MY SUMMARY'S

TOPIC 8 -

Intermediate filaments -

There are six different types 70 genes encode for them, there are 210 cell types meaning three cell types have intermediate filaments. they are highly specialized

The basic unit of a intermediate filament is going to be a dimer that in the middle has a coiled coil region that is going to be an a helices. both monomers that make up the dimer have their own a helix, and both helices wrap around eachother. it as well has a head and tail. They dont bind nucleotides, atp, gtp. They are equilibrium polymers becuase they dont use energy at all so nothign to conver them to steady state polymer. (steady state uses energy)

Lamins are the first family members that evolved. they are nuclear intermediate filetms in the nucleus and some prentylate and attach to the membrane to provide a system for the nuclear enveloep. so basically the laminas were the first they are the progeniators. Then the cytoplasmid intermediates are only in animals because they evolved after plants and animals divereged. Basically they lost their nuclear localization singal and their ability to prenylate. that is why they arent in the nucleus.

a turn on the alpha helix is 7 amino acids. so both of the helices every time they turn their 7th amino acide reside is going to face other and they are hydrophbic so this is perfect because they are going to bury inside the a helices. occasionally the residue is negative and the other pair will be positive. So the dimers have a head and tail regions or N(head) and C is the tail. the tail is biger than the n head. so basically when we put two dimers together into the higher order strucutre (the tetramer) then it is going to pair up in either direction. meaning that intermediate filemtns have no direcitonality. This anitparllel where the C’ ends are facing opposite directions in eveyr family member are conserved. all intermediate filements are non polar. This was confirmed using the immunogold electron microscopy that is going to use gold particles, bind them to antibodies, these bind to C terminal ends and this is going to create dark spots when shot and this is going to reveal the location of the C terminal ends. it was seen c’s to be opposite.

So tetramers are going to line up end to end to make a protofilemtn, four proto-filments lay next to each other to make a proto-fibril. and four proto -fibril wrap around each other to make a intermediate filament.

intermediate filaments are the most stable and strongest. so we check turn over by using frap flouscence photobleaching. we photobleach a segment and check how long it takes for the flouscence to come back and it takes 20 to one hour inidcating that they live long theyre stable they can turn over slowly. actin takes like 1-2 mins .

another experiment involving flouscently labeled monomers tracts the exsisting filaments. and over 20 - 1 hour there is small amount of the flouscnet labeled IF into the exisitng area showing that they are slowly tunring over. This is microinject bitoin labeled kertin into cells.

Intermediate filements dont need acessory proteins in order to help them assemble. if u put dimers, tetramers in a cell they will form the intermediate filemtns. if u take monomers that build two types of intermediate filements they will form indenptly from eachother they dont need eachother to build.

to test what happens is that they microinjected antibodies that block assembly into tissue cultured cells and in test tubes. the anitbodies stoped the assembly of intermediate filemtns but saw that the cells were perfectly fine and there was no phenotype defect. The filemts wee dirupted by cells were okie. Some animals dont have cytoplasmic IF’s meaning they arent needed for survival in vivo. but the limtiation is that this wasnt a real organism in real tissue .

So the experment on mice on kertins was done. kertin are expressed in the skin in the epithilial layer the top layer. kertin 10 is expressed in the dark brown cell in the layer at the top layer and keratin 14 in the lower layer and the goal was to dirupt one of these keratin layers so that we dont make kertins in the skin layer. So usually CRISPR is used in order to make that loss of fucntion that is going interupt that intermediate filament but this expeirment was done preCRISPR. and it was hard to make a loss of fucntion transgenic mouse because it was hard to knock out both genes . it was easier to insert a gene that was dominant to see the phenotype so what they did was they plalced a negative dominant gene that is (dominant genes are usually gain of fucniton) loss of fucniton, so an extra gene to interrupt the intermediat filament. So this gene is going to make a mutant monomer. That mutant protien is going to add onto the growign end of the polymer so no more monomers can be added after that . deoxy sequnecing is when u have a nuceltoide that doesnt have a hyroxyl group so that once ur growing ur dna chain u put it on an A and when that stops at that A it means that u have an A in that particul positon. this is the posin polymer model, u add dom neg allele make a dom neg protin that stop assembly.

we are going to put in a mutant allele to make a transgenic mouse for the dom neg alle of keratin 14. it will stop making intermediat filemt of the skinn cells. the wild tyep mouse has skin cells that are nice and tightly bound and the dom neg mouse has skin cells disrupted, no longer tight layer. they are more likely to get blister. we did this on a tissue culture and saw no effect bc the indivual cells arent movin and not likely ever to get blisters. So the intermediates of KERATIN of one cell are going to bind to the other cells intermediates through the transmembrane cells that is what makes them bound tighly and resist strain. remember that keratin is for skin.

Diseases of keratin 14 or 5 are going to cause blsites, loss of hair nails , problems with eyes.

Desmins are going to be for the muscle cells. they are going to bind the z disc’s of the sacromers together. this is going to help again the stress of contractions. and if there is no desmin or mutation then we have muscle failure because it wont resist the force of the muscle contraction rubbing together.

neurofilaments are intermediate filaments. neurons can extend three feet from your spine to ur toe. the neurofilaments is going to allow faster electriacl signal, provide integrity, and stop it from breaking from bending

keratin evolved from exoskeleton from insects that had crunch outer parts. keratin provided the mechanical integrity to now have ur skin on the outside instead of on the inside. now have a skeleton on our inside and our soft parts on the out.

migraiton pattern from africa to middle east. Our relationship to other human type indivuals like neadnderthals. we had a ocmmon ancestory with neanderthals. we mixed with them and then we went to middle east. Some of the genes are the skin pigmentation genes, in african we need darker skin for UV radiation.

