled coil tail- 2 myosin monomers can bind to each other and form parallel dimer
-dimers join into anti-parallel tetramer, the nucleus to which more and more dimers bind –> resulting assembly is bipolar thick filament, active form of myosin
-motor domain of myosin II uses ATP hydrolysis to drive conformational change
-positive feedback loop promotes PGF assembly in regions of the cell where actin is already under tension
-during cytokinesis, feedback is first initiated through chemical signals from mitotic spindle and resistive stresses to cell expansion happening at the poles
-myosin contextual actin network continues to contract, deepening the cleavage furrow until eventually most of the myosins lock in
-after this, division of the cytoplasm is driven by fluid physics
-cell is pinched in enough that the most energetically favorable shape is total split into 2 spheres (2 daughter cells)
