Meeting 25, 26, 27

  1. lamellipodium
    Image Upload 1
    • thin leading edge; part of a migrating cell (ex. fibroblast) found at the front; cell moves the direction where the leading edge is facing
    • - the large cell body falls behind it
  2. components of cytoskeleton (3)
    • Image Upload 2
    • (1) Microfilaments: 7-9nm, made of actin (~smallest)
    • (2) Microtubules: 25nm, made of αβ-Tubulin dimer (biggest)
    • -found everywhere in the cell
    • -organization is tightly controlled
    • (3) Intermediate filaments: 10nm made of globular monomers (INTERMEDIATE SIZED!)
    • -more structured (less dynamic) than other 2
  3. the point of this picture:
    Image Upload 3
    is to show how the same filaments can be organized differently throughout a cell

    • microfilaments (made of actin) can be viewed using phalloidin (binds to and arrests actin)

    • • stress fibers: span entire cell body; providing ridgidity to the cell (made of γ-actin [gamma])
    • • toward leading edge you have filopodia: smaller spikes/digit-like protrusions (made of β-actin)

    • *α-actin makes up contractile rings
    • Image Upload 4
  4. Actin
    Image Upload 5
    • • a globular protein with INHERENT polarity
    • •basic unit of microfilament
    • -there’s a cleft on one side, and no cleft on the other
    • • - end: the end of a filament w/ an exposed ATP-binding cleft
    • • + end: opposite of the - end :)
    • •globular, polarized, & can bind/hydrolyze ATP
  5. g-actin and f-actin
    • •g-actin: monomeric globular form Image Upload 6
    • •f-actin: fibrous form Image Upload 7
    • •actin can reversibly polymerize from g to f type
    • •the strands of the f-actin double helix go in the SAME direction
    • •helical shape means there’s periodicity...of 36 nm
    • -36 nm later, there is one full rotation of a strandImage Upload 8
    • -important because myosin ‘walks’ on actin in 36 nm steps
  6. ___________ is the main limiting step in de novo actin polymerization
    • Nucleation is the main limiting step in de novo actin polymerization
    • -ugh I have no idea

    • -another thing:
    • Image Upload 9
    • critical concentration (Cc): concentration of G-actin monomers in equilibrium w/ actin filaments
    • •when concentration of monomers (G-actins) is below Cc, no polymerization takes place
    • •increasing concentration means at some point nuclei form & polymerization occurs; filaments assemble until steady state is reached --> [monomer] falls back to Cc
  7. actin monomers are added at the _______ of pre-existing filaments
    (+) end

    • • (+) end: contains ATP-actin
    • • (-) end: contains ADP-Pi-actin & ADP-actin
  8. G-actin monomers are added ___ faster at the ______ of pre-existing filaments
    G-actin monomers are added 10x faster at the (+) end of filaments

    • -critical concentration:
    • Cc = (disassembly rate) / (assembly rate)
    • Image Upload 10
  9. at steady-state, actin filaments grow @ (+) end & dissociate @ (-) end, leading to treadmilling
    • • treadmilling: mechanical force; how actin polymerization exerts force/moves
    • - accomplished by addition ATP actin (+ end) coupled with removal of ADP actin (@ - end)
  10. profilin & cofilin cycle(s)
    Image Upload 11
    • basic mechanism behind treadmilling of actin filaments

    • •profilin cycle: profilin binds ADP-G-actin & catalyzes the exchange of ADP for ATP
    • - the molecule is then either added to (+) end or simply dissociates

