The Cytoskeleton

1. 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

2. 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

3. 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)

4. The Cytoskeleton plus-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 minus-end

5. 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

6. 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

7. 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

8. 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

9. 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

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

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

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

14. 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)

16. 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” transport

17. 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

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

19. 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

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

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

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

23. 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

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

25. 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

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

27. The Cytoskeleton • Actin binding proteins + cytochalasin D

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

29. 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

30. 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

31. Sliding filament model of muscle contraction

32. Sliding filament model of muscle contraction