Cell Bio- Exam 2 Slide Set 5: Vesicular Traffic, Secretion, and Endocytosis Vesicular Transport Proteins are synthesized in the ER, then are moved from ER to golgi, once mature proteins are formed, they need to leave the ER (Secretory) After golgi, they have multiple different pathwaysConstitutive secretion- constant secretion of proteins from cell, golgi to out of cell regulated secretion- secretory vesicle takes protein out of cell from golgi Endocytic- early endosome takes proteins from membrane to late endosome and then sometimes to lysosome Microscopy study with GFPstudied t r a f f i c k i n g v i a G F P v i r u s p a r t i c l e s trafficking via GFP virus particles t r a ff i c kin gv ia GFP v i r u s p a r t i c l es use temperature, i f t e m p i n c , p r o t e i n m v m t b l o c k e d if temp inc, protein mvmt blocked i f t e m p in c , p ro t e inm v m t b l oc k e d you can track proteins via fluorescent microscopy results: t h e r e i s t r a f f i c k i n g w i t h i n t h e c e l l , y o u c a n g e t a r o u g h e s t o f t h e t i m e t h a t i t t a k e s there is trafficking within the cell, you can get a rough est of the time that it takes t h ere i s t r a ff i c kin g w i t hin t h ece ll , yo u c an g e t a ro ug h es t o f t h e t im e t ha t i tt ak es tracking total fluorescence signal over time Oligosaccaride modificationmannose trimming occurs when oligosaccaride moves from ER to golgi treated with endoglycosidase D which cleaves sugar from protein Vesicle Budding and Fusiontransport vesicle leaves donor compartment transport vesicle fuses with target compartment Coated Vesicle BuddingSNARE protein helps transport vesicles recognize target membranes membrane cargo protein and soluble cargo protein bind together coat proteins surround vesicle Uncoated vesicle fusionV SNARE proteins will interact with T SNARE proteins on membrane R a b s p r o t e i n − c a n h e l p r e c o g n i z e w h i c h t a r g e t m e m t h e y s h o u l d f u s e t o o Rabs protein- can help recognize which target mem they should fuse too R ab s p ro t e in − c anh e lp reco g ni ze w hi c h t a r g e t m e m t h eys h o u l df u se t oo Three types of coated vesiclesClathrin coated - helps with transport from trans golgi network to late endosome and helps transports obj entering the cell via endocytosis have heavy and light chains, as well as binding site for assembly particles soccer ball structure Functions:help form m e c h a n i c a l f o r c e t o f o r m v e s i c l e mechanical force to form vesicle m ec hani c a l f orce t o f or m v es i c l e coat subunits bind to surface of donor membrane clathrin and other proteins help form bud/vesicle and help with the mechanical force of budding off capture membrane receptors c l a t h r i n a n d a d a p t i n ( b o u n d t o g e t h e r ) b i n d t o c a r g o r e c e p t o r b o u n d t o c a r g o m o l e c u l e s i n m e m b r a n e , a n d t h e n s t a r t b u d d i n g p r o c e s s , clathrin and adaptin (bound together) bind to cargo receptor bound to cargo molecules in membrane, and then start budding process, c l a t h r inan d a d a pt in ( b o u n d t o g e t h er ) bin d t oc a r g orece pt or b o u n d t oc a r g o m o l ec u l es inm e mb r an e , an d t h e n s t a r t b u dd in g p rocess , adaptin helps transmem receptor bind to coating proteinscertain aa are carried that signals adaptin to bind, these are then phosphorylated D y n a m i n Dynamin Dy namin r e q u i r e d f o r p i n c h i n g o f f o f c l a t h r i n v e s i c l e s f r o m d o n o r m e m b r a n e required for pinching off of clathrin vesicles from donor membrane re q u i re df or p in c hin g o ff o f c l a t h r in v es i c l es f ro m d o n or m e mb r an e polymerizes around the neck and then hydrolyzes GTP, conformational change initiated in dynamin that stretches vesicle neck until the