Filaments Flashcards
(33 cards)
cells assume many shapes
-don’t have the right form –> difficult to function
-neurons have cell body, dendrites to make contacts with certain parts of the body, and long axon
-budding yeast stick out bud that is daughter cell and pinches off
-gut lined with microvilli that are densely packed with actin filaments to create fingers that get sticked out
mechanosensing and mechanotransduction are a fundamental part of normal physiology
-BP and heart pumping blood then smoth muscles sense the pressure and tune the pressure by being like a hose to help blood move throughout the body
-at a system level, same types of cytoskeletal machinery are sensing and generating stresses
Ex. when you break a bone, put weight and pressure on fluids that then intercalate into the bone and then you have osteocytes that sense this and guide deposition of bone matrix –> when final bone is healed, often stronger than OG bone
Ex. cause skeletal muscles to relax and contract- guided by the actin-myosin meshwork that helps to contract the muscles
Ex. sound is a mechanical wave and causes stereocilia to flex and when they flex, there’s an anchor that’s a cadherin family protein that anchors one stereocilia to the next
3 major filaments in most cell types
- microfilaments
- microtubules
- intermediate filaments
microfilaments
-provide strcutrual and mechanical support and provide tracks for myosin based contractility
-composed of actin monors
-strucutured as 2 start right-handed helix, 8 nm diameter
-uses ATP
microtubules
-still in the micro rangs but with tubular structure
-provide long distance transport from which the cells can build structures like mitotic spindle
-line axons all the way down
-composed of alpha-beta tubulin dimers and gamma-beta tubulin dimers, which forms nucleus that microtubule grows from
-have 13 protofilaments that form hollow tube that is 25 nm in diameter
intermediate filaments
-provide structural and mechanical roles for cells and tissues
-composed of lamins, keratins, and neurofilament proteins
-coiled-coil dimers, assemble into flexible, non-polar polymers 10 nm diameter
-no energy source- assembled through more of a thermodynamic guided process
actins assemble into a two-start right handed helical strand with 13 monomers per 36 nm pseudorepeat
-4 globular domains forming horseshoe with pocket that allows ATP to diffuse into it and bind to its binding site
-b/c it’s in that ATP state, molecule is actually ATPase that takes ATP and hydrolyzes it to ADP + Pi –> ADP + Pi comes off –> ATP can rebind
-minus end is the slow growing end and the plus end is the fast growing end
-molecules orient and you can see 2 filaments growing around each other (2 start helix) and they coil
-they don’t live separately and have to be coassembled in parallel –> happens b/c there’s a hydrophobic loop that will stick in and lay across the axis of the helix to stabilize the subunits in place
-minus end has clefts pointing downward and plus end has flat ends pointing upward with hydrophobic plug sticking across the helical axis
repeat
-start on this strand and go all the way up and back around
-how long does it take before I find a subunit that’s in the same location one repeat down the road? helical pitch process is defined by the repeat
-when you have one subunit here, you only have to go halfway around before other strand presents a subunit in the same location (pseudorepeat- not a full repeat but you have equivalent site from actin subunit on the other strand- measure length of this (36 nm) and full repeat would be 72 nm)
-molecular motors look for actin filaments and comparable binding sites 36 nm away
stiffness and modulus of elasticity
-actin filaments are coiled around forming right handed helix with pseudorepeat and there can be a cross sectional area
-geometry matters when determining the stiffness of a material
-spring constant k is proportional to how much force it takes to change the length if you’re pulling or flexing
-elastic modulus- how much stress do you apply to induce strain?
-most actin filaments in the human body are ~1 micrometer long, therefore, your body (10% actin) is constructed from tiny plastic-like fibers
actin filament polymerization is a slow process, the dimer is highly unstable
-if you take actin alone, it’ll be in free monomeric form
-plug that reaches across that’s hydrophobic –> if you want to promote hydrophobic interactions, add salt
-ability of thsoe subunits to be bound is dependent on ATP state- ATP, ADP + Pi, ADP, or nucleotide free –> need some nucleotids to help promote this since it changes the structure of these structures to help make them either favorable for forming a filament or coming off the filement
-if you have ATP going into a binding pocket, you also need a dication like magnesium –> calcium is veyr low in comparison to magneisium
-have to have 2 monomers come together to form a dimer –> add on another monomer to form a trimer –> tetramer –> pentamer etc.
