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sliding filament assay by jim, this allowed people to look at movment of myosing motors in vitro and allow them to figure out things like which end is whihc white direction does the motor move, does it move ot hte plus end to the minus ?

why its importnat another reason. people studied myosin 2 motors intially because you could ge lots of them. you could go to the grocery store and get steak and get a lot of myosin from there. there are a lot of myosin family members that are lower in abdunce, so muscle celss have more motor 2’s So this assay is used to identy motor prpoeng that were ofund in cell types.

another thing is the idea about opitcal trap and it allowed him to study a single myosin motor and ow much force it generated. this was the first time ppl studied indivual single protein molcules. it opened up a field of single molceul biocheistry.

TOPIC 8

there are six dif type s and 70 dif genes there are 210 cell types and in averate any intermeidate filemtn would be expressed in roughly three cell types. these things must be faily specialized. if theyre only being expressed ina. few cell types they must have been specialized over time to carry out the particular fintion in that realtively small fractiono f cello types.

the basic structure of the unit that is going to be repeated to generate the intermeid filemtn is a dimer. where we have two protiens come togheter . all famly fmembers have a coiled coil region that are xtednreigons of alpha helices that wrap around eachohter. and those form very stable strucutres in dimers. its a general stratgey used in many cases to form dimer between two invidial protien omoclesu. the dimers havea. head reagion and a tail region. they dont bind nucleotides, no atp, gtp, no hydrlozying nucletoides. they dont use the erngy of atp hydrolyisis at all. there are equilirium polymers because there is not input of enregy to conver them to a stead state polymer

they dont need no energy assembly or disassebly so they are NOT steady state polymers.

lamin are the first family member that evolved. they are nuclear intermeid filmetns found in the ncuelus and some are prenylated and attached to the memerbate to provude a sytem for the nuclear envelope.

the cytoplasmic intermediat filmetsn areose after naimals and plants diverge so therey only found in aneimals and as they evolved they lsot the nuclera localization singals. so unline lamits theyre going to get stuck in the cytoplasm. cytoplasmic ones los tht prenylation site so that is why theyre in the cytoplasm.

the alpha helical coiled coile region on averatge on turn of alpha helix is about 7 amino acids as the alpha helices wrap around eachother, every 7th amino acid, the side group from one of the coiled coil regions will be factin the other one. and often those ones that are facing eachother are hydrophbic and it cost a lot of enregy to have hydrophibic residues on the outside of proteisn. so what they do is bury those hydrophbic resides in between the two alpha helices. once dimized never apart. ociassional. you have a charged amino acid if one alpha helix is a negative charged amin acid it typically pairs with a psotive charged one on the other alpha helices so that these things are desinged to be paried and bascially zip up. if we look at the dimers we can ask what th earrangmetn of the monomers on the. dimers. so each o the monomers has a head region and a tial region.

so as the helices wrap around eachother, the helices wrap aroudn eaochter and every seventh amino acids resident face eachother. They residents are hdyrpobic and they are buryed between the two helices is favoraly.

in the tail region is larger than the head region and if u look at the dimer the two tail regions are on one end and the amller head regions are on the other. if u take two dimers and put them together they from a tetramer, the lowest order structure is the dimer and then the dimers can get together to form tetramers. if u look at this tetramer in the electorn microscop u cans ee that the c temrinal reigon is poining out ineither direiton this means that the tetramer does not have any poalrity.

it donest face in one particular direciton is looks the same goingin one direciton as the ohter. in both sides the c is futher out than the N. since the higher order strcture is build by putting tetramers togethera nd they are not polar then the intemedi filmetn itself will not be polar. it will look the same going on direction.

what did actin polarity allow for ? becaue actin filmetns were poalr any motors that moved. along them move in that prticular direciton. so myosin move from minus ends to plus ends. that i svery useful because it prov des a direcitonal roadway for the motor to move donw. if u have itnermediate filemtsn that are not poalr hte motor can move randmoly and any direction it happened ot be facitn. so. not motor proteins that fucntion on intedmiate filemts becuase they dont have any direcitonality and thefore motor ptoeins wouldnt be parituclary useful getton ont here becaue it would be running in ranodm direciton.

Some intermediate filmetsn got lots of fmaily members the N and C termini are about ehsame size. do all family memebrws arrance in dimers the same way?with the two C poining one way and the n the other way? this was answered using the immunogold electron micropcy. sothe wya it works is that u shoot electrons at a sample and whatver blocks those elctrons will show up as something dark. its the blocking electron that u see and the problem with most bioglical sample and protin is that they don t black x rays very well. x rays go right thorugh . what does block electrons very well is emtal, so in immuno electron miscopry make a antibody against a part of a protien and attach a gold particle, a metal bead and that gold particle will travel along with antibody. so lets say the antibody binds to the C terminus and the gold body. antibody goes there and stick it in the electro micrscopr e and ask wehre the antibody and u do that in very case that people have done this, there is the arragment of two c temreini poitning in opp direciton. all interemdi filemts are non polar and hav eno protien to mov elaong it.

