Cell Bio Quiz 6 Material

What was the experiment that used Chlamydomonas (Chlamy) to demonstrate that regenerating flagella grow from their tips and not from the base? Note: Chlamydomonas is a green algae

Flagella grows from tips Chlamy has 2 flagella that beat so algae can move -If you shear Chalmy in a blender, typically only one flagella breaks -then the longer flagella retracts and shorter flagella grows--> until they are the same size--> then they grow in unison to original length This is important to understand before actual experiment Now for the experiment: took 2 mating types of chlamy (one mating type had GFP labeled tubulin--> fluorescent microtubules in flagella and cilia and other mating type has no fluorescent microtubules) -then you blend nonfluorescent mating type and do blending experiment and you catch them in midway point where both flagella are still growing but not fully grown Then you take these nonfluorescent chlamy and mate them with fluorscent, intact flagella -when these cells mate--> fused cell with four flagella -fused cell now has a pull of GFP tubulin and it will get incorporated into regenerating flagella Then you can visualize where GFP tubulin is getting incorporated -you see fluorescent tips--> tells us flagella grow from tips

What is intraflagellar transport (IFT)? Why is it needed

For flagella to grow, all of that material that is required to grow flagella and cilia needs to be transported to the tip of flagella and cilia -there is lots of bidirectional that occurs to do this (called intraflagellar transport) Intraflagellar transport (IFT) involving flagellar-specific dyneins (IFT dyneins) and flagellar specific kinesins (kinesin-2) that carry packets (IFT particles) of materials out to tip (anterograde by kinesins) where flagellum is elongating and back to base (retrograde by IFT dyneins) during growth and maintenance

Describe the actoymyosin ATPase cycle and how that is related to the movement of actin

Here is ATPase cycle: Green subunit is G actin in actin filament that motor protein interacts w/ Motors are barbed end directed motors, but actin is what moves b/c motor protein is static and stuck toward barbed end 1. Myosin motor in prestroke state- ADP+P state -under this condition, myosin bounds weakly to actin filament 2. At some point, myosin will bind in correct orientation to actin subunit that is near and this binding--> promotes phosphate release --> key step to get myosin tightly bound to actin filament and you have power stroke where lever arms swings 3. You want to step on filament when head tightly bound to filament--> force generation and actin filament moves --b/c power stroke has forced actin to be displaced one subunit up--> ADP is released and now we are in empty state (motor not bound to nucleotide) --this is called rigor state (most tightly bound to actin) Summary: M or M-ADP bind tight (rigor), M-ATP or ADP+P bind loose, alternation allows to release and re-bind. Force produced when going from AMDP-->AMD (i.e., Pi release step). ADP released, new ATP binds-->head releases; when ATP hydrolyzed to ADP+P-->lever arm cocked, ready for another cycle.

Describe the kinesin structure (particularly kinesin 1) Compare it to the structure of myosin II

Kinesin I is the smallest known motor protein -it is packed, and very efficient -it is also a dimer--> two head/motor domains, neck linker (lever arm), a longed coiled coil region called stalk and tail region w/ light chains (Each heavy chain binds to light chain) that help kinesin I bind to cargo (like vesicles) -For kinesin 1, the lever arm equivalent is the neck linker that performs power stroke Comparison of myosin II and kinesin I: -kinesin heads bind to beta-tubulin-->need to take large steps compared to myosin II. -myosin motor head is much larger, but core part is very similar to kinesin I

Describe movement by pseudopod extension

Locomotion by pseudopod extension (ameboid movement): growth cones via pseudopods and filopodia, keratocyte via pseudopod. We've seen lots of this already with white blood cells, phagocytosis, etc. Actin filament growth associated with plasma membrane pushes it forward = leading edge. Leading edge binds to substrate via adhesion proteins. Myosin-based contraction in rear that releases the "tail" end of the cell. Put differently, here is the Basic idea of ameboid motility: lamellapodia stretches PM (due to forces), further pushing PM out then PM rebinds to substrate via adhesion proteins, then back end of cell aka the tail retracts and repeat

How does the dynein structure generate force?

Mechanical element of dynein is linker region that shifts position as a function of ATPase cycle in triple AAA 1--> weakly and strongly bound step in binding region -aka linker region is responsible for power stroke -step size of dynein is variable (8-24 nm) and can side step The ATP-dependent conformational changes are thought to be communicated to the MT-binding site (that is located far away from AAA1) by dynein-specific structural motifs like the C-term, strut and coiled-coil stalk (that has the MT-binding domain at its tip).