    • •cofilin cycle: cofilin binds to ADP-actin and makes them fragment, enhancing DEpolymerization of (-) end
  11. Thymosin-β cycle
    • *thymosin-β cycle: binds G-actin (in its ATP bound form), keeping it from polymerization until free G-actin concentrations are lowered; only then will it free G-actin so it can polymerize
    • •if actin were left alone, it would just endlessly polymerize & fill cell w/ filaments
    • •parameters suggest system is tilted toward F-actin formation
    • •thymosin-β: protein that sequesters G-actin & prevents it from random polymerization
    • • cellular levels of actin = 100-400 µM
    • • up to 50% of actin is unpolymerized, therefore:
    • - cellular unpolymerized actin = 50-200µM
    • • Critical concentration (Cc): ~0.2-0.3 µM
  13. Tropomodulin
    Image Upload 12
    • • a (-) end capping protein that inhibits disassembly & stabilizes F-actin
    • • binds to minus ends of filaments; PREVENTS
    • dissociation
    • -want this activity when you want filament that stays around for a while
    • -important for muscle physiology
  14. CapZ
    Image Upload 13
    • • a (+) end capping protein that blocks assembly of actin
    • filaments
    • • prevent further polymerizaton or decrease of critical concentration
    • -these proteins themselves can be further regulated by membrane phospholipids
    • -that’s why you have actin phosphorylation at membrane

    • gelsolin: another protein that functions to prevent F-actin elongation; is activated by increases in Ca2+
  15. Rho GTPases
    Image Upload 14
    • • small molecules that, via signaling, regulate shape + control formation of actin cytoskeleton
    • • in cytoplasm, are inactive in Rho-GDP complexed with GDI form
    • • signaling pathways bring Rho-GDP to membrane, when GEF exchanged GDP for GTP
    • • Rho is now active!
    • • in active form (@ membrane), it binds to effector proteins that alter actin cytoskeleton
    • • remains in active form until GAP exchanges GTP for GDP, returning it to inactive cytoplasmic form
  16. Foramin: one of the aforementioned effector proteins
    Image Upload 15
    • • inactive foramin is ACTIVATED by binding Rho-GTP to its RBD (Rho binding domain)
    • • this causes exposure of FH2 domain, which in turn promotes the nucleation of a new filament
    • • the adjacent FH1 domain recruits prolifin-ATP-G-actin complexes that can be added to growing (+) end of actin filament)

    • -foramins do all this by dimerizing, and therefore act w/ TWO actin monomers
    • -once they've facilitated nucleation, they sit at (+) end and PREVENT capping
  17. WASp activated Arp2/3 complex promotes assembly by mimicking (+) ends
    Image Upload 16
    to nucleate actin assembly efficiently:

    •Arp2/3 complex: binds to the side of an actin filament + to an activator, such as WASp

    • •WASp: activator that induces a conformational change in Arp2/3; the complex can now bind an additional actin subunit (2 in total!)
    • *WASp is activated by binding to membrane-bound small G-protein Cdc42-GTP (a Rho-GTP ohmigod), releasing what inhibits it to expose acidic domain for interaction w/ Arp2/3

    -the Arp2/3 branch makes a 70° angle between filaments
  18. F-actin cross-linking proteins: each contain 2 domains
    • (1) Fimbrin: protein that creates a network
    • Image Upload 17

    • (2) Filamin: protein that creates a network
    • Image Upload 18
  19. Myosin
    Image Upload 19
    • myosins: molecular markers that use actin filaments to move things around or move actin filaments

    • • head: made up mostly of a heavy chain that binds to ATP and actin; uses energy of ATP to create movement by binding to actin
    • • neck: made up of 4 light chains, which stiffen the neck so it acts as a lever arm for the head; is site for regulation
    • • tail: made up of 2 (identical) heavy chains; used to attach motor to whatever you want to move around [form coiled-coil by dimerizing]
  20. This experiment is fun!
    Image Upload 20
    • (1) attach myosin heads to a glass coverslip
    • (2) expose to a solution of visibly stained actin filaments

    • •in the presence of ATP, myosin heads walk TOWARD to (+) end of filaments
    • •b/c they're stationary, the filaments move TOWARD the (-) end
    • Image Upload 21