TOPIC 9 -

Microtubuel filaments are going to be straw shaped structures. they are going to be made of dimers alpha and beta. they can both bind gtp, alpha is in the gtp form always and beta can hdyrlozye it. These dimers can make protofilaments and thirteen lateral protofilaments can make a microtubule. alpha’s are next to alphas but the point where the alpha and beta are next to each other is called a seam. This is similar to actin because the free dimers are in the cytoplasm in the gtp form (more gtp than gdp) . B tubulincan hydrloze the gtp to gdp and then kick it out and exchange.

So the dimer will come into the plus end in the gtp form( remember or know that the dimer consists of the a tubulin and b tubulin) and then it will attach to the polymer. At the polymer the B is hydrolyzed and the gdp cannot be removed until the monomer falls off the polymer(gets removed).

Once theyr ein the polymer they cannot remove gdp while theyr ein the polymer(like actin) . the idmer is added to the plus end , its in the gtp form. Most of the utbulin in the tubulinis in the gdp form , it adds onto the plus end, hydrolyzes and cant relase. can excahnge from gdp to gtp after the monomers release from polymer.

microtubuel based structure - fillia and flagellumthat is going to be projecitno of the surface of a cell in order to move usbtance and wiglle ont he surface.

so when mitosis happens we are going to disassemble the mcitubules and rebuild to become spindle poles that are going to be used to segregate the chromosomes to become two daughter cells. centrosomes are micrubtuel organizign centers.

Microtubules are going to be nucleated and have a microtubule organizing center that is going to be centrosomes that are going to reorganize. During interphase there is one centrosome and the microtubule minus ends are going to be attached to it and the plus ends are going to be out towards the edge of the cell, for structure, shape, and trnaportation. during mitosis the centrosome duplicates and the microtubules minus ends are anchoerd to the spindle pole and the plus ends towards the chromosomes. The centorocomes are going to be equivalent to eacother when they divide. Centrosomes are for animals

Cilia and flagella have basal bodies that are going to be their microtubule organizing center that is going to have the minus ends bound to it and then the plus ends are going to grow towrads the cilia or flagella.

during mitosis we are going to have g1 s g2 and M. the g1 and g2 are the gap/the growth stages. S and M are the action phase because during the S phase the replication happens and during the M phase the chromatids are going to segregate. Although in drosophilia embryos the g1 and g2 phases are skipped because they want to generate a lot of cells os they go into s phase replicating and m phase segregating and then back to s. The spindles are going to from rapidly during mitosis and dismantle fast after.

How do we regulate the building and disassembly of the microtubules?

Alpha and beta are family members they have 40% identical amino acids and are conserved evolutionarily. So plants and animals have 75% of the same amino acids. Both bind Gtp. GTP in alpha is going to help fold. beta hydrolyzes it and when the dime is released gdp can release and bind a new gtp. The plus end is in the GTP form and most of the microtubule is in the gdp form. the polymer is linear. Microtubules have polarity.

You can grow microtubules from the plus or minus end but the plus is faster. the two ends are chemically differeiont so they have different critical conentrioant. This is a steady state. So it is possible for teadmiling. Not all mcirubtules in the cell undergo treadmiling because of their minus ends are caped and anchored in to micrubtule organizing center. growth and shrinkage can happen at the plus end. The higher CC is at the minus end and the lower is at the plus end.

So in an experiment they added monomers and they saw polymerization and they saw that microtubules were being made. as it reached steady state(the gtp hydrloyzes allows it to be steady state it is going to use that energy to make it steady state and to keep-the ends chemically different like actin). As it reaches the steady state it is seend that the number of microtubules is become less and those that remain is going to become longer. they look at an indivual microtubule and saw that the micrtubule will grow at the plus and and then suddenly shrink this is called catastrophe. and then when it starts growing this is rescue . something they will catastrophe until it dissapears. others will mainly grow. this is called dynamic instability altering cycles of growth and shrink. if the microtubule reamins the same the plus end was capped and anchored to stablize and maintin its legnth.

So why does dynamic instability happen? when the plus end is having monomer’s added, the rate of hydrloysis can catch up and make the plus end gdp form and that leads to disassembly. usually it is in the gtp form that forms like a cap that is going to make it assembled. so until a monomer attaches to the plus end it will resuce it. The thirteen proto-filaments that make up the cylindrical microtubule is going to be in a linear gtp form. the cap that is at the plus end and it being gtp bound is going to give it energy to keep steady state and to keep it i a straight strained conformation. when it looses that cap, it will got to gdp and it is goign to cause it to splay out disassemble. cuz thats what it wants to do and without the cap it will.

Microtuble Assembly Proteins (MAP) is going to be EB1 that is going to bind at the seam but only in the GTP forms so near the cap and so it is going to prevent premature catastrophe, promotes elongation, and stabilizes the cap. Since microtbules grow from the centrosomes, the growing ends are visible to EB1, but if the ends start shrinking the EB1 will not be able bind to the gtp tubulin cap because the microtubule will not have that cap.

There are other proteins that are also going to stablize the cap and promote elongation like XMAP215 and CLASP. they are modular protiens that are made of repeated domain and the domains bind to tubulin at the plus end.

On the other hand there are microtubule diassembling protiens like PINESIN 13. theyre going to bind to the protofilmetns the same as the stablizng , at the plus end, but theyre going to provide energy to bend the ocnirmation.

KIN13 is going to be a motor protein that moves along microtubules. Its going to use the energy of hydrloyzes in order to bend. KATANIN is involved in severing and then disassembling from the end of severing. the microtubules are going to be long and adding katanin is going to chop them. it disassembles from both ends the new plus end and the old one.

