Lecture #11 - Filaments

Question 1

Why can things stay on top of a cell

Answer 1

Because of life at low-rendels number molecules can stay on top of the cells instead of only falling to bottom

Life at Low Rendels number = viscous/elastic components of the cell dominate over inertial forces (gravity)
- Inertial forces are minimal compared to viscous forces

Question 2

What molecules lets out body move

Answer 2

Filaments + motor proteins

Motor proteins = use filaments as tracks to pull on ad walk across

Question 3

Filaments

Answer 3

Filaments = structures/machinery that allow cells/organisms to move and change shape

Example of movement:
1. Nuerons crawl to reach synapse
2. Ameoba – Cells change shapes and send out protrusions over and over

Question 4

Form and function

Answer 4

Form is important for function –> if something does not have the right form then it is hard to function

Examples of cell forms (structure):
1. Neurons have cell body + dendrits + have long axons
2. Budding yeast - stick out a bud
3. Fish keratyocye forming lameli so it can now glide across surface
4. Microvilli in intestine have actin filaments to create fingers that stick out to increase the surface area to be able to absrobe nutrients

Question 5

Cytskelaton at the organism level

Answer 5

At the tissue/organ/organ system/organism level the cytoskeleton is important

Example:
1. Blood pressure –> cytoskalaton machinery that can sense and generate mechanical stresses that squeeze blood
2. Bones breaking –> use the bone when it is healing because to pressure on the fluids near bone the fluids go into the bone –> osteocytes sense when fluid goes into the bone and help guide the deposition of bone matrix
3. Skeletal muscles to contract and relax –> Governed by actin and myosin meshwork
4. Sound –> stereocillia (cytosklaton) convert the mechanical wave of sound to an action potential so you can hear

Question 6

Right handed vs. left handed helix

Answer 6

Two start helix (actin) = right handed

Coil coil (Microtubules) = left handed

Question 7

Microfilaments (Overall)

Answer 7

Function – Structural support + mechanical support + tracks for myosin based contractility and motility

Components – Actin monomers

Structure – two start right handed helix (8nm in diameter)

Energy source – Use ATP to modulate assembly properties of filaments

Question 8

Microtubulues (overall)

Answer 8

Function – provide long distance transport + builds structures like the mitotic spindle

Components – Alpha/btea tubulin dimers + gamma/beta dimers

Structure – 13 protofilaments form hollow tube (Hollow tubules are 25 nm in diameter)

Energy source – GTP

Question 9

Intermedate filaments (overall)

Answer 9

Function – Structures + mechasnical

Components – Lamins + keratins + nuerofilaemnt proteins

Structure – Left handed Coiled-coil dimers assmeble into felxible non-polar polymers (10 nm diametrer)

Energy source – None –> Do no bind ATP or GTP (assembled through thermodynamic guided process)

Question 10

Location of Actin in cells

Answer 10

Use rhodamine is attached to aflatoxin molecules (toxin binds to actin and marks where actin filaments are)

Actin = throughout the cytoplasm AND have filaments align along the periphery underlying the plasma membrane in cell cortex
- Actin can form stress fibers in cytoplasm

Question 11

Is the cytoplasm liquid

Answer 11

Because have Actin and intermediate filemants and microtubulues throughout the cytoplasm –> the cytoplasm is NOT a liquid but INSTEAD it is a viscoelastic meshwork

Gives life at low rendelds number –> THIS is why molecules can go thorughout the cell and build structures where it needs to go

Question 12

Actin monomer

Answer 12

Actin monomer has 4 globular domains that form a horshore structure –> has a pocket where molecules do not cross over
- ATP can diffuse into the pocket and bind to the ATP binding site

In ATP state – actin monomer is an ATPase that can hydrolyze the ATP

Actin monomer has a minus end and a plus end
- Minus end = slow growing end ; Plus end = fast growing end

Question 13

Actin Molecular orientation/Assembly

Answer 13

Have one SU then then next then the next –> looks like have 2 filaments going around each other (WHY we call it a 2 start helix) AND the filaments are coiling (looks like 2 proto filaments wrap around each other)

NOT REALLY 2 start protofilaments because NOT building 1 filament and then the other filament and then filaments coming together IN REALITY they have to be co-assembled in parallel
- Co-assembly – SU adds then next then next

Question 14

How does co-assmebly of Actin happen

Answer 14

Co assembly happens due to a hydrophobic loop that sticks in and lays across the axis of the helix to stabilize the SU in place