    •conclusion: sliding-filament assays show that the head+neck region is sufficient for ATP hydrolysis and actin-based motility
  21. a myosin’s speed depends on:
    • the length of its neck; a longer neck means actins are carried faster
    • Image Upload 22
  22. Myosin moves step-wise [toward (+) end] on actin (Myosin V)
    Image Upload 23
    • •Myosin V has 2 head domains & 6 light chains per neck
    • •bind brown box receptors on organelles, which they transport
    • •when only one myosin head was tagged, a step size 72 nm was shown, proving it moves via hand-over-hand model (not inchworm)
  23. myosin 'walks' because of conformational changes induced by ATP hydrolysis
    • w/out ATP, myosin is FIRMLY attached to actin filament
    • (1) myosin + ATP: when ATP is bound, myosin releases from actin filament
    • (2) the head hydrolyzing ATP to ADP+pi causes it to rotate with respect to the neck!
    • -this 'cocked' state stores energy from ATP hydrolysis as elastic energy (like stretched spring)
    • (3) cocked myosin binds to actin
    • (4) when bound, the head couples release of Pi to release of elastic energy to MOVE actin filament: power stroke
    • (5) head remains bound as ADP is released until new ATP is bound to the head...process repeats

    • -unbound when attached to ATP; bound when attached to ADP or ADP+Pi
    • -energy comes from ATP hydrolysis when unbound and release of that energy coupled to release of Pi when bound causes power stroke/movement of F-actin filament
  24. Overview of Microfilaments
    • • made up of of actin monomers
    • • actin binds ATP
    • • forms rigid gels, networks, & linear bundles
    • • regulated assembly from a number of locations
    • • highly dynamic
    • • polarized
    • • actin serves as tracks for myosin proteins
    • • contractile machinery & network at the cell cortex
  25. Overview of Microtubules
    • • αβ-Tubulin dimer binds to GTP
    • • are rigid and not easily bent
    • • assembly is regulated from only a FEW locations
    • • highly dynamic (as well)
    • • polarized (like microfilaments [actin])
    • • serve as tracks for kinesins & dyneins
    • • overall purpose: to organize & transport organelles over a long-range
  26. microtubules are found in diverse structures (examples)
    • • MTOC determines cell-type microtubule organization
    • -used for support/vesicle trafficking/movement in:

    • • cilia or flagellum: microtubules make up shaft, and MTOC is called basal body
    • • mitotic cell: MTOC = spindle poles
    • • neuron: MTOC is found in cell body and then microtubules are released into axon and dendrites
    • • interphase cell: MTOC = centrosome
  27. Microtubules
    Image Upload 24
    • • are polarized polymers of αβ-Tubulin dimer
    • -α is GTP-bound; β is GDP-bound
    • • one microtubule = 13 protofilaments (made up of tubulin)
    • • unlike actin, tubulin dimers don't polymerize spontaneously in vitro; tubulins are found as dimers
    • • polarity: subunits are added to the (+) end, where β-tubulin monomers are exposed
  28. Centrosome: specialized structure required for MT nucleation
    Image Upload 25
    • • a type of MTOC involved in organizing spindle poles before mitosis; composed of 2 different centrioles
    • • 9 triplets form a single barrel, and 2 together are 90 degrees from each other
    • • pericentriolar material: surrounds centrosomes (MTOCs) & contains proteins for microtubule formation

    • *•little red circles = γ-TURC, which is (gamma) tubulin that triggers formation of new microtubules; attached to MTs at (-) end
    • -found in pericentriolar material
  29. Polymerization of αβ-tubulin dimers (is similar to actin polymerization)
    • • αβ-tubulin dimers assemble into MTs when above Cc
    • • above Cc, MTs at steady state are in equilibrium w/ free αβ-tubulin dimers
    • • there are different critical concentrations at the (+) and (-) ends (b/c addition is higher at (+) end)
    • • Cc for MTs = 10-20 µM, which is much higher than Cc (0.03 µM) of actin
    • - regulation by MAPs plays a major role in inhibiting spontaneous formation of MTs