Maps can bind to microtubules on the sides and because of this it can bind two at the same time or bind to intermediate filaments. How they were disocered is that they were pruified with microtubules.

Tau is localized to axons and Map2 is localized to dendrites. They have binding repeats, modular protiens that bind to the side of microtubules. They have arms that exend out on a second micrubtule to tie them together. Tau is one of the major proteins seen in alzheimers disease. Tau is bound to microtubule and is helping to arrange them in higher order structures and in alzhemers the tau is phorhylated and can longer bind to micrubules and then the neruons get tangled and disrupt neuronal funciton.

Maps can regulate spacing, dif maps have dif lengths and so map 2 u get wider spacing and tau u get narrow spacing.

Insect cells make litle round cells and if u express map2 in them then the cell shape wont be round, it will be lumpy and the microtubules will be parallel. these map2 are expressed in dendrites and if u add to a cell it will start to look like a dendrite.

The role of intermediate filmetns is to provide structural stability. So microtubules are weak and intermediate that are able to withstand stress can help the microtubuels become stronger.

drugs like Colchicine and colcemid are able to bind to the tubulin dimer and prevent them from assembling microtubule polymers. so at low concentraiotn is is oging to block cells in metaphase because it stops the spindle fibers from forming. this is good to count chromsomes and see how many there are or if there are any missing to see diseases or cancer. it is good because m phase is hte only phase u can see the chocmomes. in gout it is swelling of the extremeties and immune cells are going to migrate to that region, and in low concentraiot of the drug it stop the cells from migrating and reduce thesymptoms. Another is taxol that is a breast cancer drug that is going to stabalize micurbtules at M phase so that they arent able to disassemble and then this blocks the contination of the diving o f the cancer cells. in order to keep diving the microtubules need to be disassebled.

Maps are regualted by phoryalation this happens through cyclic dependent kinases that are a series of kinases turned on at different stages. So CDK’s are going to turn on proteins. In early M phase they turn on/phoshphoylate proteins that are going to build microtubules for the spindles and then late M phase is going to phoshporylate the ones that are going to promote diassembly.

Topic 10 -

So microtubule organizing centers are the basal bodies , centrosomes, the spindle poles

during the cell cycle the conetorsome is duplicated and the two spindle poles are formed. they migrate to opposite side of the cell. in interphase the microtubuesl radiate out in all direciton but in m phase they are going to radiate outwards but grabbign onto the chromosomes. so centrosomes and spindle poels are all the saem thing .

Standard microtub ule is going to have 13 protofilemtsn. the orgtanizing centers are going t ohave micurbutles that form either doublet or triplets.

The doublet is going to have A and B a has 13 protfilament and B has 10. The triplet is going to be made out of A, B, and the C also has 10. Triplets are going to be made out of basal bodies, centroils

So the basal body is going to nine sets of the triplets and there are going to be proteins that are going to arrange them in this shape. Axoneme is the name for this structure that sticks out the basal bodies(they are not nine triplets) . the axoneme is going to have nine doublets. In the axoneme , there is going to be a pair of microtubules in the center. these are going to be structural role. the plus ends are going to be at the tip of the flagellum. C isnt at the basal body. The axoneme is going to have doublets instead of triplets because the motor proteins have attach.

dyneins are going to be the motor protiens that are going to attach to one micrutbule double, They extend arm like projections to other doublets. the Dynein arms bind to the neigboring mcirubtules and attemp to walk along it. They attach specifially to the A tubule.

Becasue part of dyeing is anchored to one doublet its walkign motion on the neighboring doublet generates force and produce mvovement between the mcirubtubles. Like myosins motors walk on actin filments. If myosin is free it walk if its anchored it pulls on actin. so like lets say the dynein is free then the filment is free to mov eand slide past eachother. if the motor is fixed in placfe the motor tries to walk and the filemnt moves instead. So the dynim motors are ridlgey attach to one micrubtule doublet walkign along the neighboring doubelt, it nofhting stops them the doublets are going to slide past each other. although there are other sets of pritne that are going to lock the micrubtules togeher and wont let them slide.instead of sldiign the force generates the microtubules to bend. The bding motion is what allows sperm cell to swim. so it goes back and bends and back like a wave. this is what causes the tial the cilia flagella overall to wiggle.

centrosomes are going to have centroiles. and the centroiles are going to be a nine pairs of doubles. they are surrounded by the pCM the paracentroil matieral. the centroils are going to be cylindrical and perpericndual to each other.

genetics studies identified a third tubulin family as the gamma tubulin family and antibodies to gamma tubulin and with thsi they claolted ot enofmrt .

gamma tubulin is another type of tubulin. this is found only in the centrosomes, if u add blocking anitbidies to the gamma tubulin u dont form micrubtule networks. gamma tubulin is part of the mechanism that caps and anchors the minus end of the microtubules in the centrosomes. Gamma tubulin is part of a large complex that is Gamma-Turc . Gamma is a family member o fhte alpha and beta tubulin. gamma tubulin ring compelx forms and is stable. the compelx contain 13 gamma tubulins.

So arp2/3 complex in the actin fmaily memebr niucleat the formaiton of actin filaments. here we have tubulin fmaily memerbs part of a complex.

So this gamma ring complex turc is going to be near hte PCM surroudnign the centriles. it is going to nucleate the microtubules, cap the minus ends, anchor the miceubtuels. they are not extension of the microtuules in the centrosomes. The paracentroil mateiral is going to surround the centroils and contain the gamma complex. So the microtubuesl are going to radiate outwards from teh PCM formign a network. the two centroils are not identical .

one of the centroils is goig to be the mother and hte other one is going to be the duaghter. the motehr is more mature and has an extra appendage. The mother can organize the parecentrial material and activate the gamma complex to nucleate formation. So during interphase we want an active centersome, in mitois we will ahve to active ones. once theres two centrosomes we call it spindle poles. It takes more than a full cycle to build a fully mature cycle ot make it fucniton and active.

