Biol 3040 --> Exam 2: 16C, 16D, 16E

Formin

A dimer that is bound to the plasma membrane Has whisker like extensions terminating in a pair of clamping devices that can interact with the growing plus end of an actin filament right near the plasma membrane Promotes the addition of G-actin to filaments in concert with profilins (profilin bound to the whiskers) Whiskers (unstructured protein domains) are also staging areas for profilin near the plus end Mechanism: Clamp ratchets back and forth Recruiting fresh ATP charged G-actins to the position near the plus end Scaffolds a socket to plug the G-actins in and dramatically increases the rate of polymerization at the plus end thats ATP bound This how you get explosive polymerization

Lamellipodium

A sheetlike extension, rich in actin filaments, on the leading edge of a motile cell or growth cone. Body of the lamellipodium supported by perpendicular arrays of actin fibers Branched actin network

Latrunculin

Actin Chemical Inhibitor Effect on Filaments: Depolymerizes Mechanism: Binds free available actin subunits and inhibits polymerization

Cytochalasin B

Actin Chemical Inhibitor Effect on Filaments: Depolymerizes Mechanism: Caps filament plus end

Phalloidin

Actin Chemical Inhibitor Effect on Filaments: Stabilizes by preventing depolymerization Mechanism: Binds along filaments Used to stain actin filaments in fixed cells

Profilin

Activates ADP for ATP exchange on G-actin, does this by promoting the ejection of ADP and the uptake of fresh ATP F-actin binding releases it Docks to NPF and accelerates polymerization at the plus end

They all have 2 different actin binding domans

All the actin cross-linking proteins have one thing in common, what is it?

Myosin

Allosteric motor protein: ATP hydrolysis drive unidirectional (+ end-directed) movement along a polar filament Actin-specific motor protein and requires actin for ATPase activity

Myosin I

Anchors actin filaments to the plasma membrane and move filaments relative to the membrane Gives cells the shape they have because of the movement of cortical actin monomeric protein

Tropomyosin

Binds actin and blocks it ability to bind myosin It's a long bar like protein, and it prevents myosin from having access to the actin.

Cofilin

Binds along the sides of ADP-bound actin filaments twisting and destabilizing the structure causing breakage and depolymerization

Stathmin

Binds to 2 MT dimers and decrease available subunit concentration Inactive when phosphorylated Regulator of dynamic instability Important for the development of the neurons of the amygdala -- fearless mutant mice Teacher: It can wrap around a pair of dimers, and in that configuration they can't be used to polymerize. So it drops the available subunits. Most similar to thymosin in actin

Thymosin

Binds to free g-actin and decreases free g-actin pool/availability thus depolymerizes the actin filament Opposite of Profilin

Cell cortex

Branched and unbranched filament network

Troponin C

Calcium binding protein that works to move tropomyosin off of actin

Tropomodulin

Caps actin filaments at the minus end Prevents polymerization and depolymerization

Cap Z

Caps actin filaments at the plus end Prevents polymerization and depolymerization

9 sets of Triplet rings with no microtubules on the inside 9 + 0 orientation

Centrioles and basal bodies microtubule structures are ...

Doublet ring, fusion of 2 microtubules Nine of these doublets around a circle and then two singlets that go down the middle. 9 + 2 arrangement

Cilia and Flagella microtubule structures are ...

Myofibrils

Contractile units of muscle cells The rings Dense with actin and myosin

Alpha-actinin

Dimer Bundling protein Holds actin fibers parallel and a little but more spaced out compared to fimbrin Forms contractile bundles that are using the stress fibers to pull the back of migrating cells Allows for loose packing that allows myosin II to enter the bundle

Filamin

Dimer Gel-forming protein Holds actin fibers perpendicular to each other Plays major role in lamellipodium

yes

Does MT organization vary widely among cell type?

Catastrophins

Enhances catastrophes, more regular Kinesin-13 is a type of this

Arp2/3 complex

Enhances polymerization by nucleation and remains bound to the minus end of actin filaments Can be considered minus end capping protein Can build branched arrays of actin

Are less stable and favor depolymerization Also induce a bend in the protofilament

GDP bound subunits are what and favor what and induce what?

EB1

Highly conserved plus-end-tracking protein across all eukaryotes Special affinity for the GTP cap Binds to the GTP cap. And whenever that GTP cap is present, no matter how fast it's streaming forward, it stays associated. It seems to crawl forward staying on a cap and it helps to recruit other TIP proteins, including the catastrophins and the map 215 like proteins. It's an anchor to help guide them to the to the cap.