    Image Upload 26
  30. individual microtubules elongate progressively and shorten suddenly
    • Image Upload 27
    • • at dynamics are at the + end

    Image Upload 28stochastic
  31. GTP hydrolysis by (+) end capping β-tubulin = catastrophe
    Image Upload 29
    • • microtubule with GTP-β tubulin on the end of e/a protofilament is favored to grow
    • • microtubule with GDP-β tubulin on the end of e/a protofilament forms a curved structure & will disassemble rapidly
  32. colchicine
    drug that gets rid of existing MTs
  33. MAPs (Microtubules Associated Proteins)
    • • required to stabilize and organize MTs
    • • influence polymerization & catastrophe rates; are regulated by phosphorylation

    • • MAP2 protein has a long arm; results in more even spacing
    • • Tau protein has a shorter arm, resulting in less evenly distributed MTs
    • - *modifying a protein that binds to MTs w/ phosphate will inhibit binding, & therefore promote catastrophe
  34. EB1
    Image Upload 30
    • • a “+TIP” MAP that binds to & stabilizes growing MTs; binds to the (+) end at the seam
    • • convinient place to bind b/c it is unstable & where catastrophes will most likely start
  35. MT destabilizing proteins
    enhance the rate of catastrophes by inducing protofilament curvature

    (1) ATP-dependent kinesin-13: enhances disassembly & is helped by ATP

    (2) stathmin: binds to curved filaments and enhances their dissociation from MT (+) end; can be inhibited by phosphorylation
  36. cellular materials are transported at different rates & in different directions
    • • microtubules can serve as tracks for transport in both directions
    • - ex. antero & retrograde transport occurs on MT tracks in axons
    • • however, for a given vesicle, transport on a MT is uni-directional

    • -anterograde: neuron nucleus to synapse
    • -retrograde: endyocytic vesicles to lysosome
  37. Kinesin1
    Image Upload 31
    • • the molecular motor for anterograde transport; use microtubule tracks to transport vesicles
    • -tail: interacts with receptors on cargo
    • -stalk: responsible for dimerization
    • -Head+linker: serves as ATPase, & motor

    ATP is required for movement

    • • vesicle to attached to kinesin receptor
    • - they walk toward + end ONLY
    • -is centripetal: move from inside to outside (he says centripetal but I think it's centrifugal = outward force away from the center of rotation)

    •each step is 8 nm
  38. Kinesin uses ATP to 'walk' down Microtubule
    • kinesin-1 with 2 heads; if ADP is bound to both, it is NOT attached to the MT
    • 1) when one head binds to a β-tubulin subunit, it is induced to release its bound ADP --> now it's strongly bound to MT
    • 2) leading head then binds ATP
    • -this causes a conformational change (!) that makes the linker region point forward and therefore thrust the trailing head forward
    • 3) the new forward head, now bound to the MT, releases its ADP
    • - this induces the new trailing head to hydrolize it's ATP to ADP + Pi
    • 4) Pi is released, and now that a head is bound to ADP, it dissociates from the MT

    then the cycle repeats...ADP is released, blah blah blah
  39. problem of coupling ATP hydrolysis to mechanical work
    • • myosin and kinesin heads have come up with two similar (but unrelated) solutions to this same problem, indicating convergent
    • -they have the same catalytic core (no amino acid conservation) with a fold that uses ATP hydrolysis to generate work
  40. Dynein motors
    Image Upload 32
    • • also transports cargo along MTs, but this time moves toward the (-) end of microtubules & are organized differently than kinesins
    • • Dynactin: protein that helps bind (therefore transport) cargo to Dynein