So in the g1 phase we start with our centriols. we have one cell, two centroils. and then in the S phase it is going to become replciated so the mother make and immaure and the daughter makes an immaure and again this is in one cell. Then in M phase the oringal daughter beocmes mature and the new immaure from the orginal daughter beocmes a duaghter. in the other cell the old motehr sayts the same and the new immature from the orignal mother turns into a daughter. and now we have tow cells with two full centroils in Mphase. it repeats again. it repeats twice ecause they dont beocme mature unitl the following cycle m phase.

So there was an experiment that was done that cut a fragment of a cell and the microtubules were able to rearreagne themselves so that all. the minus ends are pointing towrads the center and the plus ends towrads the edge even though they didnt have any centrosome. If u were to destroy all centrosomes u can still assemble cneotroms de novo. So mcirubtuesl can self organize without centroils or centrosomes. there is a machinem that counts and controlls duplcaiotn, and old centroils are not needed for formign new ones. Centroils are providing structure.

so there was an experiment where they took a centorosme and then they placed it in a plastic box corner and placed nucletoides and tubulin and saw that the microtubules formed and the centrosome moved to the center of hte box. so microtubule assembly is suffieint and needed to move the centrosomes from the edge to the middle.

There are two possibilities for this. The microtubules grow until they hit a wall of the chamber and then that makes a force that moves the centrosome away from the wall. micutbtules grow in mutleipl directions and this causes the centering . another reason could be that the micrubtules hit the wall so they cant elongate and so it will difuse away from the wall and now we go tht emcirubtuel to extend from every direciotn. In the first possibilit the mcirubtuels continue growing even tho it hit the wall. and the second says that they stop growing .

in a typical cell the micurbutles can grow the lenght of a cell. in a neruon it cannot span that distacne.

So micrubtule skelton is going to act as a map . the minus ends are at the center and the plus ends are at the edge. when vesicles are made from the er and golgi, the er is located aroudn the ncuelus and the golgi extends out. vesicles ocm out the golgi and have to go to the edge . so the vesicles move along the microtubules. motor proteins run form minus to plus to cary the vesicls. if there are no motor protien they are going to diffuse out through diffusion and it would take a long time. so dnamic instabilitu allows u to build a map. If u have a mcirbutl grwoing in a bizzare pattern it will get removed with dynamic instability. the ones that grow in the right direction are going to get captured . This is all random trail and error.

interphase - micrubtuesl build a map - trial and error

mitosis - more microtubules are needed to build a functional mitotic spindle. the spindle must: capture sister chormids , pull them towards the opposite spindle poels. so the cell repurpose mcirubtuels from mapping to chrosmsoem segregation. CDK’s phroylate protein involed i mcirubtuel assembly. Microtubules in inteprahse are reogzanized cnetosoems beocme spindle poles.

TOPIC 11 -

So there are two types of motor kinesis that are plus end directed motors that move towards the edge of the cell and the dyenins that are going to be minus end direct and move towards the center of the cell. So since gollgi is maintined in the middle ish waht happens is that both kinesis and dyniens are attached and they battle it out to see who is going to win. it all depends on the ratio of dyenin and kinesis.

Axons are extremely. long, and microtbuels arent that long so what happens is that microubtules are going to overlap each other and make a roadway. proteins in neurons are made in the cytoplasm of the body. If u inject radioactive amino acids. they get incorporated into newly synthesized proteins and we can track the rate of movement. we will see that the vessicles in which these proteins are going to move are going to move at different rates. So different vessicles are going to have different motor proteins bound.

an experiment involving take a giant squid neuron and crushing it till the cell squishes. The cell bursts and so the cytospam sis going to ooze out onto the microscope slide. Microtubules are to appear stringy, long, and the vesicles are going to be small dots. the veiscles move along the micrutbuels, this is in vivtro.

EM - Veiscles physcially attached to the micurbtuesl, veiscles can move laong the length and they wont crash into eachother.. this is becuase we ahve 13 protofilemtns and they each go on one protofilament.

So if we were to purify a protein, add a veiscle, atp nothing will move, but if we include the squi cytoplasm there is movment meaning th cytoplams contains motor proteins. now if we were to add non hydrolyzable atp analog then the motor proteins are going to loc onto the microtubueles. Microtubuels are large and heavy and if we centrigeu the extract there would be micrubutles, vesicles, and motor proteins. the supernant will have other proteins. if we resuspend teh pellet and add real atp then the motor are going to resume walking and run off the ends of the mcirubtuels and dteach into the solution. if we centirfuge then hte motor proteins will be in the uspernant

Most of the kinesis are dimers, they are going to have two motor heads, a coiled coil, and a cargo binding tail. THe heads bind to the micrubtules and hydrolyze atp. the pi is realses and realsed adp and binds new atp. The tail can bind to other proteins. the tail is the light chain. The heads are going to have a helices and b sheets and nucleotide binding site but they have very little amino acid sequence similarity. this makes peopel to speculate that myosin and kinesis are not family members or maybe they are and were going to have the lever arm in the myosins and neck region. they divergent eovltion forma primordial motor. myosins and kinesins are distant family members.