Minus end has the cleft pointing down and round end sticking up with the hydrophobic plug across the helical axis

Question 15

Repeats in Actin Helix

Answer 15

Repeat = when start at one strand and go up –> how long does it take to find SU that is in exactly the same location 1 repeat away
- Coil-coil structures are defined by the repeat

Because both strands are built from the same type of SU –> means that when you have a SU lower in strand you can go halfway around the helix before the other strand will have a SU in exactly the same location
- SU halfway around on the other strand = Pseudo-repeat (half-repeat)

Pseudorepeat = have equivalent site from actin SU that came from the other stand
- Length to get to pseudorepeat = 36 nm ; 72 nm for a full repeat
- MY WORDS - SU in the same locaton just higher up BUT the SU is part of a different strand = pseudorepeat

Question 16

Why care about pseudorepeat

Answer 16

Why care about pseudorepeat –> because molecular motors use actin filaments and myosin motor –> Motors look for actin filaments (‘rocks’) to step on

Motors use the comparable binding sites that are 36 nm away (Molecular motors take 36 nm ‘steps’)

Because 36 nm is a pseudorpeat (would have the same binding site just 36 nm away)

Question 17

Breaking longer and thicker thing + measuring elasticity

Answer 17

Something that is longer or thicker would be harder to break

K = F/dL = E*A/L
- Stiffness = spring constant (k)
- K is proportional to how much force it takes to change the length if bending/stretching

E = Stress/strain = (F/A)/(dL/L)
- E is a material property –> how much stress do I apply to get strain for that type of material
- Stress = F/A (force per unit of area)
- Strain = dL/L

Question 18

Example of measuring stiffness

E = 2.3GPa
A = 20
L = 1

Answer 18

IF E = 2.3GPa ; A = 20 (area is 20 nm^2) ; L = 1 (filament is 1 microon long)

K = (2.3 X 20)/1 = 50
- Can see what E is for a 1 micron long filament –> if E is 2.3 then stiffness is 50nm/um

Question 19

Actin filaments in human body

Answer 19

Most actin filaments in the human body are ~1um long ; E = 2.3 = stiffness of plexiglass

MEANS actin = plastic like fibers in terms of mechanical properties BUT actin fibers can be built and taken apart readily (actin can be remodled while plexiglass can’t be)
- Body (10% actin) is constructed from tiny plastic like fibers

Question 20

Actin filament assmebly

Answer 20

Actin dimer is highly unstable ; actin alone is in a free monomeric form

To build actin filaments in tube:
Start with Actin monomer pool –> add Magnsium + ATP + salt –> need two monomers to come together to get a dimer –> add monomer to a dimer and get trimer –> get tetroer etc.
- Use salt to promote hydrophobic interactions of the hydrophobic plug)
- Use ATP because the ability of the SU to bind depends on ATP state
- Use Mg because ATP going to the binding pocket (needs a dicaton)

Question 21

What affects if Actin SU can bind to filaments

Answer 21

If actin SU can bind to polymer depends on if the SU is bound to ATP or ADP/Pi or ADP or nucleotde free state

Need nucleotide to change structure of SU to make it favorable for adding a SU to the filament (forming filemnt)

Question 22

Getting trimer of actin SU

Answer 22

To get trimer you first need a dimer

ISSUE the dimer is very unstable/comes apart fast

BUT if you have a high enough concentration of free actin then a third actin will join the dimer quickly enough so that the dimer does not fall apart
- Once have trimer –> the structure is stable because have pocket for the hydrophobic plug to reach across (the hydprophic loop coming across will stabilzie the structure –> NOW can build a polymer)

Question 23

ATP bound actin SU vs. ADP bound actin SU

Answer 23

Actin polymer assembly dynamics depend on nucleotide state of SU
- Actin is allowstic = means if you have a confirmation shift in 1 SU then it communicates the confirmational shift to the neigherbors

When ATP binds to Actin SU it causes a confirmatinon change so the actin SU is in a confirmation where it favors the SU is able to bind to the filement/other actin SU

Actin is ALSO an ATPase –> once SU with ATP gets into the filaments –> puts the SU into a confirmation where it favors hydrolysis of ATP to get ADP + phosphate –> phosphate will leave (NOW ADP bound)

ADP actin ALSO has a different confirmation –> ADP bound SU favors leaving the filament

Question 24

Where is ADP vs. ATP actin

Answer 24

ADP bound actin comes off the fast or the slow end (either end)
- ADP bound actin SU doesn’t want to be on the front growing end