vesicle pinches off C O P 1 − i n c h a r g e o f m o v i n g p r o t e i n f r o m t r a n s g o l g i b a c k t o E R COP 1- in charge of moving protein from trans golgi back to ER COP 1 − in c ha r g eo f m o v in g p ro t e in f ro m t r an s g o l g iba c k t o ER coatomer coated intra golgi traffic, golgi to ER A R F ARF A RF C O P 2 − h e l p s w i t h p r o t e i n l e a v i n g E R t o c i s g o l g COP 2- helps with protein leaving ER to cis golg COP 2 − h e lp s w i t h p ro t e in l e a v in g ERt oc i s g o l g coatomer coatedS a r 1 u s e s C O P 2 c o m p o n e n t s Sar 1 uses COP 2 components S a r 1 u ses COP 2 co m p o n e n t s GTPasesActive- when protein binds to GTP G A P − h y d r o l y z e s G T P t o G D P GAP- hydrolyzes GTP to GDP G A P − h y d ro l yzes GTPt o G D P Sar 1 initially binds to GTP, then binds to Sec 12 to hydrolyze GTP, then recruits COP2 components to have GTP bound to mem S a r 1 − c o n t r o l s c o a t a s s e m b l y o n C O P 2 v e s i c l e s Sar 1- controls coat assembly on COP2 vesicles S a r 1 − co n t ro l sco a t a sse mb l yo n COP 2 v es i c l es inactive- off, GDP bound G E F − r e l e a s e s G D P s o G T P c a n b e m a d e GEF- releases GDP so GTP can be made GEF − re l e a ses G D P so GTP c anb e ma d e A R F ARF A RF p l a y s r o l e i n c o a t f o r m a t i o n i n C O P 1 a n d C l a t h r i n c o a t e d v e s i c l e s , i n t i t i a l l y b i n d s t o G D P plays role in coat formation in COP1 and Clathrin coated vesicles, intitially binds to GDP pl a ysro l e in co a t f or ma t i o nin COP 1 an d Cl a t h r in co a t e d v es i c l es , in t i t ia ll y bin d s t o G D P What are the molecular signals on vesicles that cause them to bind only to the appropriate target membrane? S N A R E S a n d R A B G T P a s e s SNARES and RAB GTPases SN A RES an d R A BGTP a ses generate tight interactions, help vesicles fuse to the donor membrane RAB GTPasedonor mem: RAB receptor, vesicle: RAB mediate diff transport vesicles fused to diff transport membranes many diff RABs in eukaryotic cells How do transport vesicles and their target organelles fuse? S N A R E a n d R A B h e l p v e s i c l e r e c o g n i z e d o n o r m e m b r a n e SNARE and RAB help vesicle recognize donor membrane SN A RE an d R A B h e lp v es i c l ereco g ni ze d o n or m e mb r an e R A B w i l l n o t h e l p f u s e RAB will not help fuse R A Bw i ll n o t h e lp f u se Vesicle Fusion MachineryVesicle Docking: V SNARE and T SNARE associate, RAB binds to RAB receptor Assembly of S N A R E c o m p l e x : SNARE complex: SN A RE co m pl e x : SNAP 25- snare complex, includes V S N A R E a n d S y n t a x i n V SNARE and Syntaxin V SN A RE an d S y n t a x in generates strong force to help fusion to the membrane t w i s t e d v e r y t i g h t l y t o g e t h e r twisted very tightly together tw i s t e d v ery t i g h tl y t o g e t h er Membrane Fusion proteins work to untwist SNAP 25 fusion of membranes occurs Disassembly of SNARE complexes SNARE complexes disassociate and are free for another round of vesicle fusion, RAB also disassociates from the RAB effector Steps in Secretory Pathway cont Vesicular Transport from ER to Golgi protein always goes from cis to trans face of golgi cis cisterna→ medial cisterna → trans cisterna E R r e t e n t i o n s i g n a l ER retention signal ER re t e n t i o n s i g na l C i s t e r n a l p r o g r e s s i o n t h r o u g h g o l g i g l y c o s y l a t i o n Cisternal progression through golgi glycosylation C i s t er na lp ro g ress i o n t h ro ug h g o l g i g l ycosy l a t i o n removal of 3 mannose residues in cis golgi ( − 3 M a n (-3 Man ( − 3 M an protein moves to medial golgi by cisternal maturation 3 GlcNAc residues added , 2 more mannose removed, single fucose is added ( + 3 G l c N A c , − 2 M a n , + F u c o s e ) (+ 3 GlcNAc, -2Man, + Fucose) ( + 