in most

again tetramers are non polar. to build the filemtsn the tetramers are going to line up one after another to make a protofilametn. four protofimnts interact with eachother laterlay to make a protofibril. and then four of those protofibrils wrap around eachtoer to generat the overall intermedi filemtsn.

foru protfilmets tha tmake up a protfibril and four protfibrisl that make up the itnermediat filmetnso that overal we have 16 protifilmetns that make up the itneremdi iflemtns. .

since the potbirils wrap around eachother they look like braided wire . if we look at th eintermediate filemtn stability it turns our that intermeidate fielmtsn are stable, once they form they presist for a long time, other actin filemtsn. lik at hte leading edge where those filemtns may have a life or only two mins and osmething like that =they turn over very rapidly as a resuytl of reamidling , here were going to do a FRAP expeiremnt.

were gonn have flouscently labeled intermedi filmetsn and zap with laser lgiht ot phot bleach and loov over time and then track recovery time. after 20 mins or even longer maube eand hour do they fully revoer this indiates that these filemtns are realtively long lvied, theyre stabl they can turn over but slowly.

another expierment we have here is a cell that has an intermediate filemtns cytoskelton , what were doing is tht the same clel is going to be inject flouscnely labeled monomers and imer sna fter 20 mins osme of hte monomer and dimer come together to form a little itny filmetns or little tiny sectionf ofilmtsn and its only after seval houyrs that they all get incorpated into the previosuyly exsisitng cytiolsleting, they have long half lifes stable and do turn over at a long time scale.

intemiea filmetns have no accessory protein that help intermeid filmetsn assmeb l. in vitro in the tes tutb u dont need any addtionoal protein jsut take intermeid filmets the from dimers, tetramers and assemble filemtns. most cell types have only one or two types of intemediate filmtsn out of the 70 family members, those that have two types and take the mnomers of those two types in citro and throw them into a test tube theyll assembly indeptnly of eachother. sol filemtsn type 1 forms its one indepently of intermediate type 2. they have all the infro they need for self assemmnbly.

what did they do? how to answer that ? poeple aksed this quesiton intailly was to make blockign antibodies, anitbodies that would bidn ot the monoemr and dimers and prevent them from assembling into full size filemtns . in a test tubes we sho that happens. they took those anitbodies and inejcted into tissue culture cells in a petri dish i a lab and aswed what hte phenotype was they looked at hte cells and tehy saw that there were not intemediate filemtsn. so the anitbodies worked. but the surpigins resutl was that the cells were perfeclty find and there was no observalble defect in the cell.

you got these conserved protein, large family member of protiens, theyre expressed in highly specific ways in a cell types in the human body , yet u inavtive them and nothing happens. people asked what happens in a living organism. so this tissue culture is an articial sutition. non of our cells exsist by their own, u can sort of think of blood cells that way but most of our cells are in tissue sinteractin with their. neighbors so is there something about intermediate filemts that requied in a lving organims in a whole animla thats not required in indivual cells in tissue culture outside .

people wanted ot inactivate the intermediate filaments in a particular tissue and see what happens to the animals as. awhole so this was done in mice on keratines. keratins are expressed in the skin, in the epithiulium . so we have dif layers of skin and it doesnt mtter what the dif layers are and there are dif keratines. in dif layers. so keratin 10 is expressed in the dark brown cells in that layer and keratine 14 in the lower layer so the goal is to disrupt on of these kertain so that we dont make keratins in the skin layer and see what happens. so you would od it using CRISPR. CRISPR allows u to go in and delte both copies of a gene, or modifyt the gene to make amino acid changes. these expreimetns were done before CRISPR was disocered and invented a a tool. PRE CRISPR

the probelm is that we want to dirupt intermeidate filemtsn, we dont want to make any, loss of function of intermediate filemtns, loss of fucntionis associated iwht recessive allels. so we want ot make a transgenic organism in whihc we deted both copies of hte interemdiate filemtn gene, before CRISPR this was hard to do. it was easier to make transgenic animals that has. a single copy ebign added . in this case we owuld need tht to be a dominatn mutaiton. and even more that if u start with a wild type mouse and befor cirps when u added a new gene that gene would typically insert randomly in the chormsome so at the wild type locus u would have two copies of the wild tyep intermeidatefilemtna gena dn then somewher elese on that chromosome around another chormosme u would have ur mutatn intermeditate filemtn gene. which means that u would only see a phenotype if u put a dominat mutaiton into that mouse.

problem is that most dominat alles are gain of funciton not loss of fucntion. so how to put a third copy of an intermediate filemtn gene and see a pheontype iwth loss of function. there is a rare type of mutation that is dominante negative mutaiton. it is dominatnt so if u put in ane xtra copy of that gene it will have a phenotype , but its unsual that rather having a gain of funciton it will ahve a loss of fucniton phenotype so the phenotype assoicated iwth a dominatn negati mtuation is simular ot null phenotype of removing both copies of the wild tyep gene. the domain negative mutations work is that the domiantn mutant form of the gene produces a protien that interferes with the funcitono f the wild tyep protein.