Last unit, we talked about the experiment done where myosin heads were mixed with actin filaments, and this is what gave rise to the arrowhead terminology. Was this experiment carried out with ATP present? Why or why not?

Mmyosin heads bind to actin very stably in the absence of ATP (so this experiment was done in the absence of ATP) -if you did this experiment in presence of ATP--> no binding of myosin heads to actin filament Basis for arrowhead configuration: myosin heads in their M or M-ADP configuration bind tightly to actin filament

What is the directionality of the motor proteins

Motor proteins have directionality, so polarity of tracks becomes important Directionality (conferred by filament polarity) -Myosins generally move towards barbed end of actin (exception = myosin VI) -Kinesins generally move toward plus-end pf microtubules (exception = kinesin-14 family) -Dyneins move toward minus-end of microtubules.

Describe the basics of how motor proteins work?

Motors are mechanochemical enzymes -they couple chemical energy (ATP hydrolysis) to mechanical force generation (in the form of conformational changes of motor proteins) Basic steps: Motor domain needs to bind to track--> conformational change to exert force on filament/pull on it--> motor protein needs to release the filament--> rebind--> repull--> release and so on Or put another way: • Mechanochemical enzyme: energy from ATP hydrolysis drives conformational change-->movement. • Head/Motor domain: binds track and hydrolyzes ATP • Tail domain: binds cargo (or itself as in myosin II) -region in b/w of motor protein is called stalk region that varies greatly in length • Basic Steps: Grab, pull, release and repeat • If motor anchored, cytoskeletal polymer moves; if cytoskeletal polymer anchored, motor + cargo moves.

In skeletal muscle, what is myosin II attached to? How do the motor domains of myosin act in relation to each other?

Myosin II in skeletal muscle is a part of a structure called the thick filament with many myosin motors that have self-associated (ex of macromolecular assembly) Although myosin II is a dimer (with 2 motor domains), each motor domain acts independently of the other (i.e. there is no communication b/w two motor heads)

Myosin makes up a large superfamily. How are they all similar? Different?

Myosin family tree - lots of them; they all have similar head domains (whether monomer or dimer), but vary in tail domains.

Describe the structure of myosin

Myosin is a dimer There are two head domains (b/c it is a dimer) -Heads: N-term of heavy chain has catalytic domain with actin-binding site and ATP-binding/hydrolysis site. In some myosins, this is followed by a long coiled-coiled and then tail domain.

Describe the invitro motility assay with myosin and actin.

Myosin motors are attached to a glass surface and you provide fluroescently labed actin filaments so you can visualize them And if you supply ATP, then myosin motors will bind and do powerstroke to displace actin filament and you can visualize it Actin filaments move unidirectionally b/c all of myosins moved toward barbed end -so the pointed end is the leading end

Where do the motor proteins originate from? What are they similar to?

Myosins and Kinesins derived from primordial NTPase, with their fold also resembling Ras-type GTPases; dynein a different idea (AAA ATPase). In both kinesins and myosins, the core part of their motor domain (i.e. the engine) is related in sequence to GTPases (all originated from last common ancestor, and then there was duplication and divergence--> some became GTPases, some became ATPases (myosin and kinesin use ATP as fuel) Dynein motors are unrelated to other motor proteins (it is an AAA ATPase like katanin)

Describe the strength of myosin binding to actin during different types of the actomyosin TTPase cycle?

Note: this is associated and weak and binding of myosin II to actin 1. When myosin not bound to nucleotide or is in the ADP only state--> myosin binds tightly to actin 2. when myosin is in ATP or ADP+P state--> myosin binds weakly to actin

What is a popular model for intracellular movement and why?

Popular model is axon b/c it is a long projection and so efficient movement along axon is critical for well being of neuron

What does position of motor domain of kinesin with respect to the polypeptide chain tell you?

Position of motor domain of kinesins w/ respsect to polypeptide chain is very informative -motor domains can either be on N terminus or it can be on the C terminus or internal -this is informative, b/c if motor domain is at N-terminus, these tend to be + end directed kinesins (we saw this w/ kinesin I) --if motor domain is at C-terminus, kinesins are - end directed (ex. Kinesin 14) --if motor domain is internal, kinesins work to destabilize microtubules (ex. Kinesin 13)

What are primary cilium? What function do they serve? What happens if there are defects in IFT?

