Lecture #11 - Filaments Flashcards
Why can things stay on top of a cell
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
What molecules lets out body move
Filaments + motor proteins
Motor proteins = use filaments as tracks to pull on ad walk across
Filaments
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
Form and function
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
Cytskelaton at the organism level
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
Right handed vs. left handed helix
Two start helix (actin) = right handed
Coil coil (Microtubules) = left handed
Microfilaments (Overall)
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
Microtubulues (overall)
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
Intermedate filaments (overall)
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)
Location of Actin in cells
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
Is the cytoplasm liquid
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
Actin monomer
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
Actin Molecular orientation/Assembly
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
How does co-assmebly of Actin happen
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
Repeats in Actin Helix
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
Why care about pseudorepeat
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)
Breaking longer and thicker thing + measuring elasticity
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
Example of measuring stiffness
E = 2.3GPa
A = 20
L = 1
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
Actin filaments in human body
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
Actin filament assmebly
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)
What affects if Actin SU can bind to filaments
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)
Getting trimer of actin SU
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)
ATP bound actin SU vs. ADP bound actin SU
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
Where is ADP vs. ATP actin
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
On off rate of Actin SU
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
Critical concentration
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
Rate of filament formation over time
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
Actin Monomer concetration over time
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
Monomer vs. Actin vs. Polymer plot - looking at the steady state concetrations at plateu
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
Graph of filament assmebly if start with dimers
IF start with dimers (bottom graph) = skip the slow step and just quickly get polymers and then plateu out
How do you study actin polymerization in lab
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)
Actin assembly assay
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
What does actin interact with
Actin filaments often intercat with other things
Examples:
1. Sequestering molecules (Ex. Beta-thymosin or profilin)
2. Cofilin
Use of Sequestering Molecules in cell
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
Profillin and beta-thymosin
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
Cofilin
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
Where is most actin incorporated
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
Arp2/3 (overall)
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)
What creates the branching actin Network
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
Older filaments in network
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
Arp2/3 and Coffin intreaction
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
Arp2/3 and Profilin
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
Leading edge of crawling cell when add GFP actin
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)
Energy in Actin Filaments
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
Brownian ratchet model
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
Brownian Ratchet model (MY WORDS)
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
How do cells crawl
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
Forces involved in allowing cell to crawl
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
Focal points in cell movment
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)
Bacteria + Actin machinery
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
Actin Cross linkers
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
What are most proteins that cross-link/organize actin filaments
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
What do Cross linkning proteins do
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
Differential sedimentation on Actin
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
Results of differential centrifuation
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
Overall life of Actin filament
Overall life of Actin filament = monomers –> nucelation –> elongastion –> treadmilling
Ways drugs can inhibit actin polymer assembly
- 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
- Sequester monomers (Latrunculin)
- 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
Is actin a stable polymer
Actin = does traedmilling (use oyerene labled actin to study actin treadmilling)
Micritubulues undergo dynamic instability (have catastrophe)
Microtubulue structures thrughout cell cycel
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
Centrosome
Central body of the cell
Centrosome = Microtubule organzing center from which large lines of microtubules are enucleated
Why is the nucleus in the center of the cell
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
What happens to centrosomes in S Phase
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
What happens when a microtubule interacys with a chromsome
When the microtubulues find chromosomes the mirotubue interacts with the kinetochore of the chromosome which stabilizes the end of the microtubulues
Why do chromosomes align in the middle of the cell in mitosis
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
What happens with the microtubules in the final stage of mitosis
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
Microtubulue Subunits
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
Microtubulue protofilaments
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
Fully formed microtubulues
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)
Enuclation unit of Mircotubule
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
Centrosome
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)
Ends of microtubules
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
Microtubule organizing centers
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
Nucelotide states of microtubules
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
What happens when the microtubule splays and SU come off
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
Mirotubule growth and catasrophere cycle
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
What controls the frequencey of catasphorhere events in microtubulues
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)
Microtubule dynamic instability
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
What affects microtubule dynamic instability
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)
Microtubule dynamic instability when have 4 nuclei and Tubulin High
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)
What is unique about Microtubules
Microtubules have dynamic instability (do catastrophe)
Dynamic instability is critical for the mitotic spindle to capture the chromsomes and align at the metaphase plate
Drugs can inhibit microtubule dynamics
- Block addition (Ex. Colchine and Vonblastine) –> caps the tips of microtubulues and prevents growth
- Bind tubulin dimers (Nocodazole)
- Sequester free SU so microtubules won’t be rebuilt after catastrophe
- 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)
Taxol
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
Function of intermediate filaments
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
Where are intermediate filaments concetrated
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
Example Mutation in Keratin14
Shows mutation in keratin14 = get EB simplex
Building Intermediate filaments (coil coil)
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
Building Intermediate filaments (dimers binding)
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
Side to Side associations of Intermediate Filaments
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)
Mechanical properties of each cytoskelatal polymer - microtubule
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
Mechanical properties of each cytoskelatal polymer - Microfilament (Actin)
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
Mechanical properties of each cytoskelatal polymer - Intermediate Filament
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
Are intermediate filaments polar?
Intermediate filaments are not polar (no motors use IF as a track) BUT you can still levrage intermediate filaments as a track
Answer - B
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
Answer C
