1. Molecular Cell Biology and Genetics
THE CYTOSKELETON
Mitch Denning, Ph.D.
Molecular Cell Biology and Genetics
August 31, 2018
The cytoskeleton is a network of 3 intertwined, interconnected and dynamic filament systems involved in cell movement, cell shape, and cell integrity
Actin
Microtubules
Nucleus

2. Cytoskeleton Overview
Functions
Structure
Assembly/Disassembly
Drugs
Motors
Actin
Filaments
5-9 nm
Functions
Structure
Assembly/Disassembly
Drugs
Motors
Microtubules
25 nm
In my lecture, I will discuss each cytoskeletal element individually.
Actin Filaments: smallest diameter
Microtubules: largest diameter
Intermediate Filaments: Intermediate diameter
Each cytoskeletal element is assembled or polymerized from soluble subunit. Polymerization and depolymerization of cytoskeletal elements is highly regulated.
The polymerized filaments can be organized into different types of arrays localized in different parts of the cell.
Functions
Structure
Diversity
Intermediate
Filaments
10 nm

3. Actin (Microfilaments) Functions
Cell Shape
Surface area
Cell adhesion
Cell-Cell (Adherens Junction)
Cell-Matrix (Focal Contact)
Polarization
Apical vs Basolateral
Phagocytosis
Phagosome
Muscle contraction
Cortical Actin
Actin
Endocytosis, contractile ring in cytokinesis

4. Actin (Microfilaments) Functions
Cell Migration
Development
Wound Healing
Immune System
Cancer Metastasis

5. Neutrophil Chase

6. Structure of Actin Filaments
Actin monomers are the soluble subunits of actin filaments.
Sequence is highly conserved.
Actin filaments are flexible and polar. Plus end is more dynamic.
ATP becomes hydrolyzed in the filament
Actin monomers bind ATP/ADP
Helix of 2 protofilaments

7. Filament Assembly and Dynamic Structure
Dynamic regulated equilibrium between soluble and filamentous actin
Accessory proteins regulate assembly/disassembly
Accessory proteins regulate assembly/disassembly

8. Direct addition of actin “nuclei” shifts kinetics
Actin Filament Assembly Kinetics
Direct addition of actin “nuclei” shifts kinetics
Nucleation is rate-limiting
One of the obstacles a cell must overcome is that the initiation of polymerization (nucleation) is slow and thus rate limiting.
Cells utilize pre-formed oligomers or nuclei to initiate rapid filament polymerization.

9. Actin Assembly and Disassembly
• Actin nucleation at cell membrane: Cortical actin
Cell Surface Structures: Microvilli, Filopodia, Lamellipodia
• Actin Related Protein complex, ARP, nucleates at minus end
Binds pre-existing filaments at 70° angle
Subunit Binding Proteins: Profilin (+); Thymosin (-)
Cofilin bind ADP Actin filaments to promote depolymerization
End Binding Proteins (ARP/Cap Z) modulate filament growth and localization
Efficient stoichiometry
Subunit Binding Proteins modulate filament elongation
Less efficient
Filament Binding Proteins modulate filament stability and orientation
Cofilin: Actin Depolymerization Factor

10. Consequence of Nucleotide Hydrolysis
Treadmilling
High Cc
Low Cc
In addition to filament and subunit binding proteins, actin filament are regulated by ATP hydrolysis.
Treadmilling

11. Actin Protrusion in Lamellipodia
(Cap Z)
70° Angle
Binds ADP form of F-Actin to cause depolymerization

12. Actin Arrays in Cells Microvilli Stress Fibers Striated Muscle
a-Actinin
Striated Muscle
Spectrin
Filamin
Fimbrin

13. Actin Filament Cross-Linking

14. Global Actin Rearrangements
Rho family GTPases
Stress Fibers
Focal Contacts
Lamellipodia
Membrane Ruffles
Microspikes
Filopodia

15. Actin Targeted Drugs Drugs Effects on Actin
Phalloidin Binds and stabilizes filaments
Cytochalasin Caps filament plus ends
Latrunculin Binds subunits and prevents their polymerization
Death Cap Mushroom
(Phalloidin)
Phalloidin from Death Cap mushroom. Most mushroom poisoning deaths worldwide.
Clostridium difficile: TcdA TcdB disrupt Rho and thus tight junctions
Bacterial toxins also target actin polymerization (Rho Family)
Induces cell death
Disrupts tight junctions (diarrhea)