3 Gl c N A c , − 2 M an , + F u cose ) processing completed in trans golgi by addition of 3 galactose residues and linkage of N-acetylneuraminic acid residue to each galactose ( + 3 G a l , + 3 N A N A ) (+3 Gal, + 3 NANA) ( + 3 G a l , + 3 N A N A ) R o l e o f g l y c o s y l a t i o n Role of glycosylation R o l eo f g l ycosy l a t i o n p o s t t r a n s l a t i o n a l m o d i f i c a t i o n post translational modification p os tt r an s l a t i o na l m o d i f i c a t i o n helps protein become hydrophilic→ aids in folding aid in transport (rarely- targeting to lysosome) resistance to proteases (stability) protein protein interactions Vesicular sorting at trans- golgi network Vesicular Trafficking to Final Destination (golgi to ___)Endosome Plasma Mem constitutive secretion- unregulated membrane fusion regulated secretion- regulated membrane fusion Lysosome some proteins go here very acidic environment v class pumps used with ATP to pump proton inside lysosomes form a functional hub for cellular trrafficking pathwaysER→ Golgi→ lysosome Pinocytosis→ lysosome Phagocytosis→ lysosome autophagy→ lysosome H o w d o e s c e l l k n o w w h i c h p r o t e i n s a r e s e n t t o t h e l y s o s o m e ? How does cell know which proteins are sent to the lysosome? Ho w d oesce ll kn o ww hi c h p ro t e in s a rese n tt o t h e l ysoso m e ? M 6 P r e s i d u e s ! M6P residues! M 6 P res i d u es ! receptor on trans golgi network that will bind to M6P and will incorporate into vesicle and then will go to late endosome if pH low in late endosome, M6P transferred to lysosome Lysosomal Storage diseasescan be due to absence of 1 or more lysosomal hydrolases or the mistargeting of lysosomal hydrolases characterized by tissue destruction or accumulation of undigested macromolecules I c e l l − p r o t e i n s t u c k i n t r a n s g o l g i , s e v e r e t i s s u e d e s t r u c t i o n , G l c N a c D e f i c i e n c y I cell- protein stuck in trans golgi, severe tissue destruction, GlcNac Deficiency I ce ll − p ro t e in s t u c kin t r an s g o l g i , se v ere t i ss u e d es t r u c t i o n , Gl c N a cDe f i c i e n cy Endocytosisgoes through plasma mem, through early endosome then late endosome, then lysosome p i n o c y t o s i s pinocytosis p in ocy t os i s very tiny things; proteins, lipids. Goes through e a r l y , l a t e , t h e n l y s o s o m e early, late, then lysosome e a r l y , l a t e , t h e n l ysoso m e continuous process, rate depends on cell type pinocytotic vesicle forms from clathrin coated pits in plasma mem r e c e p t o r m e d i a t e d e n d o c y t o s i s − l i g a n d b i n d s t o c e l l s u r f a c e r e c e p t o r , c l a t h r i n h e l p s t o f o r m v e s i c l e , c l a t h r i n c o a t s v e s i c l e receptor mediated endocytosis- ligand binds to cell surface receptor, clathrin helps to form vesicle, clathrin coats vesicle rece pt or m e d ia t e d e n d ocy t os i s − l i g an d bin d s t oce ll s u r f a cerece pt or , c l a t h r inh e lp s t o f or m v es i c l e , c l a t h r in co a t s v es i c l e p h a g o c y t o s i s phagocytosis p ha g ocy t os i s large things like bacteria; p h a g o s o m e t h e n t o l y s o s o m e phagosome then to lysosome p ha g oso m e t h e n t o l ysoso m e feeding for lower single celled euks multi celled orgs- used as a defense against invading microbes requires surface receptors, triggered event a u t o p h a g y autophagy a u t o p ha g y from ER, if we do not need certain organelles anymore, a u t o p h a g o s o m e f o r m s t h e n t r a n s p o r t e d t o l y s o s o m e autophagosome forms then transported to lysosome a u t o p ha g oso m e f or m s t h e n t r an s p or t e d t o l ysoso m e LDL Uptake LDL- byproduct of fat transport, have ApoB protein A p o B a n d L D L r e c e p t o r b i n d ApoB and LDL receptor bind A p o B an