Kinesases heads at the N temrinus and tials at the c temrinus tha are going to tranports vesicles. the unconventioanl are going to have the heads at hte c tmerinus or middle often dont tranprto cargo. Kinese 13 is going to depolymerize mcirubtules at plus ends. Kinesisn are not always on they can fold into ianctive comriton unpon singling they unfold, bind cargo ,engaeg mcirubteul, and begin movments

kineis is a plus end directed , walks along one protofilemtn and the setp is 8nm exaclty like th elnght of hte one a/b tubulin dimer . the movmetn is processive one head always bound the motoer doesnf fall off. differnent motors use dif protofilemnts. Movmente comes from atp driven conformational changes in the neck reiogn: it has the elading head and teh trialing head which laters. So one hea dbinds apt , atp hydrlozyes changes neck conofmriotn, the neck docs onto the head, this throws the other head forwards and the rols swithc. the power stroke of one head moves the other head forward. differnet atp state corresponds to L head ithglty bound vs lossely bound neck doced vs undoced. there ar efour majr stages four ocnoftrmaitons.

there is a lot of different kinesisns, most have the head the tail the cargo . the coiled coil that allows for dimerizaiton. there are other types that are refered to as the unconventional kinesis that have thier heads at the c temrinus or somwerhe in the middle. like kin 13 that is goig to diassemble microtubules. its binding to the proofilemtns at hte plus end to help aid in diassembly.These kinesins have an inactive and active form theyre not necessarily on all the tiem so tehy can fold into an inactive fconfromation as a reuslt of a signal.

optial trap to measure step size, myosin as it tursn out the step size is 8nm. which is the length of one tubuli dimer so what these thigns do is that they get on profielmtn and they step on every dimer walking down the length of that protofilament dragging the cargo. one head is always bound so tehy dont fall off. the movmetn of the kinesis - head and neck, the neck ahs two conformtaion, one where it extends away from teh head and anotehr where it is boudn to the side of hte head. The neck gernerates the movoemtn of hte motoer. so hea dhead can bidn atp and hdrplyze and that is going to infleucne the conformation of the neck.

thereis the leadign and the trialign head. and it atlernates. as a result o fhte atp hdyrloysis the neck is going to raptoe and throw the trailing head forward. and now the trialing is the aleding and the atp cycle happens . power stroke of one head throws the other head forward. there are four stages of the atpase cycel that generate these four confromation changes that is responsible for the movement.

the neck can be eitehr docked onthe sideo fhte head or away from the head. the neck not boudn to the head is going to rotate forward and bring alongisd ehte head. no atp bound o thte head it tihgtly bound to the mcirubtuela . so when atp binds it is going to chang the direciont of hte neck. so atp nekc binds to the ehad and throws the trialing head forwardy, hdyrzlie the atp into adp and we are going to go throught the steps againg.

kinesis are regualted htey fold up into an inactive confromation and then at the right them when they are needed for singlign theyre gong to unfold and become active theyre going to connec to their cargo. so in active teh tail is going to attach to the cargo and the microtubuesl are the raodways. priamry role in interphase is to move veicsles wher ehtey need to be. there are kinesis that are specialized for mitosis and they can bind micubteules and push the mcirubtuesl past eacohter. they dont move cargo . slding the spindle poles apart is going to help seperate the sister chromatids. So kinesis 13 is going to induce curvature in protiflemtns and facilitate dpeolymerizaiton.

so we had said that myosins and kinesis are going to be similar so what they did is the dyenisn were prufied and cloned the genes and saw that they have idfferne strucutre they are part of a family known as the AAA Atpase fmaily.

there are three domains of life, bacteira, archae ,and eukartoes these aaa atpase family members ar efoind in all three domains which mens they all game from a commone ancestor many biollions of eyars away. LUCA is the last univeral common ancestory.

So aaa atpase is goind to form a hexamer. sometimes it is6 domains that ie scoded in one long polypeptide by a singel gene. or six dif gens that codes for one domain . So dyneims are going to be these AA ATPASE

remmebering poly and proteases. the polyubiquitin antion marks proteins for degradation, and the cap of the protoseoms are going to recongize the polu and unfrod the protien by theading the proiein to unfold it. there are amino acids in

the chamber in which does the degradation. AAATPASE family membera have threading activity just like the proteaes. Dif fmaily members evovled to thread dif macromocleusl. So like the aaa atpase in the cap for degratdation because it is going to thread it throguht. in dna helicases thehelicase sperates doubel stranded dna into two and helicase gets onto one strand and moves down teh dna. so its threading the dna through it .

katoanin was a protien that severed mcirubteule, SPASTIN is closely related it is a tripel aaa atapase .so looking at the spastin katanin model it is docked on the side of our microtubel, it is going to grab onto the c termainl tail and thread it throught the central aaa pore . it is going to denaure the tubuling and weaken the microutbule a the severing site.

u have ur dna pol and want to rpelciat ehte dna. u do this fast but it keeps falling off the dna and ahving to restart. it slows the whole process down. u want hte polymerase to hang onto the dna but the problem is that the tighter ht epol hodls on the slower its goign to move along becuase u are bound tightly. to get onto th next site how do we keep the pol assciated and moving fast. we use a slingidn clamp to form around the dna but not touching it so that the pol doesnt fall off and can reattahc quicker cuz the clamp is keeping it there.

the clamp isnt a family member but the clamp loader is. the clamp loader is the aa atapse fmaily member. it has five subunits instead of six. it is going to form a pentameric strucutre and it binds to the clamp. how it works. the clamp loader binds to the sliding clamp. hydrlozes atp and it changes confriamntion and opens the ring of the clamp. dna is inserted throguh the clamp open and the clamose clsoes around athe dna clamp loader is relased.

in proteims it is threading proteins through a pore for degradation

in helicases it is threading dna

and in katian/spatin it is thread tubulin for disassembly.