They are capped at both ends, CapZ at plus end/Z-disc and tropomodulin at minus end

How do microfilaments maintain length within the sarcomere?

Attached: At the start of the cycle, a myosin head lacking a bound nucleotide is locked tightly onto an actin filament in a rigor configuration. In an actively contracting muscle, this state is very short-lived, being rapidly terminated by the binding of an ATP molecule. In the absence of ATP, myosin and actin are locked together Released: A molecule of ATP binds to the large cleft on the "back" of the head (that is, on the side furthest from the actin filament) and immediately causes a slight change in conformation of the actin-binding site, reducing the affinity of the head for actin. When ATP is added, it enter the binding cleft of the motor subunit which induces a conformational change that disrupts the protein protein interaction between myosin and actin. It causes the release. ATP binding causes of the release of myosin from actin. Cocked: ATP binding triggers a conformational change in the cleft that leads to a rotation in the converter domain, causing the lever arm to swing out and the head to be displayed along the filament by a distance of about 5 nm. Hydrolysis of ATP occurs, but the ADP and inorganic phosphate remain tightly bound to the protein The energy from hydrolysis is absorbed by the protein and moves this lever arm forward towards the plus end and it becomes cocked, becomes locked in that position. And the thing that's holding it is this phosphate that's kind of the wedge that's keeping it open. Re-Binding and Power Stroke: The myosin head binds weakly to a new site on the actin filament, causing the release of the inorganic phosphate produced by ATP hydrolysis, concomitantly with the tight binding of the head to actin. This release triggers the power stroke--the force-generating change in shape during which the head regains it original conformation. In the course of the power stroke, the head loses its bound ADP, thereby returning to the start of a new cycle When that subunit in this caucus state comes in to lose or just gentle contact with another actin subunit. It has low affinity in this allosteric conformation. As it binds, that is recognized that protein-protein interaction induces the ejection of the phosphate that wedge pops out. And now that the energy can be released in the power stroke and it returns to its previous conformation in the process of returning, ADP is going to be ejected. So first phosphate out, then ADP out then we are so everything look okay here we're back to where we started.

How does myosin move the actin filament/how does it generate force against the actin filaments?

Express a protein called Act A that binds to Arp2/3 complexes These are then gonna be pulling in actin into polymerization on to nucleated fragments and depolymerizing at the back

How does the bacteria Listeria move?

The Gamma Tubulin ring complex is made up of a series of pairs, dimers. And each one can hold each dimmer can hold two gamma-tubulins. So basically we get there's going to be seven of these pairs dark blue, light blue, dark blue, light blue all the way around. Each with two gamma-tubulins. This mean 14 gamma-tubulins are being coordinated around this ring. The thing that happens here now. The Gamma-Tubulin ring complex is kind of a spiral staircase and it overlaps by one. That's where it gets the strength to. Maybe you can do this better than me to connect and become a good template. That's the perfect diameter to then build off of. But since we have 14 minus the one overlap that gives us 13. Docking sites in a perfect circle. And that's why we get a 13 proto filament circumference microtubule. Where that overlap is, we can actually visualize a seam in the side of a microtubule where the subunits meet. Generally beta laterally binds to beta and alpha to alpha, but at the seam they're staggered by one.

How does the gamma-tubulin ring complex function to nucleate and form a MT?

Myofibrils are encased in a sarcoplasmic reticulum A nerve impulse causes binding of neurotransmitter on the muscle cell membrane and opens channels that lead to depolarization of the membrane Depolarization travels to the T-tubules and triggers Ca2+ channels to open in the sarcoplasmic reticulum, releasing Ca2+ throughout myofibrils Ca2+ changes the conformation of troponin and tropomyosin, allowing myosin to bind to actin Teacher: Myofibrils are surrounded by a structure called the sarcoplasmic reticulum. They are closely in contact with plasma membrane derived tubes called transverse tubules that permeate the inner spaces of muscle cells. Basically making the plasma membrane continuous with the inside of the cell and the extracellular space is continuous with the inside of these tubes. Extracellular is rich in calcium and the sarcoplasmic reticulum is rich in calcium. And these transverse tubules, as they go through, they're in close contact with the sarcoplasmic reticulum. And so that comes when you have an action potential mediated by a neuromuscular junction. Voltage gated channels on the t tubules can open. They release a small amount of calcium, but they are in contact with calcium release channels on the sarcoplasmic reticulum that release a bunch of calcium. And that calcium can then bind to. Troponin C, that is a calcium binding protein that works to move tropomyosin off of actin. As soon as that calcium floods in the troponin complex uses calcium bound state to have this inhibitory protein move the t sub unit and that moves tropomyosin out of the way and the contraction can happen.