d L D L rece pt or bin d vesicle begins to form with help of clathrin coat transported to early endosome→ late endosome→ lysosome Disorders- LDL receptor missing, receptors do not associate with clathrin coat Fate of cell surface receptors after endocytosis recycling of receptor to same domainreceptor transported back to surface of membrane and pH will change→ receptor ready to bind to another LDL particle degradation of receptor after endocytosis t r a n s c y t o s i s transcytosis t r an scy t os i s a n y p r o t e i n t h a t i s m i s s e n t t o b a s o l a t e r a l s i d e w i l l b e r e s e n t t o a p i c a l m e m b r a n e s i d e any protein that is missent to basolateral side will be resent to apical membrane side an y p ro t e in t ha t i s mi sse n tt o ba so l a t er a l s i d e w i ll b erese n tt o a p i c a l m e mb r an es i d e t h e v e s i c u l a r t r a n s p o r t o f m a c r o m o l e c u l e s f r o m o n e s i d e o f a c e l l t o t h e o t h e r the vesicular transport of macromolecules from one side of a cell to the other t h e v es i c u l a r t r an s p or t o f ma cro m o l ec u l es f ro m o n es i d eo f a ce llt o t h eo t h er Slide Set 6: Microfilaments The Cytoskeleton Functions of cytoskeletoncell shape, mvmt, and contraction organelle mvmt and organization cell division intracellular org and vesicle mvmt interacting with signaling pathways basically like the bones of the cell ComponentsMicrofilaments actin filaments, thinner Microtubules tubulin dimers, thicker Intermediate filaments various, diff proteins combined together Cell signalingsignals tell cytoskeleton abt organization and mvmt of organelles as well as changes in cell shape, mvmt, and contraction Actin Microfilaments Functionsorg of intracellular organelles and transport of vesicles (myosin) intracellular mobility (bacteria) cellular stability cellular motility muscle contraction Lamellipodiumsupported by growth of actin filaments, generates a protrusion structure to adhere to surface and move cell forward Polymerization and Dynamics1 actin filament= 2 strands one + end (0.12 M), one - end (0.6) g actin is monomer, microfilament polymer of actin ATP binding cleft in actin structure alpha, gamma, and beta actin: all associated with diff structures G a c t i n p o l y m e r i z a t i o n G actin polymerization G a c t in p o l y m er i z a t i o n g a c t i n b i n d s t o f a c t i n , e l o n g a t i n g e x i s t i n g f i l a m e n t g actin binds to f actin, elongating existing filament g a c t inbin d s t o f a c t in , e l o n g a t in g e x i s t in g f i l am e n t c a n b e a d d e d t o + a n d − e n d , a n d l e a v e f r o m b o t h s i d e s can be added to + and - end, and leave from both sides c anb e a dd e d t o + an d − e n d , an d l e a v e f ro mb o t h s i d es g a c t i n d e c t o c r i t i c a l c o n → p o l y m e r s h r i n k g actin dec to critical con→ polymer shrink g a c t in d ec t ocr i t i c a l co n → p o l y m ers h r ink g a c t i n i n c a b o v e c r i t i c a l c o n c → p o l y m e r i n c i n l e n g t h g actin inc above critical conc→ polymer inc in length g a c t inin c ab o v ecr i t i c a l co n c → p o l y m er in c in l e n g t h Actin Binding ProteinsPolymerization- Profilin and Thymosin B4 P r o f i l i n − p r o m o t e s p o l y m e r i z a t i o n Profilin- promotes polymerization P ro f i l in − p ro m o t es p o l y m er i z a t i o n T h y m o s i n b 4 − b l o c k s p o l y m e r i z a t i o n Thymosin b4- blocks polymerization T h y m os inb 4 − b l oc k s p o l y m er i z a t i o n L e n g t h − C o f i l i n , G e l s o l i n Length- Cofilin, Gelsolin L e n g t h − C o f i l in , G e l so l in N u c l e a t i o n a n d b r a n c h i n g − A r p 2 / 3 Nucleation and branching- Arp2/3 N u c l e a t i o nan d b r an c hin g − A r p 2/3 Crosslinking- Filamin Motor