so our wild type is going ot make a long polymer, to that cell were going to add a bleu gene that makes a mutant form of the protein and that mutant form of the proign can be addedonto the growing end of the polymer but no more monomers can be added after that. Dideoxy sequencing =u have nucletoides that lakc a criticalo hydroxyl group so that once your got a growing dna chain u put in an A that doesnt have a hydroxyl group and that dna stops elongating and that tells that an A was at that particular postion. so its similar. u add a blue monomer and then u cant add any more red monomers so we end up with a little tiny interemdiate filmetsn and they cant forma bigger filemtns. this is the poison polymer model where the dominant negative allele makes this dominante negative protien that once it iassmbles into the polymer it terminates .

it will have a dominant phenotype that coresponts ot he losso fo fucntion pheontype.

so we put in a mutant allele, make a trangenic mouse, but we use a domiantn negative alle of keratin 14 , that mutant form of the protien dirutped hte intermedi filemtsn in teh skin cell s and saw the pheontyp of hte mouse. wild tyep mouse has skinc ells that are nicley adhered and in the odminant negative mouse tha leyr of skin cells is compeltley direutped . there is no longer tught layer of cells laong the skina and they form blsiters. we dirutped hte integrity of the layer of skinc ells.

itissue culture had no effect because in tissue culture we have large indivual cells and there was nobody runnigna roudn mechnaically dirupting them they were just laying there . so there was no stress, in hte mice as their skin gets rubbed as they move around that littl abraison or rubbing would nt form blsiuters on us because our skin cells are tighlty held but if u get rid of the intermeidate filemts now lightly rubbing is going to form a blister.

so the intermediate filematn cytoskelon of one cell to the cytoskelong of the neighboring cell, and so all those regions of tying the cells togher are what allows these cells to resist mecaila stress it holds tehm togeher in a tight sheet of cells. if u get rid of the interemediat filemts the neighbors are no logner tighly holding out and cant resist mechanial stress and get dirupted under mechanical strain.

so the cells attach to tranmembrena protiens on the neigboring cells that also conenct to the intermediat filemtns . that tighst each cell togeher byt tightening their cytoskelong togher and tha cytoskelogn is the ridgind framowkr that gives us its shape and strucure.

so the intermeidates of one cell are coonnnected o the itnermediate filemtns of the other cell through transmembrans that is what makes them tihgly boudn togeht4er. the IF cytoskelgon acts like a rigid framwork insid eeahc clel the givn teh cell shape and strucutral uspprti

there ar ehuman disease that are base on the exact same sort of thing where we have dif kertins expressed in dif cell types and there are dom negative allels in the population that cause these kidns of diesases. so in blistering in mice are caused by allels that affect keratin 14 or 5 . we have. bunch of dif disease that ar espeific to dif tiseisses where we get blisters, lossing hair, nails, or problesm with surface of eye or esophause .

we got 70 dif genes that are in six clsses .

so we got DESMIN - demsin ties sarcomers together and this i. in sketal muscle cells . so we got the z disk, interemdiate filemts that tie the z disk togeher. so the top sarcomer we tie to the other ones /neighbors. muscle contraction is a violent thing. so rubbing the cells in ur msucle as they contract causes stress. if u make mutation then u have genraized muscle failure . we have neurfilmts the intermeidate filemts found in neruson, they do two things in neruons,

best example is the enrve that goes donw ur toes, and the cell body for that nerve is in ur spine. so three feet away. those axons thos extnsiono f ur nerve that go from spine to ur big two and are three plus feet longa nd conduct an electircal signal if u looka t a cross section of thos enueron her the neruons . u can see the black dots that are intemediate filemtsna dn neurons a full of intermeidate filemtsn but majority of neurons are intermediate filemtsn. that allows those neruons to have a bigger diamter than noramlly would because u would maek so many filemtns that they jsut get big and that means faster the electrial singal moves down the neruon . they bulk up neruon so that they can conduct electricity faster. second they provide mecahnical integrity,

if the neuron were a copper wire and lets say i bend my knee how many times would it take for the copper wire to break. these itneremdiate filemts provide emchanial integratiy to our neruons. the neruons from spine to big toe last ur entire life without breaking ebcause u walk ebcause the intermediat eiflemts provide the mechanial integrity so that they dont break.

ight so we've got these different family members

the lamins supporting the nucleus neurofilaments

supporting our neurons desmond and muscles dealing

with integrity and our keratins in our skin cells.

Everybody good with that?

All right, we've only got a few slides left,

so let's talk about the evolution of keratins.

The keratins, the ones in our skin,

seem to have arisen in evolutionary time at the

time we made the transition from animals that

had exoskeletons, like insects, that have

crunchy outer parts, and all their soft parts are

inside to us, animals, that now have a skeleton

on our inside and our soft parts on the out.

And so the idea is that the keratins provided

the mechanical integrity to now have your

skin on the outside instead of on the inside.

And so our whole body arrangement

evolved as a result of the benefits

provided by intermediate get filaments.

Okay, it allowed us to have the skin on the

outside and to integrate cells into tissues.

The last part, which hopefully we've got,

yeah, I've got a minute to get through,

which we can, is humans initially arose in

Africa and then spread throughout the world