ATP bound actin preferentially binds to the fast growing end BUT can go on either end

There are proteins that bind to filaments that have actin SU that are ADP bound or visa verca

Question 25

On off rate of Actin SU

Answer 25

When have large concertation SU can push on rate (have concentration dependency) - Because ATP actin binds to filaments – you can neglect the ATP actin off rate - Because ADP actin does not bind to filaments= neglect the ADP actin on rate MEANS the Kd (dissociation constant) is based on the off rate of ADP actin and the on rate of ATP actin (Inverse of 1/k assmebley = Kd) - THIS defines a critical concetrion

Question 26

Critical concentration

Answer 26

To build filament you need a concetration of SU that is high enough for the filament to grow Concretion that is high enough for the filemnts to be able to grow = the critical concetration - IF you fall below the critical concetration then the SU won’t associate and filament/SU will fall apart NOTE - have small on rate for ADP bound = CAN make ADP bound actin assmeble BUT the critcal concetrayon to get filament is higher than if you have ATP bound actin SU

Question 27

Rate of filament formation over time

Answer 27

Process - Adding Actin SU and ATP to tube to get rate of filament formation over time Rate of polymer formation over time – has slow phase then accelerates then platues - Slow at start – because slow to get dimer (Enucleation step = 3 filaments come together) - Rate of assembly increases– because once have 1 dimer (have nuclei) THEN SU can add on (oncae have dimer rate increases) - Plateaus at end – because have less monomers in pool at you build polymers (at plateleu the rate of addition = rate of Su falling off) Monomer pool NEVER goes to 0 because you have to be above the critical concetrayion in order to have net polymer assmbly

Question 28

Actin Monomer concetration over time

Answer 28

Monomer has inverse kinetics of polymer formation (Starts high and has slow decrease then fast depleation then slow decrease) - Slow at start because slow to make dimers - Fast depletion because the monomers are going into the polymers - Slow decrease at the end BUT does not go to 0 --> goes down to the critical concetration

Question 29

Monomer vs. Actin vs. Polymer plot - looking at the steady state concetrations at plateu

Answer 29

At start – when add monomer to tube monomer rises and have no polymer being built because you are still below the critical concetration Once at the critical concentration the polymer mass increases ; monomer pool platueus and maintains the equilibirum of constant levels of that stays at the critical concetration - Change in monomer to plateu and the polymer to increase BOTH happen at the critical concetration

Question 30

Graph of filament assmebly if start with dimers

Answer 30

IF start with dimers (bottom graph) = skip the slow step and just quickly get polymers and then plateu out

Question 31

How do you study actin polymerization in lab

Answer 31

Use a pyerene labled actin --> Add pyrene labeled actin with Mg + K + ATP --> get polymer - Pyrene = floraphore - Actin Monomer – Pyrene is exposed to slovent = has low floruence - In Polymer – Pysrene is in a hydrophobic envirnmemt = has high floruence (When SU are incorpated into actin filaments the pyerene envirnment is sufficinetley hydrophobic = allows pyrerene to florunece)

Question 32

Actin assembly assay

Answer 32

Experiment – take pyere labled actin SU and add salt + Mg + ATP --> do wL scan --> see a sudden increase in flourecence - Have 7 fold difference in flourecence dependong on if the actin SU is in polymer or if the actin SU is free in solvent Graph – can image the flourscece peaks get nuceiatiion phase --> elongation phase --> then state state phase - Detecting the the transition from Monomoner --> nuclei dimers --> polymer

Question 33

What does actin interact with

Answer 33

Actin filaments often intercat with other things Examples: 1. Sequestering molecules (Ex. Beta-thymosin or profilin) 2. Cofilin

Question 34

Use of Sequestering Molecules in cell

Answer 34

Sequestering molecules = bind to free actin SU to sequester away the SU Critical concteration of actin SU is 150 nm BUT the concentration actin to build cytoskeletal structures is 20-100 micromolar (700X higher than critical concetration) - Because have more actin than the crtical concetration then all of the actin SHOULD go into polymers (there should be no free SU) Actin does NOT go into polymers BECASUE there are proteins that sequester the SU so they are delivred in a regulated dfashion to grow the actin filemnenst ONLY when it is needed SUMMARY - NOW the SU won’t all be used up randomly because have a high concetraiin in general in cell = can actually use that high concetration

Question 35

Profillin and beta-thymosin

Answer 35

Actin Su are sequestered by profilin and beta-thymosin Porfilin delivers the actin SU (help bring ATP bound actin in) --> profilin is released - Allows the filament to grow