Proteins- myosin stability/cap end of filaments- capz and tropomodulin C a p Z − c a p s a t + e n d CapZ- caps at + end C a pZ − c a p s a t + e n d T r o p o m o d u l i n − c a p s a t − e n d Tropomodulin- caps at - end T ro p o m o d u l in − c a p s a t − e n d o r g o f f i l a m e n t s / m u s c l e c o n t r a c t i o n , b i n d s t o s i d e o f f i l a m e n t s − n e b u l i n org of filaments/muscle contraction, binds to side of filaments- nebulin or g o ff i l am e n t s / m u sc l eco n t r a c t i o n , bin d s t os i d eo ff i l am e n t s − n e b u l in Actin based MotilityF o r m i n − l e a d s t o a s s e m b l y f o r l o n g a c t i n f i l a m e n t s Formin - leads to assembly for long actin filaments F or min − l e a d s t o a sse mb l y f or l o n g a c t in f i l am e n t s will form d i m e r s t r u c t u r e dimer structure d im ers t r u c t u re actin binds to structure and elongation commences A R P 2 / 3 c o m p l e x c a n b e u s e d f o r p o l y m e r i z a t i o n t o p o w e r m o t i l i t y , m e d i a t e s b r a n c h i n g ARP2/3 complex can be used for polymerization to power motility, mediates branching A RP 2/3 co m pl e x c anb e u se df or p o l y m er i z a t i o n t o p o w er m o t i l i t y , m e d ia t es b r an c hin g l i s t e r i a m o n o c y t o g e n e s u s e s a c t i n p o l y m e r i z a t i o n t o m o v e t h r o u g h c e l l s listeria monocytogenes uses actin polymerization to move through cells l i s t er iam o n ocy t o g e n es u ses a c t in p o l y m er i z a t i o n t o m o v e t h ro ug h ce ll s A c t A p r o t e i n a c t i v a t e s A r p 2 / 3 t o n u c l e a t e ActA protein activates Arp2/3 to nucleate A c t A p ro t e ina c t i v a t es A r p 2/3 t o n u c l e a t e f i l a m e n t s g r o w a t + e n d filaments grow at + end f i l am e n t s g ro w a t + e n d a c t i n r e c y c l e d t h r o u g h c o f i l i n , actin recycled through cofilin, a c t in recyc l e d t h ro ug h co f i l in , this process p r o p e l s b a c t e r i u m f o r w a r d propels bacterium forward p ro p e l s ba c t er i u m f or w a r d Toxins that perturb pool of actin monomers C y t o c h a l a s i n D − d e p o l y m e r i z e s a c t i n b y b l o c k i n g f u r t h e r a d d i t i o n o f s u b u n i t s Cytochalasin D- depolymerizes actin by blocking further addition of subunits C y t oc ha l a s in D − d e p o l y m er i zes a c t inb y b l oc kin g f u r t h er a dd i t i o n o f s u b u ni t s L a t r u n c u l i n − i n h i b i t s g a c t i n f r o m a d d i n g t o f i l a m e n t e n d Latrunculin- inhibits g actin from adding to filament end L a t r u n c u l in − inhibi t s g a c t in f ro ma dd in g t o f i l am e n t e n d J a s p l a k i n o l i d e − Jasplakinolide- J a s pl akin o l i d e − l o w e r s c r i t i c a l c o n c b a lowers critical conc ba l o w erscr i t i c a l co n c ba P h a l l o i d i n − p r e v e n t s a c t i n f i l a m e n t s f r o m d e p o l y m e r i z i n g b y l o c k i n g F s u b u n i t s t o g e t h e r Phalloidin- prevents actin filaments from depolymerizing by locking F subunits together P ha ll o i d in − p re v e n t s a c t in f i l am e n t s f ro m d e p o l y m er i z in g b y l oc kin g F s u b u ni t s t o g e t h er A c t i n a l s o i n t e r a c t s w i t h i t s e l f Actin also interacts with itself A c t ina l so in t er a c t s w i t hi t se l f types of lateral attachment of microfilaments to membranesankyrin- binds to Band 3 and then spectrin, forms network band 4.1 Actin Motor Proteinsm y o s i n myosin m yos in can bind to actin and help generate contraction in muscle cells c o m p o s e d o f h e a v y c h a i n s a n d l i g h t c h a i n s , composed of heavy chains and light chains, co m p ose d o f h e a v yc hain s an d l i g h t c hain s , myosin heads can bind to ATP and actin M y o s i n 1 