-dimers of actin come apart quickly –> need to stabilize thme
-if the []s of free actins are high enough, the actins will come apart but once in awhile, a third one will come together fast enough that the dimer didn’t come apart in time
-when the. trimer comes together then it’s stable b/c you have pocket for the hydrophobic plug to stabilize then add the subunits
actin polymer assembly dynamics depend on nucleotide state of subunits
-b/c ATP likes to bind, neglect the off rate
-b/c ADP actin doesn’t like to bind, neglect the on rate
-critical []= if you want to build filament, need constant [] of subunits that’s sufficiently high
ATP when bound to actin subunit puts the actin subunit in a conformation where it kind of favors the ability of subunits to bind
-actin is an ATPase –> takes ATP and puts the subunit in conformation where it now favors hydrolyzing ATP to ADP + Pi
-eventually Pi comes off leaving the ADP bound
-ADP actin has different conformation than ATP actin –> aDP bound subunits favor coming off (don’t like to be on growing end)
-b/c of this ability to be ATP bound, ADP bound, or ADP + Pi bound, the actin takes on different conformation depending on nucleotide state
-actin is very allosteric- if there’s a conformational shift in one subunit, it tells the neighbors
-on rate/off rate- larger [] of subunits, the faster you can push the on rate
A critical (minimal) [] of actin monomers is required for incorporation into polymer
-polymer formation- take tube and stick in subunits with ATP and monitor formation of polymers over time
-slow phase –> accelerates –> plateaus
-slow phase- need 3 subunits to come together
-accelerates- start building nuclei and subunits can add on
-plateaus- monomer pool goes down as you make polymers- still need Cc that system has to be above
-plot the [] of actin vs [] of monomers- monomers initially increase then it plateaus and polymer [] increases- build more polymers that you maintain at equilibrium
-shape of curve changes at Cc
how do you study actin?
-pyrene label the actin- pyrene is a fluorophore and when exposed to solvent, has low fluorescence but incorporated into environment surrounded by hydrophobic residues, it likes to fluoresce
-local environment in actin filaments where pairing takes place is sufficiently hydrophobic and fluoresce level goes up
-reactive cysteine near the C-terminus of the myosin subunit that is very amenable to being pyrene labeled
-now you can take your samples, G actin, or take same [] of actin and add salt, Mg, and ATP –> sudden increase in fluorescence
-directly image the fluorescence peaks and get the nucleation phase, the growth in the steady state –> actively detecting transition from monomers to nuclei to polymers
modulation of actin dynamics by actin-binding proteins
-sequestering molecules like beta-thymosin and profilin- bind free adctin subunits to help sequester them
-b/c of the Cc of the actin subunits is down ~150 nM but the [] to build the cytoskeletal structures of our muscle or structures to build migrating immune cells then []s will be 20-100 micromolar –> sequester subunits using proteins so they can be delivered regularly
-cofilin protein, severing protein that loves to bind to actin filaments that are ADP bound (old filaments and breaks them) –> exposes ends so the subunits can come off the filaments
Arp2/3 complex helps nucleate actin and mediates formation of branched networks
-7 subunits- 2 of them are actin related proteins 2 and 3
-5 subunits help dock the Arp2/3 complex to side of actin filaments
-2 subunits are actin-like and if you have actin subunit that is now escorted into the complex through scar or wasp, it will help anchor these to the other 5 subunits
-2 subunits plus actin is magic number of 3 –> stable nucleus so actin subunits flow in and bind
-profilins like to deliver these and help to bring ATP bound actins in and get released –> filament grows
-older filaments towards the rear of this meshwork, Arp2/3 complex binding to the sides, and filaments are growing outward for more Arp2/3 to bind
-cofilin frees up more ends so ADP bound subunits can come off and profilin is nucleotide exchange factor that binds to the subunit and allows it to kick off ADP and ATP is high enough it will flow back in and refill pocket with ATP actin
-profilin-actin diffuses and if it encounters a front again it can now bind and release the actin subunit and grow filament outward –> net assembly of meshwork moving towards membrane
brownian ratchet model incorporates diffusion with actin polymerization to drive advancement of membrane front
-elastic energy is stored in filament and it wants to flex back –> subunit pops on and filament gets longer and helps propel membrane forward
-diffusion of actin subunits with polymerization to drive advancement of membrane
expansive forces generated at the front of the cell by actin polymerization and contractile forces generated at the rear of the cell are coordinated to propel cell forward
-actin dense meshwork at the leading edge that are growing
-Arp2/3 mediated growth and membrane fluctuations and filaments are growing to stabilize the membrane –> pushing front forward
