topic 8 -

INTERMEDIATE FIlaments

there are six diffenre tueps ofitnermediate filetnms - theer a re u7- didifner egena. 210 cell tyesp

there are six dif types and 7- dif genes and therea re21- clel tyesp thsi mean on averge any intermeidnf ilemt souwld he xpressin roughly three cell types.

there are six dderinet ytepw na dseventy dif gene s are are 210 cedllo tyepsina verage any itnemedi fieltms owudl experss rouglth thee in eveyr clel. things mus bt

these ar ehighly xed i if hteyre only expression in a few cell they sub ecaislxe ov titme eo carr out pairtlfuintos in that regliong o

210 cell types 70 dif genes there are 6 di f tyeps. highly spexialized .

219 cek tes oabd thgere are 70 genes and there are 6 di ftyeps .

thes ethings msut be spaxleid there ar ehtree cell tyoes.

the basic sturcutre of hteunit hta is going ot be reatpedaed to gener dis a simder

itnermedial fiemtsn are oging otbe a idmer inteirmid ifelmts are going ot be a dimer. where we have to priod that ocme otghet. all famil mebre

intemedita filemtsnm are oign ot be a dimer, the dimer is going ot two protien that ocme oteht. all family memrebs have a coield coil reiognt hat are

htei nuclear localizing singal and hte prenyaltion therefor ethey arent gong ot bne un teuncuelus theyv voeld afte rnaimalsna dplants divered o saplants have it not aniamsl.

the lamins wer he irs to evolte they ar ehte progneiatos .

the lamies wer hte fist to eovle afte ranimsla nd plants idverd therefore they ar eoufn only naimsl during eovlito thes eycotpams intemra lost thei rnuceliziaotn singal

cytopalsms initermeid fiuletms evoedl afte ranimals and plants only in fnails during oeltiont ehs eyctoasm lsot their

re atramer n thei si soign go tb enonpoalr so it eodmes mater wher it is poitning. the \

ac itniflemtsn are poalr and mcinrutles have no poarlitmenaing no direicbntoaluy

Cytpplamis itnerme after \

so firs wer eht almsin then the cytoplasmis intermedia filemtsn los t

dimer the tail is C and the head is N the dimers are the lower sturcu

in most intmeriedl iflemtns fmaily memerb have their c ptoistnin inp opsti and n thei emena the idmer is antiparllela meanign the ptiotn i opena dn

msot intemreial fiemrls thei c ‘s are anitparlle poitign did reiocnt. if some mfailm mebrehave more merbsin ins ome the N and C are forughly ts ame but hte anitparlle dimer is arrengment is ocnsierved. to dimer forma. temtrarp in a n ainteirmamelreawl maner

beucaso ehte mre teinot fb

so basicdally di doens tmatter whta the siz eo the c and n the anitparll fo the C’s piotonignin dif directions are oging to be convied. two dimer associate to froma. atememta in an anitplrle lametn, beuca os thei arroudmnet temetr amerb ahve no paorlity. tetramerbs have no poarlitu tetramers havce o. poalrity teteramers hav eno porlity tetramer hav eno poarkit tetramer shav eno poarlity fiodling inteor e

immunogodl eodlorn mciriot foncomed this

it uses colg aprtilc attahced to anitbodie that are goingot bdint ot he C tmerial domaisn. the ogld aprticl es are going ot attach, and theyare going ot reveal as dakr psots to revela the locaiton fo teh C temrianl. this showed the c tmerinal poitning in opp

so it goes dimer - tetramenre - protofilament - protofibril -intermediate filametn

dimer - tetramer protiflemt protifbil itnermedia fimetns

dimer tetramer protieil progvibil intermedial fielmts

dimer tramil protifilmetn proitbirl interimeital idlemtne.

so basically the probfils are aoign og brian and htey is adds onto the stheri sineg and tubusl

fi if are stbale than other cysotkel aicntim may turn over 1 dmind due ot rpaidl rmeovin gin irnetmed ifelmtsn preseif o

for hours or venve longer

so intermeid fieltsm are hte stronges and more sitbaile.