How is muscle contraction regulated?

13

How many protofilaments are arranged to make a hollow microfilament?

Kinesin-13

It's a motor protein, but the motor domain is right in the middle of the protein. It clamps down on microtubules and uses that motor activity ATP hydrolysis to pull a protofilament out of the tube. Induces catastrophes and depolymerization.

Nocodazole

Microtubule Chemical Inhibitor Effect on Filaments: Depolymerizes Mechanism: Binds free tubulin subunits

Colchicine

Microtubule Chemical Inhibitor Effect on Filaments: Depolymerizes Mechanism: Caps both filament ends

Taxol (paclitaxel)

Microtubule Chemical Inhibitor Effect on Filaments: Stabilizes Mechanism: Binds along filaments

The ER network

Microtubules are necessary for the organization of what?

Microtubule Plus End-binding Proteins (+TIPS)

Modulate microtubule polymerization/depolymerization dynamics Mediate attachments to membranes and other proteins Includes MAP 215, kinesin-13, and EB1

Fimbrin

Monomer Bundling protein Holds actin fibers parallel and close Found in filopodium and microvilli Allows for tight packing that prevents myosin II from entering the bundle

singelt

Most microtubule structures are ...

multi

Muscle cells are _____ nucleated

Amoeboid Cell Migration

Neutrophil chosen mechanism of movement Ratchet mechanism-> moves something in one direction and it can exert force and it can't really be reversed easily Actin polymerization creates force by each filament being like a tiny spring that is continually bent and deflected away from the membrane by random thermal forces When the filament bend far enough away from the membrane to permit the addition of new subunits, it grows longer When it bends back towards its equilibrium position, this longer filament pushes against the membrane Has pseudopods

Augmin

Nucleation and Branching Binds to the side of one microtubule, and then it's going to nucleate, a new microtubule in a branched conformation. Interacts with the Gamma Tubulin ring complex and causes branching There is no contact between original microtubule and the branched microtubule. Reminiscent of Arp2/3 complex in actin

MAP2

Parallel Bundling In the cell body Creates a broader bundling network.

Tau

Parallel bundling Diagnostic protein in Alzheimer's disease. Abundant protein in axons and it can form an aggregate. Found in axonal projections when it's non phosphorylated, when it's phosphorylated, it can move into the cell body.

Mesenchymal Cell Migration

Protrusion of the leading edge is driven by actin polymerization which is also forming focal adhesions Unpolymerized actin is being swept forward via cytoplasmic streaming to these forms of attachment as free subunits are being concentrated, there is enough to maintain polymerization, satisfying the critical concentration At the back, actin is below critical concentation therefore disassembling and pulling the back of the cell off the surface and breaking those connections is dependent on myosin II which is working to pull and gather up the back of the cell

Nebulin

Regulates microfilaments length This green helical protein has a dimer called this term and provides a measuring stick for the length of an actin that is perfectly suited to be placed in a sarcomere.

Titin

Regulates the position of thick myosin filaments Attached to the bare zone, to the thick bipolar filament on both ends, and that keeps the thick filaments centered within the SARCOMERE and so that it doesn't lose grip to actin on one side or the other.

Katanin (Japanese for sword)

Severs MTs Must slice across 13 bonded surfaces 2 subunit complex: Small subunit conducts ATP-dependent severing Large subunit slides to the minus end and severs kinetochores MTs in metaphase of mitosis it can bind to microtubule and slide towards the minus end and when it gets there it can't go any further. So it exerts its severing activity and it can disconnect minus ends from organizing centers, which becomes important.