-cortex of actin meshwork that goes all around the cell and myosin IIs that are contractile around the cell
-if you stretch the cortical tension wants to restore that shape and help propel the cell forward
-combo of outward pushing and inward tugging that propels cells forward and cell can move forward if it’s coupling with all of this –> makes contact with substrate
we have vesicles all around the cytoplasm and they use similar machinery to help propel them forward
-lysteria shoots up to the front and go outward into extracellualr space and if it happens to be next to a tissue then it engulfs it
-actin comet tails- lysteria uses all the same machinery to trigger actin filament assembly
-actin fibers get nucleated and grow then propel the bacterium forward
-actin filaments are hydrolyzing ATP to ADP without cofilin coming in and taking apart actin filament- tapering off since they’re getting disassembled
actin cross-linking proteins control the viscoelastic properties of an actin network
-cross-link into meshworks and cause network to take on gel-like characteristic
-others will take these and orient them into parallel arrays creating bundles
differential sedimentation is one assay for distinguishing actin binding from actin cross-linking
-purify the proteins in a test tube (actin and actin binding protein) –> you can have ABP alone, actin alone in polymer form, ABP if ABP does not cross link and actin, and ABP with cross links
-high speed 100,000 g spin –> all of the actin filaments sediment out into the pellet and ABP stays in supernatant but if you mix them, can be pelleted out
-if you have molecule that you can cross link the actin filaments into gels or bundles, then you don’t have to go to 100,000 g and can spin slower at 10,000 and ABP will stay in solution but if you mix them, you form structures that are big enough to spin out and both proteins go into pellet
-middle panel for 100,000 g is showing the F-actin not cross-linked with the ABP1 but for 10,000g it is cross-linked and that is why you are able to get F-actin in the pellet form –> F-actin alone is a small molecule so at the slower speed it will only be in the supernatant, so you need it to be cross-linked for it to go into the pellet form
-in the middle panel at 100,000 g the ABP1 is able to be in the pellet form also because at high speeds small molecules can go into the pellet
three ways drugs can inhibit actin polymer assembly
- stabilize polymers and inhibit treadmilling ability of actin subunit growth
- block barbed-end addition- inhibits addition of subunits and eventually the ATP gets hydrolyzed to ADP and cofilin comes in and severs this to disassemble actin meshwork
- sequester monomers- bind free subunits and overwhelm the profilin component and sequesters subunits awaym
microtubules form a variety of structures throughout the cell cycle
-interphase- microtubules are displayed all throughout the cytoplasm and radiate outward from a central point –> this is where centrosome lives
-centrosome- microtubule organizing center
-nucleus lives near the center of the cell b/c it’s microtubule based motors pull the nucleus towards the centrosome
-as microtubules grow outward, they’re hitting membrane and in brownian ratchet style model, trying to push against it and little by little the centrosome gets shifted around until forces balance out
-cell goes through G1 to S where it replicates DNA and centrosomes- centrosomes live near each other in G2 until cell needs to divide ad it’s going to separate centrosomes (define 2 centroids with microtubules)
-some microtubules go towards the membrane, while others find the chromosomes
-when they find the chromosome, interact with kinetochore and stabilize the microtubule end
-now all the chromosomes have stabilized microtubules at the kinetochore
-they get pushed around until they gradually do a force balance and get aligned at the middle b/c of the force balance
-b/c each of the sister chromatids Is attached to the fellow sister chromatid with their kinetochore and each kinetochore is getting bound by opposing microtubules and they’re doing tug of war until they balance out and the chromosomes are centered
-once they’re centered, chromosomes are happy and trigger onset of metaphase and anaphase where sister chromatids get pulled into each hemisphere of the cell where each hemisphere defines the centroids of 2 daughter cells
-these microtubules send cues to the cortex to set up contractile machinery to pinch inward and squeeze microtubule bundle together and snaps everything –> 2 daughter cells
microtubules assemble from a simple dimer
-heterodimer- alpha beta tubulin dimer- neither is stable alone
-each one has a Walker P loop to bind to nucleotide GTP
-actin has Walker P loop where it binds ATP
-myosin has Walker P loop where it binds ATP
-alpha tubulin doesn’t hydrolyze GTP but it does need it to be stably structured
-beta subunit at the surface has more freedom of motion- hydrolyzes GTP to GTP + Pi –> get rid of Pi and GDP bound
-alpha beta tubulin is allosteric- whne it’s GTP bound, it liks to organize into protofilaments
-in full microtubule, you have alpha beta alpha beta
-typical microtubule filament has 13 protofilaments to use to go around perimeter