Primary cilium: MT doublets but no outer/inner dynein arms or central pair (9+0), so immotile. On most differentiated cells. Often serve as sensory organelles; olfactory and rod/cone receptors are specialized examples. -Nearly all cells have these sensory cilia (non motile b/c they lack central pair of microtubules) -Assembly of these cilia also need IFT to grow Defects in IFT -->can't construct cilia, including primary cilia -->diseases e.g. vision loss, polycystic kidney disease.

What is the rigor state for myosin II?

Rigor state- when myosin II is tightly bound to actin -happens when myosin is not bound to a nucleotide (Not ATP or ADP) When animals die, they get stuck happens when animals die b/c there is no ATP synthesis when we die--> myosin stuck in empty step

Does the speed of cell movement vary a lot? If so, by how much?

Speeds of cell movement vary a lot depending on type of motility (cilia or pseudopod) and cell type. -velocities of moving cells vary by >4 orders of magnitude Fast movers are cilia/flagella powered cells -ex. sperm In our body, WBCs move fast Nerve cells are slowest b/c they not only need to move to project axons to form synapses, but they have to know where to go

What is the dendritic actin model for pseudopod motility?

Steps: 1. Cell decides which movement to move (ex. Leukocytes perceive bacteria derived chemoattractins)-->signaling events that activate WASPs--> activate Arp2/3 complex in a localized manner--> 2. Activated Arp2/3 causes a huge burst of branched actin assembly, their growing barbed ends push on the plasma membrane extending it 3. Capping proteins keep growing barbed ends short to enable pushing force (because longer actin filaments are floppy). 4. Cofilin cuts older actin filaments at base--> those subunits become reactivated (i.e., bind to ATP) by Profilin and are used to sustain the continued branched actin formation. Repeat again and again-this is cyclic

Describe the structure and key features of dynein.

Structure: -One heavy chain has six ATP binding sites (AAA domains) but only one is important for motility (AAA1) --b/c head regions aren't tightly connected to each other--> dyneins take side steps -Long narrow stalk projects from AAA ring that has MT binding site on its tip. -The linker domain that drapes across the AAA ring is the lever arm that moves during the ATPase cycle. Dynein consists of 2 heavy chains, multiple intermediate and light chains—big multi-protein complex! Microtuble binding sites are far removed from engine (ATP binding site)

Describe what is meant by filopodium and pseudopod/lamellum? What generates these?

Terminology: Filopodium = narrow extension (formin involved) -Finger like projections due to linear bundles of actin filaments (formins concentrate at tips of filopodia--> linear filaments) Pseudopod or lamellum = broad extension (generated by Arp 2/3 complex) -Lamellipodia are thin, sheet-like membrane protrusions found at the leading edge (front) of motile cells

What is the role of actin crosslinking? What happens if you knockout actin corsslinking proteins?

To make actin mechanically resilient, you need to keep them short and crosslink it If you knockout filamen (crosslinking protein), then lamellopodia don't form effectively -crosslinking by filamin is important for forming proper lamellaopodia Actin cross-linkers like filamin help maintain integrity of pseudopods.

Which motor protein is processive: myosin II or kinesin? Why?

Unlike myosin II, kinesin is very processive (takes lots of steps upon binding to microtubule); this is b/c two heads are coupled

What is the function of myosin V? Is it processive? How big of a step does it take and why is this significant?

Vesicles etc. can also be transported along actin tracks via myosin V (a processive myosin unlike Myosin II) to achieve intracellular transport. Mysoin V is processive -myosin V is very interesting b/c it takes a 36 nm step, and this is interesting b/c 36 nm matches helical pitch of actin --> motor stays on same side of actin so it doesn't swirl around actin

What kinds of motors do intracellular, membrane-bound cargo tend to have?

We have these diff tracks (actin filaments for myosin and microtubules as tracks for dyneins and kinesins) and lots of diff cargo It turns out that many of membrane bound cargos have all types of motors attached to them (i.e. cargo can have all 3 motors) -cells coordinate motor activity to regulate directionality of cargo transport

What happens to myosin's structure (myosin II) during the power stroke?