Question 36

Cofilin

Answer 36

ATP SU are newly added onto the filaments and then have hydrolysis of ATP --> get ADP --> filament grows --> the ADP bound SU moves to the back of the polymer as add more ATP bound SU to the front of the polymer - ADP bound = way to define the age of SU (because old ADP bound are towards the back/end of the filament) Cofilin = severing protein --> binds to actin filmenys that is have SU that is bound to ADP and breaks filaments - Breaking filaments exposes ends so that the SU can come off the filament

Question 37

Where is most actin incorporated

Answer 37

Image - Green = lables pool of actin SU - most SU are all incorporated at the leading edge of the crawling cell EM image - Shows the leading edge has network of actin dendritic branching that have a net up direction of the filaments pointing towards the membrane (pointing/directed towards the leading edge of the membrane) - Angle of the branches = 70 degrees - When have net incorporation the SU are binding to the front end of the actin filaments

Question 38

Arp2/3 (overall)

Answer 38

Arp2/3 complex helps nucelate actin and mediates formation of branched networks - Actin related proteins 2 and 3 = Arp2/3 Arp2/3 complex has 7 SU - non-Arp2/3 5 SU in the complex help dock the Arp2/3 complx to the side of an actin filament --> NOW have Arp2/3 (blue SU) docked on the side of the actin filament --> a actin SU can be ecorted into the Arp2/3 complex using scar or wasp --> NOW have a stable trimer (Arp2/3 and actin) --> New actin SU can come in and bind - Scar/Wasp anchor the actin SU (yellow) to the Arp2/3 (blue)

Question 39

What creates the branching actin Network

Answer 39

Arp2/3 and Actin SU will bind in middle of a difefrent actin filament = creates branching - Often actin polymr is branching at the - end (Arp2/3 is at the minus end) Arp2/3 binds to sides of the filaments = filaments growing outwards and makes more interface where more Arp2/3 can bind and form more branches outwords

Question 40

Older filaments in network

Answer 40

Older flaments are at the back of the meshwork - Older filaments move towards the back as new filaments are made by Arp2/3 bidning onto the side of the filaments

Question 41

Arp2/3 and Coffin intreaction

Answer 41

Cofilin (serving molecule) binds to ADP actin and cut filaments = frees more ends so ADP actin SU can leave END - Arp2/3 is buidling the molecules to have more branches AND Cofilin is cleaving to break the built filaments

Question 42

Arp2/3 and Profilin

Answer 42

Porfilin is a sequestering molecule BUT it is ALSO nucleotide exchange factor Profilin = binds to ADP bound SU to make ADP leave --> ATP can come back in --> get ATP actin - Profilin encounters a front again and binds and releases actin SU (make SU ATP bound so that it can be added to the filament or won't be cleaved) --> Can grow filament outward leading to a net assembly of the meshwork moving towards the membrane - I THINK saying that because profilin makes the actin SU bound to ATP now cofilin wont cleave = get net assebly of meshwork moving towards the membrane

Question 43

Leading edge of crawling cell when add GFP actin

Answer 43

Limiting concentration of GFP actin reveal actin dynamics of the leading edge of cell High GFP actin - See Membrane pushing outwards BUT can’t confrim that there is a rearword flow Less GFP actin - See indiviual actin SU coming in and being incorpated at the leading edge and then flowing back towards the rear of the meshwork (membrane is continuously being pushed outwards)

Question 44

Energy in Actin Filaments

Answer 44

Actin have cross linking filemntes + have ATP + have thermal energy At the high temperature in cell the actin filaments are wiggly (have vibrations) - Because know filaments spring constant and because the actin is small thin rod = the thermal and energetic components causes the actin filaments to fluctuate

Question 45

Brownian ratchet model

Answer 45

Brownian ratchet model incorporates diffusion of actin SU with actin polymerization to drive advancement of membrane front IF the filament is bending and the SU happens to pop on when the filamnt is bent --> THEN filemnt got longer --> the elastic enegery from the filament bending is NOW stored in the filemnts so the filament wants to flex back --> when the filemnt snaps back it will push the memrane foward More complicated because have instances where membrane can be inserted to help expand and actin filemnts come back and fill in gaps

Question 46

Brownian Ratchet model (MY WORDS)