Myosin 1 M yos in 1 s m a l l , s i n g l e h e a d small, single head s ma ll , s in g l e h e a d s t e p s i z e 10 − 14 n m step size 10-14nm s t e p s i ze 10 − 14 nm w o r k s w i t h m e m b r a n e a s s o c i a t i o n a n d e n d o c y t o s i s works with membrane association and endocytosis w or k s w i t hm e mb r an e a ssoc ia t i o nan d e n d ocy t os i s M y o s i n 2 Myosin 2 M yos in 2 d i m e r ( 2 h e a v y c h a i n ) dimer (2 heavy chain) d im er ( 2 h e a v yc hain ) 8 n m s t e p 8nm step 8 nm s t e p b i p o l a r f i l a m e n t s bipolar filaments bi p o l a r f i l am e n t s works with contractions M y o s i n 5 Myosin 5 M yos in 5 b i g g e r , 2 h e a v y c h a i n s a n d m o r e l i g h t c h a i n s bigger, 2 heavy chains and more light chains bi gg er , 2 h e a v yc hain s an d m ore l i g h t c hain s 36 n m s t e p 36nm step 36 nm s t e p r e s p o n s i b l e f o r o r g a n e l l e t r a n s p o r t responsible for organelle transport res p o n s ib l e f oror g an e ll e t r an s p or t M y o s i n m v m t p r o c e s s Myosin mvmt process M yos inm v m tp rocess A T P b i n d s t o h e a d g r p , h e a d g r o u p n o t a s s o c i a t e d w i t h a c t i n y e t ATP binds to head grp, head group not associated with actin yet A TP bin d s t o h e a d g r p , h e a d g ro u p n o t a ssoc ia t e d w i t ha c t in ye t A T P h y d r o l y z e d ATP hydrolyzed A TP h y d ro l yze d h e a d g r p b i n d s t o a c t i n head grp binds to actin h e a d g r p bin d s t o a c t in power stroke occurs, P i r e l e a s e d a n d m y o s i n s t r a i g h t e n e d , m o v i n g a c t i n f i l a m e n t l e f t Pi released and myosin straightened, moving actin filament left P i re l e a se d an d m yos in s t r ai g h t e n e d , m o v in g a c t in f i l am e n tl e f t A D P r e l e a s e d , t h e A T P b o u n d a n d h e a d g r p r e l e a s e d f r o m a c t i n ADP released, the ATP bound and head grp released from actin A D P re l e a se d , t h e A TP b o u n d an d h e a d g r p re l e a se df ro ma c t in Step size vs neck lengthis myosin step size/velocity proportional to neck length? Y E S , v e l o c i t y i n c w i t h i n c n e c k l e n g t h YES, velocity inc with inc neck length Y ES , v e l oc i t y in c w i t hin c n ec k l e n g t h contractile ringmyosin 2 takes a large part in forming when cells are splitting, myosin 1 is on outside of cells Sarcomere (not protein, just structure of skeletal muscle) vertical component is Z band, in between is A band, myosin in between actin filaments actin end facing inside is - end sarcoplasmic reticulum- specialized region of the ER, regulates and stores Ca (Ca helps muscle cells to contract) Cap Z- binds to + end of actin Tropomodulin- binds to - end of actin Nebulin- binds to side of actin filaments Titin- binds to myosin and Z disk proteins R h o G T P a s e s Rho GTPases R h o GTP a ses m e m b r a n e b o u n d R h o p r o t e i n s c a n b i n d e f f e c t o r p r o t e i n s t h a t c a u s e c h a n g e s i n t h e a c t i n c y t o s k e l e t o n membrane bound Rho proteins can bind effector proteins that cause changes in the actin cytoskeleton m e mb r an e b o u n d R h o p ro t e in sc anbin d e ff ec t or p ro t e in s t ha t c a u sec han g es in t h e a c t in cy t os k e l e t o n dominant active rho- always keep making actin C d c 42 Cdc42 C d c 42 w o r k s a t t h e f r o n t o f c e l l , a c t i v a t e s R a c works at the front of cell, activates Rac w or k s a tt h e f ro n t o f ce ll , a c t i v a t es R a c guys see a Rac and are activated R a c G T P RacGTP R a c GTP l e a d s t o a c t i v a t i o n o f A r p 2 / 3 a n d R h o leads to activation of Arp2/3 and Rho l e a d s t o a c t i v a t i o n o f A r p 2/3 an d R h o R h o G T P − RhoGTP- R h o GTP − leads to m y o s i n 2 a c t i v a t i o n myosin 2 activation