in clamp loader it is open an assembl a protein ring around dna.

dynein is a aatapse member it has three parts, the head, stem and stlak. the head is the aaa atpase. part, the stem , the stalk. the stem is going to bidn ot he cargo and the stlak binds to the microtubuesl. stalk does the walk. most of them aer dimers. the classic one s that walk down the mcirubteusl need two dimers. the stem is the dimerizaiton reigon and binds additonal protien that can help it attach to whatver the carg ois. the stalk are going to contact with the mcubteusl.

so the dimers are large proteins/ the entire protien is part of a single gene.

so we used electron micropscopy in order to visualize walking for dyneins . they used atp vandate that cant be hydrloze in order to trap the dynein in the pre power strojke. vandate is replacing the phosphate.

so remeber that in kinsin when atp bind it isgoing to strenthgthen the hold onto the micurbtule from the head. in the case of dynein when atp it is weak and with no nucletoide it is strong binding so this is how we saw the post power stroke.

so we image through a electron miscrosocpy and we see that we are going to have thousands of iamge and we take the averge for the pre and post and we are going to ssee the conformation. it also is moving 8nm the lenght of a dimer.

so how does dynein connect veiscles to the stem. it is going to attach to a dyneactin. the dyein is the cab and the dynactin is the trailer attached. dynactin is going to connect the vessicles. the reason why we cant grab the membrane of the veisscle directlh because it is going to stretch out. so we use akryin and spectrin in order to help up build a shell around it. so this was orignally was for the spring like of the blood cells. the oriiginal role was used to move veiscles by stabilizing the membranes.

there is also going to be the of NUDE and LIS1 that is going to help and stablize the processvity of moving a heavy load. it slows down the dyenin but it is going to make it stronger. mutations of the LIS1 is going to cause brain defects because dynein cant work and u get smooth brain.

trafficking - the er is continous with the nuclear envelop the er goes to the golgi. from the golgi cargo is sorted and then sent out. motors move all veiscles and organells. by kineins and dyneins. humans have 45 kinesins genes and 16 for the dynin. many veiscles use both. this is so that we can move things that are part way between the edge and the middle . dynein and kinesis are going to fight for a happy medium.

so our cells are full of density they have a lot of stuff. cells evolved organelles. so that we can orgnize the cell and reduce dneisty because otherwise the vessicles would not be able to get around. so if it werent for micubtules and motors we coudl not have tranprto in an crowded cytplasm. organelles could not be maintained.

so animals change color,fish change color by incorproating pigment into their veiscles. to make a dark cell u contribute those veiscle through the cytoplasm and to make a light cell u clump them near the center. we use vessicles for this. do dynin and kines are going to mov ehtem. animals have colored piemgents.

aggregation means towards teh center. the veiscles move smoothly and disperison is toward the cell edges and veiscle move more irreugalr. so fish control the pigment movment through their nervous system. so dynein is going to moves in middle , kinesins, etc. we got. post tranlational modificaiotn of the mcirubtuesl, like adding tyrosines, glucose, other amino acids to affect their stibaility.

topic 12 -

so again the role of microtubules in interphase is to build map. inmitosis we are going to rebuild and generate two spindle poles. the chormosomes are going to be condensed there are two types of movment of chocomes. first they are going to move at the center of the cell and line up . and then the sister chroamtids will detach form eacohter and get reeled into the spindle poles.

remember that u can see chromosomes in mitosis only. naked dna has a diamter of 2nm. it is going to be packed by histone into 30nm. packing is important. because if u take a bowl of 200 mile sphagetti then pull on one it iwll break becuase it is tangeld. that is why the dna is ocndensed and packaged into choromsomes.

microtubules are reugalted by the cell cycle because we make more micubrutle in misotis than we need in interphase. in mitosis there are three classes of microtubules. there are kinetochore that bind to the chromosomes, polar which are going to extend towrad the choromosome sbut dont touch them. they overlap in the zone of interdigitation in the middle and then were going to have astral that are going to reach out towards the edge of the cell and provides structure.

the kinetochores are going to have hte inner layer that is going to have proteins that attach to the chromosomes. and the outer layer is going to face the spindle. the kinetochores do not anchor into the centromere/choromsomes

so before anaphse it is lined up and after ana the chromosomes is going to be seperated. so the cell doenst have eyes to know where chromosomes are at. So it sends out microtubules every way and stiablizes the microtubules that do attach to the chormsomes. so once it is attached the kinetocores are going to start reeling it in only when u have the proper attachment they pull apart. Then u pull them apart so theres a gap in between , if hte connections are improper they are mestable , the microubutels attach ot hte kinetic core but they wont lock on tightly, theyll try to pull but not stablize so they fall off. only when u conenct to both right then u can seprate

there is a protein complex that holds the sister chroamtids together. the complex has a kinase attachment that phorylates outer kintechore protiens. this is going to cause the mcirubtuels to bind weakly, to fall off. they only form tight connectio when not phosphoylated. so when they pull in both directions we disrup the complex and the kinases are inactive and the proteins get dephosphorylted

there are multiple motors asscoiated with the movment of chormsomes. ther eis an experiment that shows that there are som assciated iwth chocomroems arms . so if we set up a chrosomes attached to a spindle kinetechore. we cut the kinethcore microtubuels with a laser. so lets say we cut the part of the chromosome arm that isnt attached to the kinetocore it is going to move away from the spindle pole. this si prior to anaphase. prior to anaphase the motors on the arms are going to push them towards the center. once at anaphase the motors. turn off and then the kientocre wones will wind and reel teh spindle poles. so before anapahse there are motors that are going to push the chromosomes towards the middle.