Answer 46

My words – the filenst are wiggly = they bend = when add a SU on when teh filemnt is in that net state you made the filemnt longer so that when the filemnt goes back to being stragight it is longer than it was when it was before so NOW that longer filemnt (like the only way to add teh SU was by bending the filament so that the SU can fit then when straughtne the filaent is longer so that longer filemnts will push the membrane) - video makes it clear

Question 47

How do cells crawl

Answer 47

Overall - Expansive forces generated at the front of the cell by actin polymerization and contractile forces generated at the rear of the cell propel the cell foward At the leaidnhg edge have Arp2/3 meduate gorwth of actin meshwork --> through the Ratchet monition/membrane fluctuations the filaments growing and help stabilzie membrane (when have fultuation and stabilize mmbrane farther you push the front foward) While ratchet is happening - Have cortext of actin that goes all arond the cell and have mysoin (contractile) all around the cell --> when streatch the cortext of actin around the cell the cortical tension wants to restore the shape = cell moves foward

Question 48

Forces involved in allowing cell to crawl

Answer 48

Combination of cortical tensino (actin cortect) + myosin contracting in bacl + actin polymerization plus end pushing the membrane foward at the leading edge+ focal points= helps move the cell foward - 2 actins at play – actin in the coretct + actin at the leading edge END - Combination of outward pushing and inward tugging = that propels the cell forward

Question 49

Focal points in cell movment

Answer 49

Need the cell to make contacts with the subtrate to be able to move foward - Cell needs to propel outward and plant foot again to be able keep moving (using focal points to be able to 'plant foot' then can grow outward)

Question 50

Bacteria + Actin machinery

Answer 50

Bacteria use the same force generating actin machinery to move through the cytoplasm and spread between cells (Ex. Listeria crawls in cytoplasm) - Using Arp2/3 Listeria trigger actin filaments to assemble at the base --> Actin filaments grow and use ratchet model to propel the bacteria fowards --> THEN Actin hydrolyzes ATP to ADP --> cofilin takes apart the actin (it looks like comet tails that are being taken off because being disassebled) - Bacteria are just using cell machinery to be able to do this - IF Listeria are next to another cell when hit the membrane then it can go to the other cell

Question 51

Actin Cross linkers

Answer 51

Actin cross-linkers organize actin filaments into larger structures - Protein organize actin into a meshwork under the plama membrane or stress fibers or sarchameres or bundles - Proteins includes cross linkning (Ex. vilin and Screwin) and membrane anchoring proteins (ex. spetrins + dystropins) List of slides shows what processes the proteins are involved in

Question 52

What are most proteins that cross-link/organize actin filaments

Answer 52

Many of the proteins are monomers with two binding sites (can hold 2 filaments together) or dimers that are antiparrael or parallel so that they can bind and hold 2 filaments togetehr Proteins can be coupled to membrane anchroing domains so they can be linked to the membrane

Question 53

What do Cross linkning proteins do

Answer 53

Cross linkning proteins cross link actin into meshwork --> cause actin network have more structure (have gel characteristic) - Actin cross-linkning proteins control the visoelastic properties of an actin network OR Some cross linkers orient actin in parallel arrays = creates bundeles (Ex. microvilli in gut have parrallel arrays of actin bundles) Low crosslinker = gel ; high corsslinker = Bundle

Question 54

Differential sedimentation on Actin

Answer 54

Use differential sedimentation for distinguishing actin binding from actin cross linkning - Use differential sedimentatio to be able to seperate the types of structures being built Experiment – purify actin and actin binding protein in a test tube --> add actin alone (in polymer form) or actin binding protein alone or add actin with the actin binding protein - IF ABP does not cross link you can just get binding interaction BUT if you cross link you can get gels or buncldes forming

Question 55

Results of differential centrifuation

Answer 55

At 100,000xg all actin filaments pellet --> all actin goes to the pellete BUT the binding protein alone will stay in the supernatent - WHEN mix actin and the actin bindiing protein then the binding protein will go to the pellet with the actin (ABP and actin bound will both be in the pelet) At 10,000xg only cross-linked actin pellets - IF have a ABP that can cross link the actin into gels or bundles = can spin slower - At 10,000 g ABP and actin alone won’t sediment into the pelet ; ONLY the ABP and the F-actin that forms a gel or bundle (crosslinks) are big enough to go into the pelletes

Question 56

Overall life of Actin filament

Answer 56

Overall life of Actin filament = monomers --> nucelation --> elongastion --> treadmilling