XMAP215

Suppresses catastrophes, less regular Phosphorylated upon entry to mitosis supports spindle formation Binds near the end it stabilizes the the tip by its very binding. But it also has these whiskers with high affinity for subunits that concentrate them in the local area and facilitate their addition

Spectrin

Tetramer Gel-forming protein Creates much larger structures of actin in the cortex of the cell, supporting the plasma membrane

filopodium

Tight parallel actin bundle at the leading edge of a motile cell or growth cone. A thin projection of the plasma membrane supported by actin bundles. Slender structures that need a lot of support

Sarcomere

Within each myofibril are repeating chains of contractile units Contain parallel arrays of thin actin microfilaments and thick myosin filaments

Stress fiber

contractile bundle Parallel bundles but more spread out compared to filopodium

Provides ATPase activity and motive force Is the motor subunit

What does the head (globular) of myosin II do?

Binding site for light chains, which regulate the activity of the heavy chain head

What does the neck (alpha-helical) of myosin II do?

activates it

What does the nucleation promoting factor (NPF) do to the Arp2/3 complex?

Contains "cargo" -specific binding site, therefore determines the specific function of the myosin molecule

What does the tail (coiled-coiled, varies) of myosin II do?

Both alpha and beta-tubulin bind GTP, but GTP hydrolysis only occurs on the beta-tubulin

What is bound to GTP in microtubule subunits and what hydrolyzes GTP in the subunit?

The motor domain

What is conserved in myosin?

Describe plus ends of MTs Not all MTs behave identically under identical conditions Occurs at concentrations near the critical concentration Is due to (slow) polymerization when GTP-tubulin is present at the + end of MT's and (rapid) dissociation when GDP-tubulin is the plus end Can be regulated in cells (increased during mitosis, and decreased in a differentiated state, such as in a cilium)

What is dynamic instability, and what undergoes this process, and can it be regulated?

alpha/beta dimer

What is the basic building block of microtubules?

GDP subunits pulled out can be replaced with GTP subunits in vitro, creating new cap opportunities

What is the explanation of katanin mutants don't increase the number of MTs when that is expected but rather MTs increase in knockouts?

Arp2/3 complex is the facilitator of rapid growth at the leading edge

What is the facilitator of rapid growth at the leading edge?

Plus end is fast Minus end is slow

What is the fast and slow growing end of microtubules?

Exposed beta-subunits are at plus end Exposed alpha-subunits are at minus end

What is the plus and minus end of microtubules?

Centrosome

What is the prominent MT nucleation site?

9 + 0, 9 sets of triplets with nothing in the middle. However is not made out of microtubules but rather out of SAS-6

What is the structure of a centriole?

A protein complex containing gamma-tubulin

What nucleates MTs?

Pericentriolar material

Where are the gamma-tubulin ring complexes located?

Next to plasma membrane because it is a plasma membrane associated protein

Where do you find the NPF?

Minus ends generally remain associated with Microtubule Organizing Centers (MTOCs)

Where is nucleation most common in microtubules?

Myosin V

Transports organelles along actin filaments Takes steps towards the plus end and at the cargo binding side its gonna be attached to vesicles and organelles, its a + end directed motor Exerts a lot of force, but doesn't go that far dimer

Cofilin is at the back of the leasing edge of a mesenchymal cell

What actin-binding protein is at the back of the leading edge and at the back of a mesenchymal cell, and trails Arp2/3?

Rapid growth with GTP-capped end Random loss of GTP cap Catastrophe happens and rapid shrinkage then occurs Regain of GTP cap b/c a lot of free subunits now available aka Rescue Rapid growth with GTP-capped end again

What are the component activities of dynamic instability?

Contraction of muscle cells/tissue In nonmuscle cells: Cytokinesis Apical construction of epithelial cells and folding of epithelial sheets into tubes, e.g., neural tube formation Cell locomotion (also involves localized polymerization and depolymerization of actin)

What are the contractile function of actin and myosin?

Mesenchymal Cell Migration Amoeboid Cell Migration Blebbing Cell Migration

What are the three modes of actin (and myosin) dependent cell motility

Myosin I and V

What binds to membranes though their tail regions and are important for vesicle movement

Create a stabilizing cap and individual dimers can be added all the way around the 13 proto filaments, they're added cooperatively and you get rapid growth. Creates linear protofilament

What do GTP bound subunits create

Sculpted actin structures in front for force Focal adhesions via integrins below Myosin dependent contraction at the back which pulls the cell together and also helps push free actin subunits, sweeping them forward

What do the three modes of actin (and myosin) dependent cell motility have in common?

MTOCS tend to stabilize MT minus ends, so most instability (polymerization/depolymerization) occurs at plus ends

What does MTOC do to the minus and how does this affect the plus end?