FRAP can measure filament dyanmics - floursncely label IF proitne photblea a reiogn iwth alster track how logn it teka so frolsout to reutnr.

if If

rover of IF is slwo often teanekign 320 mint oa nd over meanignthe IF turno ver is slwo and

onter,eoda; foe;,stm

inermdiatl fialmets are highlyst able long live dcytoskel structure whos brian oriniaon contiroes t theri strnea nd grpa apiexmr e

anoteh expaeirment using flouclsel abele dIF moneroms , all monerms ar eoign ot be IF flosucley labels and this allwos us to see if new IF incortio ito the cystkel ove ritme

mcibtuels are tubes a shaw cshape ifemts made up of alpaha dn ebta tubulin tboth combiend can bind

mcibtuesl are asically a tube a straw shape cytkile filemt mad eof dimer sof alpha and beta utbnlign both cpmbidn can bind gtp .

mcibuteusl are basically a tube a strawa

MICURBTUELs are

so intermeidat eiflmetns - itis two dimer

Micbtuesl is a tube a straw shaped cytoelit mad eup fo hte idmer alpha and bet botcombined can bidn gtp .

mcibtuesl are tube straw shapenc cytkeotli mad eup of idmer alpha beta tubling both combined cna bind gtp.

mcitbuesl are a tube shwa shaped cytkepl fielmts up of idmer alpha and betal tublign botj can bidn gtp but only ebta nc ahdyrlzoe gtp to fdp.

were going t ot alakbatout butlgin

so the mibutesl are baiscdlal a tube a stra shap cytleil fieltm mad eup fo dimer alpah and eta tibling b th can bid n grp ony beta cna grpaj gtp

were toign

ALpha is always int h gtp fomr

thes ubtul idimer can attach ot each other in ane dn to end faion to make paroiflemtsn thrith firoant cna intera laterlay tomake a tube

So there ar etwo dimer alapha ad ebt an the thrithmemna emed a tueb thalpha is lawya sint he gtp and the bteta nc abydrlzi it

it si a stra whsong

thes eubtsiulst rimd ercna pinl ot make a proteimfiel and thrithteen prteesm can interc tiwht oenaother altlery if u looka the

alsph tubul i snex to another aphs anotuen

so there are six dfferen tyeps of interemdiate filametns 210 cell tyeps 7-0 genes that encode for this emaning that there is 3 per cell type. they are highlty sepcialized

the basic unit of these are stupid ahhh dimers the dimers are going to have a hea dna tail region. another thing is that the dimer are going to be n and c terminus and are going to be hea dna dtail the tail is the C and tha tials ar elarger andother thing is that htey have a ahelics so the a helics of eahc monoemr are oging ot o form the coiled coled reiogn and they are going to have amino acid rsides and their 7th amino aci resie are going ot be hhydrophobic and are gong to face each other and hide. another thinsi s that they dont need energy no nucletides no gtp no atpin order to assemble and sinc ehtey ar enot using enegryg they are oging to be in equlaibrium state. another thing is that they are going dimer + dimer is going ot form a tetramer, tetramer to tetramer side by side is going to form a protofilemnt and 4 of those on top are going toform a protofibril and four of htose are going to form a intermedial fialment.

The first intermediate filament to evolve is the stupid add lamin with thier nclearization

so after animals divered from anila si shwen they lost htier nuclarilzoaiton singal dn their prenyaltion ability so that is what that is

oh i forhot to mention that htey hav eno polarity intemredial filaments hav NO polarity they ahve no poalrity they hav eno poalrity because when the C and N attach to other C and n’s they are going to attach oposudte of eachother and that is going to lead to them not facing eachother and another thing is that faster let faster

os no polarity this was seen hrtough the goldmaen expeiremnt where they used a gold partile and antibody iwht an electron mciropty adn saw that the c’s are opposite and this is convserv ed through all faimilesyes

immunogold immuno gold

four protilfment smake. protofibile and foru protifbils wrpa aroudn eachother ot make and intermediate filament.

intermeidal filametn are most stable and strongest

4 4

the FRAP flosune recover after photo bleacing they were ablet o photbleac a segment of the sutpid ad ifnetiemralmetn and they saw that basially what happeeisn is that

frap photo bleaching - frap photo bealching is igong to allow us to see the intermeidla itelmtns recoer and it takes 20 -1 hour

acitn iflemns take 1-2 mins so we did the raph phtobtleachign in order ot see how long it takes foru flosucnece to come back another thing is

so they did the FRAP - was used to see how long it takes for them to recover

ad the flousce labale dmonoemrs to track if monoerms area ddign to exisitng areas. it toike 20

so they microinjected buotion lavele dkertine into clels

so frap and mcirubinected labeled keritn into cells

this is micronijected biotinc labeled keratin into cells

so anotehr

so we got the FRAP and the microinjected labeled flouscent monomers.