Question 57

Ways drugs can inhibit actin polymer assembly

Answer 57

1. Block barbed end addition (Cytochalasin) - When inhibit the growth (no addition) --> Eventually ATP is hydrolyzed to ADP --> coflin cuts the filiment and disassemvles the actin meshwork 2. Sequester monomers (Latrunculin) 3. Stabilize Polymers (Phallotaoxins + Jasplakinolide) - Inhibits the treadmilling activity of actin SU growth - Stabilized so much that polymers can tolarete having SU pool lower that crtical concantration and won't fall apart

Question 58

Is actin a stable polymer

Answer 58

Actin = does traedmilling (use oyerene labled actin to study actin treadmilling) Micritubulues undergo dynamic instability (have catastrophe)

Question 59

Microtubulue structures thrughout cell cycel

Answer 59

Microtubules come in a varierty of structures throughout the cell cycle Interphase – have Microtubules all throughout the cytoplasm - Microtubulues radiate outwrad from a centrosome near the nucleus

Question 60

Centrosome

Answer 60

Central body of the cell Centrosome = Microtubule organzing center from which large lines of microtubules are enucleated

Question 61

Why is the nucleus in the center of the cell

Answer 61

The nucleus is near the center of the cell because microtubule based motors pull nucleus to the center of the cell Centrosome knows where the centroid is because it is sending microtubulues out in all directions --> as Microtubules grow outwrads they will hit the membrane --> in ratchet model this hitting the membrane pushes against the membrane --> by pushing the membrane the centrosome gets shifted around until the forceses coming from all directs balance out

Question 62

What happens to centrosomes in S Phase

Answer 62

S phase – Cell replicates DNA AND replicates the centrosomes When the cell divides it separates the 2 centrosomes so you define 2 centroids in the cell When have 2 centroids in cell = send microtubulues in all direction (because centrosome sends Microtubules in all directions) --> When send the microtubules in all directions some of the microtubules will capture the chromosomes

Question 63

What happens when a microtubule interacys with a chromsome

Answer 63

When the microtubulues find chromosomes the mirotubue interacts with the kinetochore of the chromosome which stabilizes the end of the microtubulues

Question 64

Why do chromosomes align in the middle of the cell in mitosis

Answer 64

When all of the chromosomes are captured the microtubules are pushed until the forces are balanced --> chromomes get aligned at the middle because microtubules pull in all directions until the the force is balance - WHEN forces balance all sister chromatids are attatched at a kinetchore and each kineticore is bound by oposing micritubulues Once at the chromosomes are all in the center trigger metaphase to anaphase transition --> sister chromatids can be seperated and gets pulled into each hemishoere of the cell where each hemisphere defines the centroids of the two daughter cells

Question 65

What happens with the microtubules in the final stage of mitosis

Answer 65

Once have daughter cells in the two hemispheres the microtubules send cues to the cortex helping set up the contractile machinery to pinch inward (squeezing Micrtubulue bundle togetehr as it pulls the cortext in) --> cut everything so now have 2 daughter cells

Question 66

Microtubulue Subunits

Answer 66

Alpah tubulin and beta tubulin are NOT stable on their own (no free alpha tubulin or beta tubulin) - Alpha and beta tubulin = allosteric (like actin) Alpha and beta tubulin both have a walker P loop that can bind GTP - Similar to walker P loop that binds ATP on Actin and Myocin Alpha tubulin does NOT hydrolyze GTP because it is buried BUT it needs GTP to be stabilized Beta Tubulin has GTP that is exposed on the surface = can hydrolyze GTP to get GDP/Pi --> THEN will release the phosphate and will be GDP bound

Question 67

Microtubulue protofilaments

Answer 67

When GTP is bound to alpha and beta tubulin --> alpha and beta tubulin are organized into protofilaments In protoifilamnets = alternate alpha and beta tubulin (aloha --> beta --> alhha --> beta) aligned in a straight chain Have plus and minus end of the protofilaments

Question 68

Fully formed microtubulues

Answer 68

Protofilaments coil to form the microtubulue END - In fully formed microtubule have alternating alpha and beta AND have a helical pitch (coil) as the alpha/beta tubilun dimers come around - End microtubules has 13 protofilemnts to go all the way around the perimeter Microtubules = hollow on inside --> small proteins/molecules can go into the tubule (Ex. taxol can bind to inside of microtubule)

Question 69

Enuclation unit of Mircotubule

Answer 69

Get beta tubulin/gamma dimer for nucleation center (similar to the Arp23 for actin) Nucleatiion center = centrosomes 13 gamma/beta tubulin looped around = creates a nucleation center for alpha and beta tubulin to bind --> can now grow the microtuvulue outward