When myosin in ADP+Pi state, it is bound to actin at an angle When Pi is released--> dramatic conformational change (the power stroke) that generates force of myosin motors -involves significant swiveling of myosin's lever arm -this is what generates force on actin

What happens when you put microtubules on a glass surface and couple this with kinesins bound to bead (with ATP present)? What happens if you do this same experiment in the presence of myosin II?

You can also couple kinesins to a mock cargo (like a bead) that you can monitor easily How it works: -take microtubules and put them on a glass surface (track is immobile) -then you put beads labeled w/ kinesin attached and supply ATP--> bead moves as motor walks on protofilament (Now bead moves toward + end) You can also do this second experiment with myosin (but w/ myosin II, not much would happen b/c bead would fall off after indiv steps but w/ myosin V, bead would be displaced toward the + end)

How is dynein different than kinesins?

-kinesin stays on one protofilament and stays on it -dynein is a larger, floppier motor (Dynein is huge compared to kinesin) -dyneins are a part of a completely different family from myosins and kinesins

What are the two ways to measure kinesin motility?

1. In vitro motility assay aka gliding assay 2. You can also couple kinesins to a mock cargo (like a bead) that you can monitor easily

What are the 4 ways to get intracellular movements

Four ways to get intracellular movements: 1) transport cargo along MT by kinesin or dynein 2) cargoes linked to polymerizing/depolymerizing MTs (we will see an example of this during mitosis next week); 3) transport cargo along actin by myosin (where cargo can be another actin filament -->contraction); 4) cargoes linked to polymerizing/depolymerizing actin

Kinesin makes up a large superfamily. How are they all similar?

Kinesins are part of a super family (hundreds of kinesins encoded by eukaryotes) Usually homodimers. Most move in the anterograde direction (kinesin-14 moves in the retrograde direction).

What is the Myosin II ATPase cycle in the absence of actin? Presence of actin?

Without actin: 1. ATP binding: If you have free myosin and you provide ATP, it binds readily to myosin (way higher rate than reverse reaction, not a RDS) 2. ATP hydrolysis: Myosin also readily hydrolyzes ATP (this is a reversibly reaction) 3. *Phosphate release: Release of phosphate is extremely slow (RDS) -When phosphate is released--> myosin+ADP state 4.ADP dissociation: ADP readily dissociates from myosin--> empty myosin that can bind to fresh ATP If actin is present: 1. ATP binding: Myosin bound to actin will readily bind to ATP (ATP binding is still not RDS) 2. ATP hydrolysis: actin bound myosin can still readily hydrolyze ATP to ADP and P 3. Phosphate release: Big/only difference: phosphate release is a lot faster when myosin is bound to actin than in the absence of actin 4. ADP dissociation: myosin motor in ADP state w/ actin--> ADP readily released

What are distinctive features/important points to remember about the kinesin ATPase cycle?

1. 2 motor domains work together in a cooperative fashion -kinesin moves along single protofilament -processive, coordination between heads leads to long movements (100s of steps) in contrast to myosin where heads act independently 2. They have reciprocal affinities -At least one head is tightly bound in any point of ATPase cycle -They have motor efficiency b/c they remain on track (One head is always on track so rebinding not needed) 3. One ATP is consumed/step 4. Large step size for a small head

Describe the kinesin ATPase cycle in terms of how it causes motion

1. Leading motor head is bound to beta tubulin (in MT state) -Lagging head is in ADP bound step (we will track this lagging head 2. When ATP binds to leading empty head--> induces stronger binding and neck linker will zipper along motor domain 3. This zippering along motor domain--> repositions lagging domain in forward position (now lagging head is leading) -by virtue of bringing ADP bound kinesin next to the next tubulin subunit, it gives this head chance to bind to beta tubulin--> ADP released and head binds tightly to microtubule track In mean time: new lagging head will hydrolyze ATP, and Pi is released--> ADP state kinesin binds weakly to microtubule and lets go One head is always tightly bound and one is always weakly bound Summary: Two heads influence each other: one binds tightly, the other binds loosely. ATP binding to the leading head causes the neck linker to zipper along the head-->causing the lagging head (that's loosely bound or unbound to microtubules) to swing forward.