m yos in 2 a c t i v a t i o n microfilamentsactin binds ATP form rigid gels, networks, and bundles tracks for myosin contractile machinery and network at cell cortex m i c r o t u b u l e s microtubules mi cro t u b u l es t u b u l i n b i n d s G T P , tubulin binds GTP, t u b u l inbin d s GTP , t r a s k s f o r k i n e s i n s a n d d y e s i n s trasks for kinesins and dyesins t r a s k s f or kin es in s an dd yes in s organization for long range organelles Intermediate filamentsgreat tensile strength, less dynamic, unpolarized no motors cell and tissue integrity Microtubules play a role in….organization of organelles and transport of vesicles mvmt of cilia and flagella nerve cell, RBC, and flagellar structure alignment and separation of chrom during mitosis T u b u l i n Tubulin T u b u l in m o n o m e r o f m i c r o t u b u l e s , a l p h a a n d b e t a m a k e u p m o n o m e r monomer of microtubules, alpha and beta make up monomer m o n o m ero f mi cro t u b u l es , a lp haan d b e t amak e u p m o n o m er Two populations of microtubulesUnstable short lived- assembles and disassembles rapidly stable and long lived- remain polymerized for a long time (sperm flagella, RBC, nerve cells) Polymerization and Structure of MicrotubulesStructure tubulin has alpha and beta parts b i n d t o 2 G T P bind to 2 GTP bin d t o 2 GTP a l p h a T G T P i s n e v e r h y d r o l y z e d alpha T GTP is never hydrolyzed a lp ha TGTP i s n e v er h y d ro l yze d b e t a T G T P c a n b e h y d r o l y z e d beta T GTP can be hydrolyzed b e t a TGTP c anb e h y d ro l yze d o n e e n d i s b e t a T e x p o s e d → + e n d one end is beta T exposed→ + end o n ee n d i s b e t a T e x p ose d → + e n d o n e e n d i s a l p h a T e x p o s e d → − e n d one end is alpha T exposed → - end o n ee n d i s a lp ha T e x p ose d → − e n d microtubules made up of 13 protofilaments → singlet can have doublets (cilia/flagella) and triplets (basal bodies and centrioles) as well Polymerization m i c r o t u b u l e s a s s e m b l e d f r o m M T O C microtubules assembled from MTOC mi cro t u b u l es a sse mb l e df ro m MTOC MTOC-any structure used by cells to nucleate and organized microtubulescentrosome falls into this category n e g e n d o f m i c r o t u b u l e s a t M T O C neg end of microtubules at MTOC n e g e n d o f mi cro t u b u l es a tMTOC gamma tubulin ring nucleates microtubule assembly Dynamics of MicrotubulesLength over time: Assembly stage→ Catastrophe stage→ Disassembly stage→ Rescue Stage P o l y m e r i z a t i o n o f t u b u l i n i n t o m i c r o t u b u l e s Polymerization of tubulin into microtubules P o l y m er i z a t i o n o f t u b u l inin t o mi cro t u b u l es p r o t o f i l a m e n t f i r s t f o r m e d protofilament first formed p ro t o f i l am e n t f i rs t f or m e d a l p h a T f i r s t b i n d s t o p r o t o f i l a m e n t , t h e n b e t a T alpha T first binds to protofilament, then beta T a lp ha T f i rs t bin d s t o p ro t o f i l am e n t , t h e nb e t a T s h e e t sheet s h ee t then form t u b e f o r m a t i o n tube formation t u b e f or ma t i o n G T P c a p a t t o p , GTP cap at top, GTP c a p a tt o p , GTP cap bc a l p h a a n d b e t a T c a r r y G T P , a d d i t i o n o f a n o t h e r A l p h a a n d b e t a T w i l l c a u s e h y d r o l y s i s alpha and beta T carry GTP, addition of another Alpha and beta T will cause hydrolysis a lp haan d b e t a T c a rry GTP , a dd i t i o n o f an o t h er A lp haan d b e t a Tw i ll c a u se h y d ro l ys i s \end more smooth (assembly), - end more rough (disassembly) Disassembly and reassembly of microtubules cool to 4 deg, microtubule will disassemble warm to 37 deg the microtubule will repolarize Drugs that disrupt microtubule dynamics c o l c h i n c i n e − b i n d s b t w n a l p h a a n d b e t a T d i m e r s o