Question 70

Centrosome

Answer 70

Nucleatiion center = centrosomes (has pair of centrioles) Centrosomes have rings --> ring is the gamma tubulin ring complex - Complex has other proteins associated with it that help hold 13 of the gamma/tubulin dimers together (looped dimers around like in protofilaments)

Question 71

Ends of microtubules

Answer 71

All microtubules have a plus end pointing outward ; minus ends are buried Microtubulues plus end are pushing outwrad from the centroid - By push outward the microtubules come out and find the periphery of the cell --> When find to periphery have a push can do push/pull back and forth to find where the center of the cell sits

Question 72

Microtubule organizing centers

Answer 72

Microtubule organizing centers are essnetial for microtubule nucleation Microtubule organization centers can assemble without centrioles (can build microtubules without centrioles) - 80% of microtubules are nucelatsed off the centrosome (Microtubulue orgnizing center) BUT over time if a cell is clipped can have enough material that can help reorgnize the residual micrtubulues --> creates a microtubulue organzing center - THIS is different from the centrosome because this organizing center does NOT have centrioles

Question 73

Nucelotide states of microtubules

Answer 73

Nucleotide state affects the confirmation of the MT --> determines if the MT grows or shrinks GTP bound alpha/beta tubulin = the protofilament stays straight (growing phase) As Microtubule grows --> the SU are incorporated into the filament and the pool of SU is shrinking --> As pool of free SU shrinks --> GTP on the beta SU to is hydrolyzed to GDP (only beta SU hydrolyzes GTP) GDP causes a confirmational change that affects adjacent SU --> causes the protofilament to bend Bending causes the protofilaments to splay --> splaying will cause the protofilament to separate away --> NOW the protofilaments/SU will come off

Question 74

What happens when the microtubule splays and SU come off

Answer 74

When the microtubule splays and the SU come off --> NOW GTP can rebind and can return the protofilament/SU to the first state and SU can be incorpated (go back to growing state) - I THINK when bind new GTp net New GTP cap = get straight protofilimanet = growing state

Question 75

Mirotubule growth and catasrophere cycle

Answer 75

Enucleate a micritubule and have a pool of SU the microtuvbule can grow (gets longer) --> As grow the microtubules the pool of free SU is dpeleted so it becomes hader to maintain growth (growth is slower) --> eventually intrinsic GTP hydrolysis catches up --> remove GTP cap --> NOW the microtubuleis unstable and can fall apart --> GTP cap rebinds repeat cycle When have a GTP cap the end of the SU are bound to GTP and the structure will remain straight and have net growth

Question 76

What controls the frequencey of catasphorhere events in microtubulues

Answer 76

Frequencey of catastropjhy = based on race between GTP hydrolysis rate and rate addition of new GTP dimers - GTP hydrolysis rate = first order reaction (does not care about concentration) - GTP dimer binding rate = second order reactions (depends on the concertation of free SU)

Question 77

Microtubule dynamic instability

Answer 77

Initially have a net growth of the microtubules THEN have lose GTP cap and have loss of SU --> Microtubules shortens - Losing SU/Microtubules shortning = catastrophere When microtubule shortens the SU are released --> THEN the GTP can bind again to teh released SU and NOW have GTP bound SU = can rescue and regain cap and grow outward again

Question 78

What affects microtubule dynamic instability

Answer 78

Tubulin concetration and number of nuceli inflnce microtubule dynamic instability (seen in in silco experiment) Tubulin low: Have free tubulin at start --> quickly see growth --> then see instability (Quickly see cycle of growth and catastrophe) Tubulin High: microtubulues growth everythwhere AND takes a long time for dybamic instability to happen - Eventually get depletion of SU and can see catastrphete and rescue BUT it takes longer for that phase to happen (net growth for longer)

Question 79

Microtubule dynamic instability when have 4 nuclei and Tubulin High

Answer 79

NOW have more microtubules coming out (higher pool of microtubulues) because 4X nuclei BUT they enter dynamic instability faster and over time get fewer microtubules - Get fewer microtubules BUT the ones that are there are longer Mimics what is happening in a dividing cell (2 centrosomes separate and fire microtubules in all directions) Need rapid tranistion to instability becuase you don’t want non-productive microtubulues (IF the microtubue is captured by the kinetorchore it is stabilzied BUT if it does not bind then can quickly derade it because it is not useful)