    • -prestroke state: ADP-Pi bound
    • -generating force for movement involves a change in orientation of the head relative to the stem, causing movement of the MT bound stalk
  41. dynactin complex link dyneins to cargo
    Image Upload 33
    • made up of:
    • • Arp1 capped by CapZ: binds cargo
    • • p150glued: attaches dynein to the dynactin complex; also contains an MT binding site
    • • dynamitin: holds the two above parts together
  42. Dyneins and Kinesins are required for the transport of organelles throughout the cell
    • •vesicles coupled with kinesins: sent OUTward
    • •vesicles coupled with dynesins: sent INward
    • Image Upload 34
  43. Intermediate Filaments
    Image Upload 35
    • they're formed from fibrous monomers

    • • its subunits don't bind nucleotides (ATP or GTP?)
    • • great tensile strength
    • • assembled onto pre-existing filaments
    • • LESS dynamic
    • • NOT polarized
    • • don't have motors
    • • used for cell & tissue integrity
    • • made of very different types of monomers

    • • form parallel dimers through coiled-coil domain
    • • a tetramer can be formed by antiparallel, staggered side-by-side aggregation of 2 identical dimers
    • -can create higher order assemblies of them until you get protofilabents or protofibrils
  44. There are 5 major classes of IFs in mammals
    • Class I & II: keratins make up the 1st 2 classes; found in epithelia
    • III: found in cells of mesodermal origin
    • IV: make up neurofilaments found in neurons
    • V: llamins; found in nucleus lining of all animal tissue
  45. Essential steps in directed cell migration
    • • analogy of a rockclimber; extension of the arm and then
    • grabbing (=adhesion); once the cell is attached (adhered), you need to translocate the rest of the mass forward, usually involves microtubule cytoskeleton and movement of nucleus

    • this subsequently involves endocytosis, vesicle trafficking, and recycling of vesicles

    • all of these processes involve interplay between cytoskeleon and membrane itself and extracellular matrix and how cell interacts with it
  46. PAR proteins
    • • localize at opposite poles of the fertilized egg
    • • PAR 4/5 are important to maintain mutual antagonism; aren’t localized, are cytoplasmic
    • Image Upload 36
    • • PAR proteins are conserved & diverse; form distinct mutually exclusive complexes
  47. aPKC/PAR6/PAR3 and PAR1/PAR2 mutual inhibitory phosphorylations contributes to a dynamic mutual exclusion: is this important?
    Image Upload 37
    mutual antagonism established and then maintained: model for epithelial cells and roughly apply to migrating cells too (ex. neurons)

    • • top R: PAR 3; absence on L side of cortex, due to PAR1
    • -upon phosphorylation, PAR3 interacts with PAR5
    • -wherever you have PAR1 activity: it’s going to remove PAR3 form cortex, interact with PAR5, and move into the cytoplasm
    • • activation of aPKC results in local phosphorylation of PAR1
    • -PAR5 titrates both PAR1 & PAR3
    • -PAR1 more favorably binds to PAR1
    • • PAR4: releases PAR1 from PAR5 titration and allows it to return to the cortex
    • • this is how you get 2 mutually exclusive domains in the cell cortex; used to establish and maintain polarity
  48. the ________ complex is used to establish polarity in various asymmetric systems
    • aPKC-PAR complex
    • -important for epithelial cells and neurons
  49. Rho GTPases + other actin regulators have been studied using wound-closure assays
    • • take a plate with a population of cells that cover the plate; scratch it, and create a 'wound'
    • • cells at the margin sense there is no cell next to them, polarize toward 'wound' and fill the gap
    • -wound will close in ~3 hours
    • • can use this to quantify how fast wound area is reduced compared to mutants
    • Image Upload 38
  50. Dominant-active proteins induce different actin-containing structures
    • • mutant of small GTPases can be either dominant active, or dominant negative; unable to exchange GDPs or GTPs
    • -negative = constitutively bound to GDP (could still interact with GEF)
    • -acTive = constitutively bound to GTP (could still interact with GAP)