9intermeidate filemtns dont need acessory proteins in order to help them assemble

intermediate filetns dont need acessory proteins in order to help them assemble

so they used frpa adn the flouscnent labale dmonomer so tcheck how long it takes for repair to happen s

so these arent assembled with no help of no protiens awaht they did is that they injected intermediat eiflmetns dont neeed accessory protei in order to help them assemnle if u put dimer tetramer sin a cell they willfrom the inermediat eiflemtns if u take monoerm that build two tpes of intermeidl filemtnthey will form indnetluy form eahc other they dont need eac other to build.

thsi si micdrfinjected into

intemre dont need acessory protien in order ot help dsasmebl putting a dimer a temetra

if u inejct antibodies that block the asembly nothing will heppen to the cells showin

so in order to check their turn over they did the frap pothobelaching and then they did the fluonen tmonomer

to chekc whathappens to a cell if they stop intermediate assmebly hey to buil tw

they wanted to test what hapepns to a real orgnaism so what htey did was . is

Keratine i saother Intermediate filament -

what happens is that ekratine the intermediate filametn

there is keratine 10 on top and kertin 14 and we want to dirupt ekratie 14 to see what hapens in a live animal and basically before the CRISPR theu are going to going to make a trangenic animal with a dmonintant negative nc it shar do tget recssive and knowut both genes os htey se a engative domainant nege that is going to caus e a loss of ufnciton.the gene is going to bind to the fialment to sto p the addito of another monomer and this is goin gto amek a mutant protien that is going to stop and mke bad non funciotnal intermeidate filemtns an itnermeial fimetns fro one cell are going to bind to the other iF’s through transmembrean chanensl and that is going to hodl them tightly with eachother and if we basially mess with this we see that th emcie are more liel to get blsiters. and when we did this to a culture pobsvoisu teya rearont gonn gag emblsiters uc z thneya rent moing. Keraitn is going to bascialye ovlve form outside hard shelle insects.

diases of keratin 14 or 4 are going to cause blstier kera nail and ee probeslm

so back to it again

]

so we go tkerait 10 on top and 14 the bottom layer and we dirutp keratine 14.

pokei two more we got demsisn desmins are for muscle cels. demsins are formuscle clels. demsins are for msucle clel desmisns are for muscle clel desmisn are for muscle clels. demsisns are for msucle cells desmisn are for mucle clels.

desmins are for muscl cell they are going to bind to z disc fo sacromers togehta rnw tihtout hte se demsins not holding them together the msucle ownt be abelt ot withoodl form mcontiroan and ead te msucle fialure. nerosm are intemredi filemtsn neruon can exted

so demsin are oging ot be for sucle clels demsis ar efor msucle cells z disc scacromer a

and wrestanc efor oncitorn rubbing ogeher. dmeisn are oging to be for msucdle clel.

keiatn for hari

DESMINS - a man named desmin is ugly

demsins

desmisn are gong to be holding z disc’s and help agin t the sress ofcontriaontand if there is no demsin or mtuatino then wehv emuscle fialture because it wont resit the orce o fhte msucle contriaton rubbing together.

msucle fialure because it wont resit hte foce of the muscle ocntiraon rubbing otgehter. nuerofilmtsn ar eintemeid fielmtsn the are ging ot help with fast isngaling becuas oe hte nreil and sotp it form beindign and breaking. it prvides integrit

keraitn evovled form exosketlon form insects that had curnhc otuer parts keriant provided the mech integrity to now have u skin on the outside inetead of on the inside.

migraiton pattern from

from afir co tmiddle east we hav egeneshra tare commonot neaderthal s we mized with them and thenw e went ot middle ast sof te ghe ensare the ski peigmetn gen ein afirca we nened

demsins are going to b efor stopud ahh musle an dhten the neriflemet ae ogng ot be ofr neruson

micrubtuels re goig to be tube like strucutres and they are going to be fromed from dimers. the dimers are going ot have alpha and beta and the laphna is always going ot be in the grp forma nd teh beta is oigng to be abel to hdyrlzoyes hte gtp to gdp.

