intermediate filaments

Biochemistry – Lecture14 -cytoskeleton  strength and shape and movement of cells -3 filaments: -intermediate filaments - mechanical strength - microtubules  intracellular transport, organelle location -actin filaments shape, locomotion -accessory protein control assembly -the regulation of these = helps build the cytoskeleton -motor proteins movement Actin filaments  also known as microfilaments

-they are 5.9 nm thick -all eukaryotes -conserved -they form flexible filaments  form a network  bundles -these are generally found in the cortex  just beneath the plasma membrane Microtubules - all eukaryotes, conserved -25nm thick

-rigid and hallow tubes -attached MTOC Intermediate Filaments -only in some metazoans some cell types -10nm thick -rope-like fibers that span across a cell -general principles of dynamics dynamic, constantly in flux assembled from small subunits -non-covalent interactions -rapid changes -accessory proteins control assembly and disassembly -small subunits protofilaments helical filaments -nucleation is rate-limiting -special nucleation proteins

Microtubules -subunits= tubulin -globular, alpha and beta tubulin non-covalent binding -bind GTP -alpha-GTP never leaves -beta-GTP hydrolyze and exchanges - the protofilaments have alpha and beta alternating microtubules = 13 protofilaments -microtubules = 13 protofilaments -alpha = minus end and beta = plus end -this gives us polarity Actin Filaments -subunits =actin -one globular binds ATP -protofilaments  actin subunits = head to tail -filament = 2 protofilaments -polarity: plus and minus Polymerization -plus end = rapid addition of monomers -minus = slow addition -conformation changes

-nucleotide hydrolysis  ATP or GTP hydrolyze after binding -the T form goes to the D form  this means that there is a lower affinity -the D form leaves -high monomer concentration  T form to plus and low concentration -T form to plus end -low concentration: D leaves -actin treadmilling  intermediate concentration -microtubles  dynamic instability -rapid shrinking for plus end  intermediate concentration **video 16.3 Microtubule_Dynamics** -Dynamic: requires a supply of energy  ATP/GTP hydrolysis exchange the ADP or GDP for a fresh ATP or GDP

-Intermediate filaments - subunit= alpha helix monomer -coiled – coil dimer -tetramer  8 tetramers = filament -NO ATP OR GTP -NO polarity -assembly involves phosphorylation energy from ATP -many different kinds -some are dynamic while others are stable

-Keratin= intermediate filaments -epithelial and toughness -they join at junction and are stuck together -neurofilaments  nerves

Question: If each type of cytoskeletal filament is made up of subunits that are held together by weak non-covalent bonds, how is it possible for a human being to lift heavy objects? Answer: There are many weak bonds in a large number of filaments so the stress is distributed just as a single thread is weak but a rope is strong.

Regulation Nucleation- microtubules  gamma-tubulin ring complex -Gamma- tubulin and other proteins starts minus end grow to plus end -MTDC= microtubules organizing center -location in the cell where microtubules start -animal cells = centrosome -gamma-TRC and centrioles -fungi and plants: no centrioles -MTDC and gamma-TRC around nucleus

-nucleate actin filament ARP = actin-related proteins

-ARP protein in cell cortex form webs, branches - Nucleatin protein: formin= makes straight bundles

Other Accessory Proteins - actin polymerization -thymosin= bind free subunits

-this prevents them from polymerizing -profilin= free the subunits and allow polymerization -activate profiling  make filaments -microtubule polymerization -stathmin  binds subunits prevents polymerization Question: Taxol stabilizes microtubules and causes free tubulin to assumble into microtubules, while colchicin has the opposite effect and presents microtubule formation. How can they both be anti-cancer drugs?

Actin Stability -tropomyosin binds to filaments stabilize -gelsolin breaks actin filaments -filamin crosslinkes Microtubule Stability -katanin break microtubules -MAPs = microtubule associated proteins stabilize -plectin cross-link to intermediate filaments **video: 16 Cytoskeleton Motor Proteins - bind to cytoskeleton -use ATP to move along filament -bind to actin or tubulin -caw and organelles or generate force -found in all eukayrotes Myosin -general properties -motor domain = head ATP binding -determines identity of trade - direction Tail= identity of cargo

Myosin - moves on actin -1 or 2 heads -most move to plus ends -some  minus end -vesicle transport contraction  muscle -cell migration