Question 80

What is unique about Microtubules

Answer 80

Microtubules have dynamic instability (do catastrophe) Dynamic instability is critical for the mitotic spindle to capture the chromsomes and align at the metaphase plate

Question 81

Drugs can inhibit microtubule dynamics

Answer 81

1. Block addition (Ex. Colchine and Vonblastine) --> caps the tips of microtubulues and prevents growth 2. Bind tubulin dimers (Nocodazole) - Sequester free SU so microtubules won't be rebuilt after catastrophe 3. Stabilize polymers from catastrophere and treadmilling (Taxol/Paclitaxel) - Goes to the center of microtvukes and bind to protifiliemnts so GDP structure is stable (doesnt fall apart)

Question 82

Taxol

Answer 82

Taxol binds to the center of the microtubulue which stabilzies the lateral contacts between protofilaments which inhibits dynamic instability - Maintain microtubule but remove dynamics Anti cancer drug that inhibits mitosis because it stabilzies microtubules

Question 83

Function of intermediate filaments

Answer 83

Function of intermediate filaments = needed for mechanical and structural integrity Example that shows importance of intermediate filaments for structural integrity: Mutations in keratin causes the skin to be more prone to blistering - Skin ruptures because have defects in ability of epithelium to maintain connections to each other, to the matrix and to the desmosomes/hemidesmosomes

Question 84

Where are intermediate filaments concetrated

Answer 84

Intermediate filaments are concentrated around the nucleus and anchor at hemidesmosomes and desmosomes - Can also anchor to the nuclear envelope (have IF in the Nuclear envelope) Image – shows Intermediate filaments are throughout the cytoplasm and anchor in desmsomes and hemidesmosomes in the periphery

Question 85

Example Mutation in Keratin14

Answer 85

Shows mutation in keratin14 = get EB simplex

Question 86

Building Intermediate filaments (coil coil)

Answer 86

Intermdieate filaments are assembled from monomers into rope like non-polar filaments Get tranlsation of intermediate filament with N and C terminus Filament has an alpha helix region that can form a coil coil wtih a second filament/sister moleucle (get left handed coil coil dimer) - In coil coil - N terminal are next to each other and C terminals are next to each other

Question 87

Building Intermediate filaments (dimers binding)

Answer 87

To make filament --> the N and C terminus of two different dimers come together and orient end to end (N-C) THEN side to side End to end = staggered tetramer of 2 coil-coiled dimers END = have no polarity even though the SU (building block) have polarity - No polarity because staggered and opposite directions

Question 88

Side to Side associations of Intermediate Filaments

Answer 88

Side to side and end associations allow intermediate filaments to assemble into ropey non polar filaments END – get eight tetramers twisted into a rope like filament (10 nm role like filament that have coil coil structures extending the full length)

Question 89

Mechanical properties of each cytoskelatal polymer - microtubule

Answer 89

Microtubule mixtures are flexible BUT easily rupture Chart (deforming force vs. Amount of deformation) - Does not take a lot of force to deform the microtubules because they are big enough - When cross a minimal threshold microtubules will snap

Question 90

Mechanical properties of each cytoskelatal polymer - Microfilament (Actin)

Answer 90

Actin filaments are more rigid and rupture easily Chart (deforming force vs. Amount of deformation) - Actin = very rigid/won't bend (takes a lot of force to get deformation) - BUT more prone to breaking Longer filament = more you can bend it --> Actin is on 1 micron long so it is rigid BUT if add too much force then they break

Question 91

Mechanical properties of each cytoskelatal polymer - Intermediate Filament

Answer 91

Intermeidate filaments are easily deformed (flexible) BUT withstand greater strains without rupturing - Built to be flexible and resistent to breaking THUS intermediate filaments are well suited to provide mechanical integrity to cells (durablililty in skin) - IF you have mutation in intermediate filaments = NOW have tissue that is susceptible to being damaged upon small mechanical inputs

Question 92

Are intermediate filaments polar?

Answer 92

Intermediate filaments are not polar (no motors use IF as a track) BUT you can still levrage intermediate filaments as a track

Question 93

Answer 93

Answer - B

Question 94

Answer 94

Answer C NOTE bacteria - have actin related protein that has dynamic instability like MT --> that actin separates plasmids in bacteria (doing role of Microtubules but is an actin related protein) - FTSC protein - microtbules ancetsral protein that does not have dynamic instability (used to divide the bacteria ) END - bacteris took actin and microtubulues and fliped roles

Question 95

Answer 95

Answer C