i t c a n n o t b e u s e d f o r p o l y m e r i z a t i o n , colchincine- binds btwn alpha and beta T dimer so it cannot be used for polymerization, co l c hin c in e − bin d s b tw na lp haan d b e t a T d im erso i t c ann o t b e u se df or p o l y m er i z a t i o n , t a x o l − b i n d t o s i d e o f t u b u l e s taxol- bind to side of tubules t a x o l − bin d t os i d eo f t u b u l es Binding ProteinsM A P s MAPs M A P s c a n s t a b i l i z e m i c r o t u b u l e s , can stabilize microtubules, c an s t abi l i ze mi cro t u b u l es , s i d e b i n d i n g side binding s i d e bin d in g MAP2- longer Tau- shorter +TIPScan regulate + end of microtubules Motors K i n e s i n s Kinesins K in es in s ferry cargo around the cell f e r r y t o w a r d s + e n d ferry towards + end f erry t o w a r d s + e n d have light chain, bind to ATP for energy resource bind to microtubule with head groups, bind to vesicle via kinesin receptor hydrolyze ATP to drive mvmt Kinesin 1 and 2- organelle, mRNA, and chromosome transport K i n e s i n 5 − b i p o l a r s t r u c t u r e , 2 h e a d g r p s , c a n b i n d t o 2 d i f f m i c r o t u b u l e s , m i c r o t u b u l e s l i d i n g Kinesin 5- bipolar structure, 2 head grps, can bind to 2 diff microtubules, microtubule sliding K in es in 5 − bi p o l a rs t r u c t u re , 2 h e a d g r p s , c anbin d t o 2 d i ff mi cro t u b u l es , mi cro t u b u l es l i d in g K i n e s i n 13 − c a n r e g u l a t e m i c r o t u b u l e e n d d i s a s s e m b l y Kinesin 13- can regulate microtubule end disassembly K in es in 13 − c an re gu l a t e mi cro t u b u l ee n dd i s a sse mb l y Process first head group, ( l e a d i n g h e a d ) , (leading head), ( l e a d in g h e a d ) , b o u n d t o m i c r o t u b u l e bound to microtubule b o u n d t o mi cro t u b u l e l e a d i n g h e a d t h e n b i n d s t o A T P leading head then binds to ATP l e a d in g h e a d t h e nbin d s t o A TP c o n f o r m a t i o n a l c h a n g e i n d u c e d , f o l l o w i n g h e a d s w i n g s f o r w a r d s conformational change induced, following head swings forwards co n f or ma t i o na l c han g e in d u ce d , f o ll o w in g h e a d s w in g s f or w a r d s f o l l o w i n g h e a d b e c o m e s l e a d i n g h e a d following head becomes leading head f o ll o w in g h e a d b eco m es l e a d in g h e a d n e w l e a d i n g h e a d r e l e a s e s A D P w h i c h i t w a s o r i g i n a l l y b o u n d t o , a n d n e w f o l l o w i n g h e a d h y d r o l y z e s A T P t o A D P new leading head releases ADP which it was originally bound to, and new following head hydrolyzes ATP to ADP n e wl e a d in g h e a d re l e a ses A D Pw hi c hi tw a sor i g ina ll y b o u n d t o , an d n e w f o ll o w in g h e a d h y d ro l yzes A TPt o A D P D y n e i n s Dyneins Dy n e in s f e r r y c a r g o a r o u n d t h e c e l l t o w a r d s − e n d ferry cargo around the cell towards - end f erryc a r g o a ro u n d t h ece llt o w a r d s − e n d Power stroke of dynein- ATP hydrolysis causes change in orientation of head→ mvmt of MT d y n a c t i n − b i n d c a r g o , m a k e d y n e i n m o r e p r o c e s s i v e dynactin- bind cargo, make dynein more processive d y na c t in − bin d c a r g o , mak e d y n e inm ore p rocess i v e LIS1 protein- interact with ATPase domain of dynein to elongate power stroke heterogeneous great tensile strength no known motors use them as tracks more stable than filaments or tubules no intrinsic polarity made up of protofilaments that can form diff structuresh a v e N t e r m a n d c t e r m a n d h e a d a n d t a i l e n d have N term and c term and head and tail end ha v e Nt er man d c t er man d h e a d an d t ai l e n d k e r a t i n , l a m i n , v i m e n t i n keratin, lamin, vimentin k er a t in , l amin , v im e n t in Knowt Play Call Kai 
