CYTOSKELETON (II) Intermediate filaments and microtubules

Readings and Objectives Reading Reading – Russell : Chapter 1 ( incomplete information ) – Cooper: Chapter 12 Objectives Objectives Intermediate Filaments cytoskeletal scaffolding Microtubules Dynamism Microtubule Motors and Movement Organelle trafficking Cilia and flagella Mitotic mircotubules 2

Intermediate Filaments Intermediate filaments (8-11 nm) not directly involved in cell movements provide mechanical strength Supporting scaffold: for organelles and cytoskeleton Diverse fibrous proteins Size: kD Not dynamic Not polar Regulated by phosphorylation Six types I-VI 3

Intermediate Filaments Tissue specific expression Type I and II: keratins, small size (40-70 kD), epithelial cells; Type III: expressed in many cells – Vimentin (54 kD), forms a network extending from nucleus to the cell periphery – Desmin (53 kD), connects Z-discs in muscle, stabilize actin-myosin Type IV: Neurofilaments; variable size 67, kD (Light, Medium, Heavy  NF-L, NF-M, NF-H), expressed in mature neurons Type V: nuclear lamins Type VI: Nestins, stem cells, during embryonic development 4

Structure and Assembly Structure: Structure: α-helical rod domain of about aa Globular Head and Tail domains, variable size Head and tail domains determine the specific functions 5

Structure and Assembly Assembly Dimer: central rod form coiled coil Tetramer: staggered antiparallel Protofilament: tetramers assemble end to end Filament: 8 interwound protofilament Filaments are stable and nonpolar 6

Form a network in most cells Extending from a ring surrounding the nucleus to the plasma membrane Associate with cytoskeletal elements Provides a scaffold to organize the internal structure of the cell Cellular arrangement of Intermediate Filaments 7

Plectin binds actin filaments and microtubules Plectin is 500 kD protein N-terminus domain binds Keratin C-domain binds cytoskeleton bridges them to int. filaments and areas of cell to cell connections Increases the mechanical stability of the cell Interaction with cytoskeleton microtubule Plectin Int. filament anti Plectin A fibroblast cell stained with labelled anti plectin antibody 8

Known that mechanical stress trigger development of intermediate filaments Transgenic mice expressing mutant Keratin gene (Elain Fuchs lab) severe skin abnormalities Rubbing skin causes blisters Evidence for Cellular Role Elain Fuchs 9

Human diseases Connection Epidermolysis Bullosa Simplex (EBS) – have a keratin gene mutation – patients develop skin blisters after minor trauma Amyotrophic lateral Sclerosis (ALS) – abnormalities of neurofilaments – involving progressive loss of motor neurons – muscle atrophy and paralysis 10

Microtubules are rigid hollow rods (25 nm) Dynamic structures, undergo continual assembly and disassembly Function: cell movements and determining cell shape, organelle transport, mitosis Tubulin, globular protein is the monomer α-tubulin and β-tubulin dimers make up microtubules γ-tubulin in the centrosome plays a critical role in initiating microtubule assembly Microtubules 11

Microtubules Assembly Tubulin dimers polymerize to form microtubules consisting of 13 protofilaments assembled around a hollow core Protofilaments composed of head-to-tail arrays of tubulin dimers arranged in parallel two distinct ends: a fast- growing + end and a slow- growing minus end 12

Microtubules Structure Microtubules can undergo treadmilling Tubulin dimers with GTP bound to β-tubulin associate with the growing end GTP is hydrolyzed, tubulin gets less stable, minus end dimers disassociate 13

Dynamic instability High [tubulin-GTP], dimers added more rapidly than GTP is hydrolyzed  microtubule grows Low [tubulin-GTP], GTP at the plus end is hydrolyzed and dimers are lost 14

Microtubules: assembly and dynamism 15

Microtubules and Centrosome Centrosome Centrosome ( microtubule organizing centre, MTOC ) centrosome initiate microtubule growth γ -tubulin as part of γ- tubulin ring complex in centriols required Centriols, Not necessary Microtubules extend to cell cortex Might be stabilized locally MTOC 16

Regulatory MAPs Modulation of function and stability Post-translational modification of tubulin, eg phosphorylation interactions with microtubule-associated proteins (MAPs) – Capping, Severing, disassembling/assembling, end tracking Example: Neurons MT organized differently Axon: - to + Dendrites: both direction 17

Association with motor proteins Kinesins and Dyneins responsible for powering the movements involving microtubules Kinesin: 2 heavy chains, 2 light chains Dynein: 2–3 heavy chains a number of light and intermediate chains kinesins move toward the plus end dyneins move toward the minus end See experiment movie 18

Association with motor proteins 19

Higher order molecular machines Cilia and flagella are microtubule-based projections of the plasma membrane; cell movement very similar structures Cilia beat in a coordinated back-and-forth motion either moves the cell through fluid or moves fluid over the surface of the cell 20

Higher order molecular machines axoneme of cilia and flagella consists of microtubules in a “9 + 2” pattern: a central pair surrounded by nine outer doublets. Each doublet has a complete A tubule and a B tubule with 10–11 protofilaments, fused to the A tubule Nexin links the microtubules, two arms of dynein are attached to each A tubule 21

Higher order molecular machines The minus ends of the microtubules are anchored in a basal body, It contains nine triplets of microtubules. Basal bodies serve to initiate growth of axonemal microtubules and to anchor cilia and flagella to the surface of the cell 22

Higher order molecular machines Movement of cilia and flagella: sliding of outer microtubule doublets relative to one another, powered by motor activity of the axonemal dyneins Dynein bases bind to A tubules, while the head groups bind to B tubules of adjacent doublets 23

Mitotic Microtubules Microtubules reorganize during mitosis. Interphase microtubule disassembles free tubulin subunits are reassembled into the mitotic spindle Centrosome duplicates, MTOC forms at opposite poles of the mitotic spindle 24

Mitotic Microtubules 1. Kinetochore microtubules attach to the condensed chromosomes at the centromeres 2. Chromosomal microtubules connect to the ends of the chromosomes via chromokinesin Polar microtubules stabilized by overlapping with each other in the center of the cell. 4. Astral microtubules extend outward 25

Mitotic Microtubules Anaphase A— chromosomes move toward spindle poles Anaphase B kinesins cross-link polar microtubules and move them toward the plus end cytoplasmic dynein moves along astral microtubules in the minus-end direction Poles kept apart 26