centrosomes are the MTOC. when they divide they are going to make psindle poles and they are going to stay in the middle after divion so they dont go towards the edge. so when chocmoeosm are in the middle they will push on the micubruels but the spindle poles will remain at hte middle. if we were to have no spindle poles but we do have a centorsomes and mibrutels hey are still going to form a type like psuedospindles. so we dontneed a centromes ot make spindle’s.

so we have a bunch of motors that act on mcirubtuesl we have dyenins kinesis, here we have kiensin that bind to two anitparllel mcirbutesl it walks toward the plus ends of both and the mcirbutels slide past eachother and this helps the microtubules elongate for spindle’s. two dynins can bind to on mcirbutel and theu walk togehter and pull the mcirbutels towards the minus ends. the motors on the chromosomes interact with he micurbutels.

there are mechanism that help the micrubutles find the kinetchores. so its not as random. this invovles RAN. ran in nucelus cytoplasm trnaprot is how protiens get in and our of the nucelus. in mitosis there. is no nucleus and it breaks down so the mcirbutesl can reach out and grab the psindle poles. so ran has a new job. ran is in the gtp form and its going to stabalize mcirbutesl. So ran gef is bound and this is why we have ran at the gtp form suroundin the chormosmes. gap is around in hte cyospalms so as ran difuses from the chocmomes it is converted to ran GDP.

so ran gtp is around the chormsome and ran gdp is somewhere else.

RAn gtp is going to stablzi ehte mcirbtuel so that they keep growing in any way. ran is going to help stablize the ones that are growing in the right way. ran gtp facilates search and capture .

how do we mechanically reel in the chocmeosm and part of this sotry has to do with microutuel dynamics in particular prior to anaphase the mcirbeustl are going to treadmill, just like actin filemtsn treadmill. once we hit anaphase theyre going to depolymerize from both end and this si going to help move the chocmoes. we used a spekcly microopsy and wiht that we added flouscne tubulin and saw that move through th mcirbutels from the plus end to the minus end.

we are going to see the the tubuling over itme is going to add a the plus end at the kinethcore and over tim it moves towards the spindle poels. we depolyermize form the minus end a the spindle pole. we add tubuling at the plus end and that moves trhough until ithits pindle pole and tgets realsed at the end . if we go tthe minus end anchord in the spindle pole and the plus ends attached ot the kintecores then how can we have tremaidlling going on. if hte plus end is attached to the kintecore. isnt it

So in anaphase A there is going to be motor proteins that are going to pull the chromatids apart, and they are also going to depoalreymize btoh ends at hte same time. another thing that hapepns is anapahse B , the anitparllel mcirbutesl are going to push off and then the spinels are going to push away further to increse the idstna ce betweent. So the energy o fhte dyanins that are going to pull the chofmeosm toward the psinelds come s from the depolymerizaiton. Dam1 is going to form a ring around a mictbuel the ring grpst hemcietuel and slide along lattic. so it generates a forc etha is going ot ocnitneue pulling ht choemsone toward the spindle even tho there is no more track left. So dam1 is connected to the kinetocore and as the degradtion happens the curved splay out is going to push it further. The ring along is enough to carry out sepration no need for motor proeitns.

topic 15 -

so the point of cell cycle is to faithfully replcate and segregate your dna. So we have s pahse when we replicate and M when we segregaet. u can only look at mitosis and see tha tht chocmeosm are condens. but in g1 s and g2 u cant see thc hcomesone. so how do we study the cell cycle, we use flouscne acitivated cell sorter that i sgong to add dye to the cell that flouscne when bound ot dna. the amount of dna is prioptal to the flournsce so then we can divie the cells into groups based on flournce. tehnw e can seperate thos eclel into plots and figure out which one has x amounf odna and 2x maount.

okie so cells tha hve two copeis o feveyr choemsons have a 1c amount o fdna and afte rhtey udn er go repicaiton tehy have 2c. 1c is g1 and 2c is either in g2 or m. stuff betwenn is s underoign replciaotn.

a human cell takes 24 hours to divie. g1 would last 12 hours.

if u take cells from g1 and smoosh to s then the g1 is going to also repkciate meaning that s has factors . if we take g2 with s and fuse them the g2 does not under go replciation suggest that thers a block to a second round of replciton in g2. how do u replcia the dna once per cell cyel.

If u take a cell that in m phase and rfse any other cell to it liek g1 s or g2 the cells will also go into mitosos, itll breakd down it nucle3ar envelop, condense chormoems. there some component in the m phase clel that can drive any other stage o f the cell cyle into mitosis.

so xenopus eggs, frog eggs, is that these eggs are fertalized externally. they ar estuck in g2 untul they get ferizlied an donce they get ferizled they start diving. u can trige the event to cause them to begin to divide. theyre also useful biochemistry because theyre very large. they are 100 tyhousnad x timet he size of a normal human cell. u can see with naked eye. the cells are oign to divide synchronsly theyre all diving at hte same tiem as eachother that usefull because then u can make extrac tform dif stageso fhe cell cyel.

so all the cell cyels a the same time, theyr all in s pahse same time etc. the eggs are refered to as oocytes while thyer developing and hten they are an egg. durin gthis growth pahse which si nin montsh they are arresting in g2 . so the way the cell gets to be really big is it dones under go diviiosn, they arest in g2 and then grow and gro and gro and then later trigger hte evets of dsivon whe ferilzioatn. some evetns are triggered by hormones , so the oocytes arrest in g2 and grow until its ets a bigger size. and then hromoens form mon proesor is going ot caus ethec ell to go form g2 into m phase. protiens that dirvc ethe cell into mitosis is the same as meiosis. m pahse is either or.

