The Cytoskeleton Functions Structural scaffold creating and supporting cell shape Framework positioning organelles within cytoplasm Network of molecular “roads” for intracellular transport of materials Framework for whole cell movement Framework for cell division

The Cytoskeleton Three major structural components Microtubules Major role: support, intracellular transport Intermediate filaments Major role: mechanical strength to resist physical stresses Microfilaments Major role: muscle contraction, motility

The Cytoskeleton Microtubules (MTs) Major role Intracellular transport Motor proteins drag cargo along them Structural support Resist compression forces Resist shear (bending) forces Hollow, rigid 25nm diameter, 4nm wall thickness Radiate outward toward plasma membrane from near nucleus (MTOC)

The Cytoskeleton plus-end minus-end Microtubules (MTs) Unit = alpha / beta tubulin heterodimer alpha subunit + beta subunit Heterodimer is asymmetric Beta end is called “plus” end Alpha end is called “minus” end Not referring to a charge difference plus-end minus-end

The Cytoskeleton Microtubules (MTs) alpha / beta (a/b)-tubulin heterodimer Beta subunit is a GTPase Assembly Polymer grows by addition of units at the “plus” end GTP-bound tubulin can add GTP form hydrolyzes to GDP form GDP-bound tubulin cannot add GDP-bound tubulin can release only from “plus” end GDP-bound tubulin cannot release from “minus” end or from central region

The Cytoskeleton Dynamic instability MTs can assemble/disassemble at different rates in different locations within a single cell Various proteins can bind and stabilize MTs

The Cytoskeleton Microtubule-associated proteins (MAPs) Form bridges crosslinking adjacent MTs for parallel alignment Increase MT stability Promote assembly Regulated by phosphorylation state Anti-tubulin antibody stain

The Cytoskeleton Microtubule organizing centers (MTOCs) GTP-bound a/b-tubulin spontaneously assembles into MTs very slowly GTP-bound a/b-tubulin add to an existing MT very rapidly MTOCs are the nucleation points for MT assembly Centrosome Basal body

The Cytoskeleton Microtubule organizing centers (MTOCs) Centrosome 2 centrioles at right angles to each other near nucleus Contain gamma-tubulin subunit Nucleate “minus” end of a/b-tubulin Plus-end is oriented outward toward plasma membrane

The Cytoskeleton Microtubule organizing centers (MTOCs) Basal body Single centriole at the base of cilia and flagella

Eukaryotic cilia and flagella Hair-like motile organelle projecting from cell surface Covered by plasma membrane

Eukaryotic cilia and flagella Central protein core is called an “axoneme”

Eukaryotic cilia and flagella Central protein core is called an “axoneme” Composed of 11 MTs arranged in a “9+2” array 9 outer MTs 2 central MTs Connected by various MAPs Locomotion caused by sliding outer tubules past each other Action of motor proteins (dynein)

The Cytoskeleton Motor proteins that “walk” on MTs Kinesin gene family Plus-end directed Outward or “anterograde” transport Dynein gene family Minus-end directed Inward or “retrograde”

The Cytoskeleton Kinesins are composed of 2 heavy and 2 light polypeptides Cargo-interaction domain “tail” Different kinesins have different specificities ATPase “head” Binds to MT ATP hydrolysis propels heads forward Highly processive

The Cytoskeleton Kinesins are composed of 2 heavy and 2 light polypeptides ATPase “head” Binds to MT ATP hydrolysis propels heads forward Highly processive

The Cytoskeleton Motor proteins that “walk” on MTs Dynein gene family Minus-end directed Inward or “retrograde” transport Very large (1.5MDa) Involved in cilia/flagella movement

The Cytoskeleton Three major structural components Intermediate filaments (~65 genes) Major role: mechanical strength to resist physical stresses Hemidesmosomes and desmosomes

Intermediate filaments (IFs) Animal specific Strong, rope-like

Intermediate filaments (IFs) Animal specific Strong, rope-like Bridged together with other cytoskeletal elements (e.g. plectin crosslinks MTs and IFs)

The Cytoskeleton Intermediate filaments Composition and assembly Monomers form dimers Dimers form tetramers lacking polarity Tetramers form larger fibers Incorporation into existing filaments not limited to end regions

The Cytoskeleton Three major structural components Microfilaments (MFs) Major role: muscle contraction, motility Solid, branched 8nm diameter Molecular unit= actin

The Cytoskeleton Microfilaments (MFs) Actin molecule is asymmetric “plus”-end versus “minus”-end Actin is an ATPase ATP-bound actin can be incorporated into growing MFs plus-end of MFs grows 10x faster than minus-end Higher dissociation rate from minus-end leads to treadmilling

The Cytoskeleton Microfilaments (MFs) + cytochalasin D Drugs Cytochalasin D blocks plus-end addition leading to complete MF depolymerization Phalloidin blocks turn-over locking MFs into polymerized state + cytochalasin D

The Cytoskeleton Microfilaments (MFs) + cytochalasin D Drugs Cytochalasin D blocks plus-end addition leading to complete MF depolymerization Phalloidin blocks turn-over locking MFs into polymerized state + cytochalasin D

The Cytoskeleton Actin binding proteins + cytochalasin D

The Cytoskeleton Motors that walk on Microfilaments (MFs) Myosin gene family ATPase “head” domain Cargo-interacting “tail” domain

The Cytoskeleton Motors that walk on Microfilaments (MFs) Myosin gene family Type V can walk on actin filaments carrying a bound cargo Type II forms bipolar filaments via tail - tail interactions

The Cytoskeleton Myosin type II in muscle contraction Muscle fiber Large cell, 100mm long, 10-100 microns thick Contain >100 nuclei Derived from the fusion of many myoblast cells Myofibrils thin protein strands composed of repeating units called “sarcomeres” that give muscle its “striated” appearance Sarcomere Z, I, A, H and M regions

Myosin II

Sliding filament model of muscle contraction

Sliding filament model of muscle contraction