Describe the Kinesin ATPase cycle

1. Microtubule+kinesin (strongly bound) readily binds to ATP--> microtubule+kinesin+ATP (kinesin tightly bound to MT) -ATP binding step is when the power stroke occurs (power stroke is when motor binds tightly to track) 2. Kinesin readily hydrolyzes ATP to ADP+P--> MT+Kinesin+ ADP+ P (kinesin weakly bound to MT) 4. Pi is readily released (not rate limiting as is the case with myosin)--> MT+kinesin+ADP (kinesin very weakly bound to MT, prone to breaking) 4. ADP release is RDS -MT bound to kinesin has modest influence on ADP release rate.

What are the kinds of motor proteins and what do they use as cytoskeletal tracks? What do all motor proteins have in common?

Actin based motor protein= myosin Two types of MT based motor proteins= kinesin and dynein They all use ATP

What is the motor protein associated with axonemes called and how does it work?

Axonemal dynein motors bound to A tubules of doublets on the outside -Axonemal dynein walking on side tubule--> sliding movement--> causes cilia and flagella to move -Dyneins walk towards minus end of adjacent doublet leading to filament sliding which is somehow converted to bending and bend propagation that is not fully understood. Doublets slide with respect to each other (dynein from before was cytoplasmic)

What are the aspects of cilia/flagella structure to remember?

Axoneme = cilia/flagella without the surrounding membrane. -Nine outer doublet microtubules + central pair of regular microtubules (9+2) -Microtubules usually very dynamic, but MT in cilia and flagella are very stable b/c they are post transitionally modified (w/ acetyl groups)--> fixed in length >200 other proteins including axonemal dynein motors bound to A tubules of doublets on the outside

Describe the in vitro motility assay for kinesin

Binding assays take fluorescent microtubules, motor domains, and nonhydrolyzable ATP--> kinesin locked irreversibly to microtubule -you do this in presence of ATP (unlike with actin and myosin b/c myosin is tightly bound to actin, aka rigor state) but with kinesins, rigor state is ATP state -Kinesins decorate each protofilament (they bind to one head per beta tubulin) -kinesins walk on a single protofilament If you incubate MT w/ full kinesin motors along w/ ATP and put kinesin motors on a glass surface, they will randomly bind w/ one of dimers, and once they land on protofilament, they stay on protofilament -kinesin back motor domain needs to take larger step to get to 2 beta subunits later that is unoccupied (this ends up being 8nm for the center of kinesin, but 16 nm for a single kinesin head—distance from one beta tubulin to the next is 8nm) -kinesins move toward + end, but kinesins bound to glass--> - end leads in these assays

What motor protein mediates anterograde trafficking? Retrograde trafficking?

Centrosome acts as a MTOC b/c it has lots of gamma-tubulin ring complexes -therefore you have microtubules emanating from that point - end is at centrosome and + end radiates outward Anterograde trafficking mediated by + end directed kinesin and retrograde trafficking is mediated by dyneins

What do cilia and flagella do? What happens if cilia are nonfunctional?

Cilia/Flagella: Move organism or, if cell tethered, move fluids (e.g. trachea and bronchi). Kartagener's syndrome = inherited disorder when ciliary motility impaired: immotile sperm, respiratory problems. - nonfunctional, immotile cilia -results in sterility (b/c sperm can't move in females and in females, cilia in oviduct can't move--> can't create fluid flow to steer sperm) -also results in respiratory problems b/c cilia can't move mucus

What does dynein frequently associate with? Describe the characteristics and functions of this protein

Dynein frequently requires association w/ dynactin, which is also a large multiprotein complex Functions of dynactin: 1. Recruits dynein to cargo (dynactin binds to cargo like organelles and recruits dynein to cargo so cargo can move in retrograde direction to - end of microtubule) 2. Dynactin improves processivity of dynein (part of dynactin has microtubule binding ability--> another binding site that keeps motor close to track) "Actin" part of dynactin is actually actin-related protein Arp1 (the other Arp besides Arp2/3 associated with actin polymerization) and there's a protein called p150 glued that links dynactin to dynein.

Which states is kinesin most tightly bound to microtubules? Least tightly bound?

Empty Microtubule-bound kinesins and ATP bound MT-kinesin bind tightly to microtubules (ATP is rigor state for kinesins) -ATP is rigor state for kinesins Kinesins bound to ADP+Pi bind weakly to microtubules, and ADP alone bound to kinesins is very prone to release b/c it is bound the weakest to microtubules Summary: K alone or with ATP binds strongly to MT; K in ADP+P form binds weakly; K-ADP binds very weakly.