    • dominant-active RhoA: huge contractile stress fibers everywhere

    • dominant-active Rac: peripheral membrane ruffling

    • dominant-active Cdc42: filopodia develop
  51. traditional molecular functions of small Rho GTPases are coordinated for directed cell migration
    Image Upload 39
  52. FRET-based biosensors detect Rho GTPase activity in living cells
    Image Upload 40
    • • FRET: fluorescence resonance energy transfer
    • - when 2 molecules are close (ex. CFP and YFP) you can excite one w/ a specific wavelength (high energy, lower wavelength) and then look at the other's excitation
    • -energy comes from UV laser on CFP --> CFP gets excited --> energy is transfer to YFP --> YFP fluoresces

    • • when small GTPase is active, its able to bind to effector domain; that’s what’s used in unimolecules FRET sensor
    • •RBD (RhoA binding domain)
    • • if Rho isn’t active, there’s no incentive for fluorescence (the two complexes aren’t brought near each other)

    • • dominant active mutants have HIGH FRET intensity
    • • dominant negative mutants have low FRET intensity
  53. RhoA is activated at:
    the LEADING edge in spontaneous migration

    • -when a cell is treated with PDGF, concentration of RhoA is reduced
    • -when the cell is activated, there's a negative effect on RhoA (more according to the model)
    • -PDGF activates GEF which in turn activates Rac, which INHIBITS RhoA
  54. the activity of Rac (a small GTPase) can be manipulated in vivo using light
    Image Upload 41
    • • bind a constitutively active form of Rac & attach to it plant proteins that will fold in a way to block RAC activity
    • • however, when you shine light on plant protein, it changes its conformation, making it releases RAC
    • - this means constitutively active Rac is free to interact with effectors; plant protein doesn't inhibit in light

    •combination of FRET and photosensitivity study: RAC INHIBITS RhoA LOCALLY
  55. Relationship between membrane protrusions and small GTPase activity
    Image Upload 42
    • • researches focused on leading edge and broke it into small positions
    • -for each window, they measured activity of small GTPases and whether they're there @ time of protrusion

    • • *results*: RhoA activation correlates best with beginning of protrusion, while Rac and Cdc42 are activated later
    • -doesn’t boil down to Rac inhibiting RhoA...then
  56. phsophoinotisol's role in chemotaxes
    • • neutrophils (immune cells) show chemotactic behavior toward fMLP (produced by bacteria)
    • • phosphoinositides (phosphatidylinositol?): membrane phsopholipids; long fatty-acyl chain attached to hydrophilic groups

    • Study looked @ neutropils in the zebra fish (wounded with laser)
    • 1) In the WT, upon wounding, neutrophils just migrate to wound
    • 2) In LY294002 mutant: neutrophils wander and DON’T get to wound
    • -LY294002 is a chemical that reduces production of PiP3

    • determined that there's production of PIP3 at leading edge of migrating neutrophil; at the leading edge, part of what activates Rac (proliferates cell edge) is PiP3 kinase
  57. integrins
    • • Transmembrane proteins; heterodimerized alpha and beta chains
    • • basically, dimers have specificity for ECM matrix components
    • • cell type preference for a given matrix versus another will depend in part on the composition of integrins they express
  58. Image Upload 43
    • • actin filaments in leading edge propel cell forward
    • • contractile fibers in the cell cortex squeeze cell body forward
    • • stress fibers terminating in focal adhesions pull the bulk of the cell body up as the rear adhesions are released
    • • structure of the focal adhesions involves the attachment of the ends of stress fibers through INTEGRINS to underlying ECM
    • • focal adhesions also have signaling molecules important for locomotion
    • • dynamic actin meshwork in leading edge is nucleated by Arp2/3 complex
  59. cells crawl (make protrusions) using basal lamina (black) secreted by exoderm; use it as a substrate
    Image Upload 44
    • • basal lamina is COVERED with fibronectin
    • • in WT embryo; have all these cells, are mesoderms cells that have migrated and covered basal lamina; USE fibronectin to do this
    • • matrix (aka fibronectin) is: both informative and something to adhere to
Card Set
Meeting 25, 26, 27
ActinMyosin, MicrotubulesKinesins, CellMigration