Meeting 25, 26, 27

• 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

• 
• (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

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
• 

• • 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

• •g-actin: monomeric globular form
• •f-actin: fibrous form
• •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 strand
• -important because myosin ‘walks’ on actin in 36 nm steps

• Nucleation is the main limiting step in de novo actin polymerization
• -ugh I have no idea

• -another thing:
• 
• •critical concentration (C_c): concentration of G-actin monomers in equilibrium w/ actin filaments
• •when concentration of monomers (G-actins) is below C_c, 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 C_c

(+) end

• • (+) end: contains ATP-actin
• • (-) end: contains ADP-P_i-actin & ADP-actin

G-actin monomers are added 10x faster at the (+) end of filaments

• -critical concentration:
• C_c = (disassembly rate) / (assembly rate)
• 

• • treadmilling: mechanical force; how actin polymerization exerts force/moves
• - accomplished by addition ATP actin (+ end) coupled with removal of ADP actin (@ - end)

• 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
• -OVERALL: PROMOTES BOTH ASSEMBLY & DISSOCIATION

• •cofilin cycle: cofilin binds to ADP-actin and makes them fragment, enhancing DEpolymerization of (-) end
• -OVERALL: PROMOTES DISSASEMBLY

• *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 (C_c): ~0.2-0.3 µM

• • 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

• • 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⁺

• • 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

• • 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

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

• (1) Fimbrin: protein that creates a network
• 

• (2) Filamin: protein that creates a network
• 

• 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]

• (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
• 

•conclusion: sliding-filament assays show that the head+neck region is sufficient for ATP hydrolysis and actin-based motility

• the length of its neck; a longer neck means actins are carried faster
• 

• •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)

• 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

• • 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

• • 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

• • 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

• • 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

• • 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

• • αβ-tubulin dimers assemble into MTs when above C_c
• • above C_c, 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)
• • C_c for MTs = 10-20 µM, which is much higher than C_c (0.03 µM) of actin
• - regulation by MAPs plays a major role in inhibiting spontaneous formation of MTs

• 
• • at dynamics are at the + end

stochastic

• • 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

drug that gets rid of existing MTs

• • 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

• • 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

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

• • 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

• • 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

• 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

• • 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

• • 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-P_i bound
• -generating force for movement involves a change in orientation of the head relative to the stem, causing movement of the MT bound stalk

• made up of:
• • Arp1 capped by CapZ: binds cargo
• • p150^glued: attaches dynein to the dynactin complex; also contains an MT binding site
• • dynamitin: holds the two above parts together

• •vesicles coupled with kinesins: sent OUTward
• •vesicles coupled with dynesins: sent INward
• 

• 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

• 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

• • 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

• • localize at opposite poles of the fertilized egg
• • PAR 4/5 are important to maintain mutual antagonism; aren’t localized, are cytoplasmic
• 
• • PAR proteins are conserved & diverse; form distinct mutually exclusive complexes

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

• aPKC-PAR complex
• -important for epithelial cells and neurons

• • 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
• 

• • 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

• • 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

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

• • 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

• • 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

• • 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

• • 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

• • 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

• • 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