so progesorin is going to trigger from g2 to meisois 1 and hten they can go through meiosisn 2 and ten ghey ger arrest and thant when teheyre an degge.

so emberu cell cyel sso we ant t. go vo ehe

cell extrac tna cells to do expeirme tand we use. xenopus. we gro th large egg cells oocytes and they are growing large because theya re arrested in g2. so then they get frezlied. the tranimtion from oocyte to egg is triggerd from protgestorone. while cells are grwoing they are restisatn to progestorne.

cell fusion, we take stages of the clel cycle and made interfeces based on dif properties . we are going to take a needl and take osme cytoplasm from one cell and inject it into anotehr cell. we take the mphas eyctoplasm and place it into the g2 site. we are going to transfer 1/1000000 volume of hte cytoplasm. this is going to. make the g2 to go the M. something in the cytoplasm is going to trigger it.

what is triggering it? on possibel there is a factor in m phase cell. anotehr possibility is that there is giong to be progestorne in the m phase egg. we can take a littel bit out o fhte new mphase cel that was oringally g2 again 1/100,000 and inject itinto a new g20 site and that also is going to beocme mpahse . u keep doing that and that tells us that it is not progesterino because it keeps getting diluted by 100,000 fold. so there has to be some ocmpoentne. this somethign is going to be maturation promoting factor MPF. two possibilits the MPF can eitehr be an enzyme or reugalte and enzyme . MPF is going to be inactive or active and present in g2 in inactive and present in mphae active. this maybe an autocatlytic because a little bti of MPF trigger the activation of a lot of MPF. one smiple model is that mpf turns itself on.

at what pahses of hte cell cyel is mpf active. we take extracts from g2 and then inject into our ooctytes and say that extract from g2 cell are not going to trigger that transiton. g2 cells do not have active MPF. as cells go into meiosis 1 they have mpf. if u injec the cytoplasm from the m into the ooctyes they will go into mphase.

if u injec the mphase yctoplasm and put it into the next s pahse they dont have mpf. mpf is aorund anytime when cells are in mphase but not when they are in any other time in the cell cycel. mpf is conserved from frogs to humans. i fu inject human nmphase to oocytes tehy are going to be in mphase.

tim -

when does tranciptn and trnalation start what ar ehte earliest protien that are made . he took ferilized eggs and added radioactive methionein to get incorpated ino th periona dn ran those on a gel over dif time points. after ferizliaotn.

some protiens liek b and c tht get tranlated and get mad emore over itme. there was one protien he saw that the amount increased and then decreased and isappeared. . and then increased and disaspeared . this is called cyclin. so the question became what is the proiten doing. two possibiltes is that it is a reugalto of the cell cycel and only made during that time at the cell cycel and its doing something to trigger the cell cycel trnaitons. other reason is that its just a protien needed during cell cycle. maybe those protein in s phase are used to replciate. this doenst mean theyr ereuglating the clel cyel it just menas theyre used for a part. reugaltion is builogical decision making. is it decing to change tranision or is just being used.

so they took frog eegs and then they dilted hte cytoplasm. they put the eggs in oil and they are big enough that if u centrifue them they are going to spill their cytoplasm. u spin and popopen and a cytoplasm comes out. theyre in oil and the cytopalsm is aquious u end up with pure cytoplasm at the exact same Ph the exat same clel conetrion its just pure cytoplasm as it was in the cell. u cant ehn add sperm cell that u can remove the memberan . u form a nuclera membera aroudn the sperm dna and the sperm dna goes dna replcaiotn, u get s pahse and the nucel3ar envelope break down, teh chromeosncondense and u form psindles.

u go througj M phase. and the whole thign goes into s pahse and the M pahse.

what is the role of cyclin? is cycling a reugalto or not? he took the extract that undergeso cycling and added small RNAase it degraded the messenger RNase. messenr rna is easy to destroy, so he drested hte mRNas but not the t or the r rnase. so the tranlaiton machienr is still intacte. he added back a mRNA fro cycling. so the only mRNA was cyclin. when he treated the extracs with RNAase they stoped cycling. he destoryes the mRNA the cycling stop some some mRNA is needed fo rthe cell cycle.

so mRNA is needed of rhte clel cycle, he then asked i cycling required of rhte clel cyel and its the only mRA that requied o fhte clel cycle? so what he did is that after he inactive the RNAse he added back cycling mr rna and hwen the adde dback the cyclin mRNA nowt thextract started cycling agian. so that told him two things . one that trnaltion of a protien is requied fo ryccling and that cycling needs. otehb tranlated and that the only thing tha tneed to be tranlated is cycling. that reveald that the cycling was in fact a regualtor o f the clelcyle.

so u produce cycling u get the trnaiton from g2 to m.

Cucling is produced and then degraded. he saw increase in level and dissapea3r and then again. he deted prtion of hte yccling genes to make truancated cyclign proteins and if u get rid of hte 90 amino acdsi a thte n tmeirnal end of the cycling u can undergo the g2 to m pahse but u cant get out of mitosis. u get stuck in M. so colelcitely these to told him that u have to produce yccling to undergo the g2 to M trnatiosn. and hten ger rid of cyclin to get out of mphase.

mitoti cell contain acivity that drives clels form g2 o m and that is ocnsived amon uerkatueos. cycling ellvels atelrnat eduring clel cyel and cyclin levels correalt with MPF. we get rid of cyclina as we exit m and we loose MPF as we exit MPhase.

MPF and cylin - so

topic 16 okei so geneti canyalsis

SO peopel are going to use pahges in order to build genetic pathways. so they are going to look atht assembly of pahges and wathcmthem get assmebled

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