so the alpha nad beta the point wher ehtey

so the dimers make protiflema dn then protilfme

the dimer make protielfmet and then mcirbutbesl

the alpha enc to lapha dn tehbet ana dbe tthe potn wher eht lapha na beta touch are giong ot be the seams. this is simiarl to acitn ebcaus eht eifmere dimera in tint ehcytpla sma in the grp form

so the intermdilemt aifelmts

make protiflmetn profbiel then intemridal

the mcirbutl jsut make protienile sna dhte mcintiels tueb the b is abel tho dbryaoen

onc ehteyr ein the polyer they cannt ofrom ve gto whiel theuyrein the polyer.

s owhen theyre in the polyermer htey cant oceo rhte gdp theyre in the polery . the idemr is added ot the plsu end, its in the gtp form most o fhte tiubulin is in the utbilina in the gdp fomr. it addsonot hte plsu end hdyrpzlyes and cant realse

once theyre in the polyer they cant remove gdp theyre in the polyer.

Once theyre in the polyer they cannot

once theyre in the polyermer they cannot remove gdp

So the monomer so once the monoemrs attahc to the polyemr the gtp is hdrlzie and hte gdp canot be removed until the monomer i srealsed . the idmer is added to the plus end. its is in the gtp form mosot. fhteublib in the utublin the gdp form it adds onto the plsu end hyrlzoya n catn relase cane chan from gdp to gtp afte rht emonoerm realse form polymer.

onc ehteyre in the polyer

ohhhh each dimer is made of a tubul and b tubulin

So the idmer ocmes into the plus end and the b is hdyrlzoed

—

so bascially the mcirutuels are oging ot be straw liek and the idmes are oigng o to ahve alapha dn ebet utbulina nd the beta is alwaus ogng ot hdrualon the alpah snever and bcaislclyw aht hahpens is tha t

the dimer is going to attach tho the plus end and the b is going ot hdyraloz and up ituunil the monerms ir elased ofmr the polymer the gdp cannot befrmove.

so the strucutre fo the filia ng glaggeum is going ot be peiciton of hte sruface of a cell in orde rot move subtanc eiwlgl eon the soie

so where is micurbutles? they are going to be in the fillia nd flagellum. the iflia nad glagellum, the fillia and flagelum; they are going ot be in the fillii and flageum that is going ot be on the sufae oc a cell in order to move usbtan gen a diwglgle ont he usrcac.e the filaiu dn teh fielmt mcibrueslar eoging ot be in the iflia nd flagme that is going to mvoe sutbance and iwll ont he surjca

So whem mtitois happen we are ogng ot disassmeblthe cmirbutels ad rebuild to beomc psinld epoels that ar going to be used to segreate thechocmoems to beocme tow dualt clels.

so when mittois shappen swe idaisbel the mcibtuel and they wer are oging ot rebudintopsible poels to segerat eh chormoe sto beom teo wduatlher.s

So micbtules ar enicleate dna have micnutbeul reognainzin cnter calle dcnetornpsme s

SO mcitneusl are going to

So mcibtuels are going to be nculeatea nd going to have micirubtul ornaginzi vnters that are going ot be ecobeoms .

So mcibtuld s aorieng o tbe nufvlea dn the citbuel orntainz vente that are ogin to help rongvian the mcibteul

these are calle dcnoetomes.

the ncuelus isnt in the cnetne ro fhe clel its aua thte mcib tue onigna cnt erht ai th ecenterosome.

All phases ecpteo mitosi

during interphase thsi si one centosme the mcinruesl mins ends are anchored athe tncoetmes and the plus ends extend outwards toward the clel perihepry. thsi orngianz cell shap eand ognaiell pions and intriaicelluar tpraiot

duroi ,tiosim the coneotms are dpilie and make two psinel poles the psinels poales eareuiqvlent and theminsu ends ar ofmeinteionrmeind anchroed ot eh psiel blso the plsu end grwo outwards.

,cotineils asspciat e[rpote

Many maps bind ot the side of mciurbtules rather than the ends. Many maps bind ot the sid eo fmcibtuels rather than the ends.

Mpa bind ot the sides f mcirubtuesl rather than the ends. Mpa sbind to hte side of omicubtuesl rathen thatn the ends some maps can cross link mciubtutle sby bdingin two mciutbeusl ate one.

microtubuesl bind do the sides rather than the ends MPA bind to the sides rathe rthan the ends maps ind to the sides rather than the ends mpa sbind ot eh sides rathe than the end map sbind to hte side rahten athn the end Some maps can corss link mciubrutel sby bidng two at one Cerina map scan also link mcirbutels to IF’s

MAPs bind to micubrutel son the sides and because of this it can ocnenct two