16. Phalloidin Binds Actin Filaments
Actin cytoskeleton stained with fluorescent phalloidin
Effects of phalloidin on actin filaments

17. Actin Molecular Motors: Myosin
Actin-based motor proteins
Myosin II
Plus-end directed
Bipolar Thick Filament +
Myosin II found in muscle
2 heavy chains + 2 copies of 2 light chains
Majority of myosins are plus end directed +

18. Myosin Motor Mechanisms
Myosin “Rowing” on Actin
High speed
Heads work independently
Attached Released Cocked Force Attached
Starts in “rigor” state.
Phosphate release triggers power stroke
Familial Hypertrophic Cardiomyopathy
Myosin b heavy chain mutations
Enlarged heart
Cardiac arrhythmia
Sudden death in young athletes

19. Movie 16.6 Myosin moving along actin (Cartoon)
Movie 16.8 Myosin motors moving actin

20. Microtubules Functions Positioning of organelles
Golgi and Endoplasmic Reticulum
Intracellular transport
Mitotic Spindles
Vesicle and membrane transport between ER, Golgi and plasma membrane
Anterograde/retrograde axonal transport of organelles and proteins
Cell motility
Flagella
Cilia (motile and non-motile)
Golgi
Microtubules
Membranes are recycled after moving from ER to Golgi
ER to Golgi: Anterograde
Golgi to ER: Retrograde

21. Structure of Microtubules
GTP Hydrolysis 13
More Dynamic
Alpha/Beta tubulin dimers are the soluble subunits for microtubules.
Centrosome
(MTOC)

22. Microtubule Organizing Center
(MTOC)
Basal bodies organize cilia and flagella microtubules.
Similar to centrioles

23. Microtubule Assembly/Disassembly
Dynamic Instability
GTP form favors assembly
Minus end protected in MTOC (centrosome)
GDP form favors disassembly

24. Microtubule Dynamics – EB-1 and Tubulin GFP
16.3 Movie
Microtubule Dynamics – EB-1 and Tubulin GFP
16.2 Movie
Microtubule Leading Edge and ER Extension

25. Higher Order Microtubule Organization
Centrosomes: Astral arrays
MAPs: Filament spacing, Stabilization
Neurofibrillary Tangles Hyperphosphorylated Tau aggregates in Alzheimer’s Disease

26. Microtubule Targeted Drugs
MICRTUBULE DRUGS
Taxol
Colchicine, Colcemid
Vinblastine, Vincristine
Nocodazole
(Mitotic Spindle Poisons)
Binds and stabilizes microtubules
Binds subunits and prevents their polymerization
Microtubule-targeting drugs useful for cancer therapy
Anaphase of Mitosis

27. Microtubule Stabilization by Taxol

28. Microtubule Molecular Motors
Kinesins and Kinesin Related Proteins
Move toward plus end
Endoplasmic reticulum
Dyneins
Move toward negative end
Cytoplasmic: Vesicle trafficking, golgi locomotion
Axonemal: Rapid movement of cilia/flagella
Both kinesins and dyneins are involved in chromosome segregation.
Both are also involved in melanosome movement in fish pigment cells (figure16-61)
High cAMP
Low cAMP

29. Hand-Over-Hand/Step-by-Step
Microtubule Motor Mechanisms
Kinesin Dimer “Walking” on Microtubules
Cargo
Cargo
Cargo
Cargo
Nucleotide exchange throws other head forward.
High Processivity
Hand-Over-Hand/Step-by-Step

30. Movie 16.7 Kinesin moving along Microtubules (Cartoon)
Movie 16.5 Organelles moving along microtubules

31. 9 doublets around 2 singlets
Flagella and Cilia
Axonemal Dyneins
Flagella: Wave-like motion
1-2 per cell
Protozoa and sperm
Cilia: Whip-like motion
Many per cell
Respiratory epithelium
Flagella: 1 or 2 per cell. Longer than cilia
Cilia: hundreds per cell. Shorter than flagella
Axoneme
9 doublets around 2 singlets

32. Basal Bodies Anchor cilia and flagella at cell surface
9 Microtubule Triplets
No center pair of singlets
Basal Bodies similar to centrioles inside centrosomes.
Kahr-tag′ĕ-nĕr syndrome
Kartagener’s Syndrome: Primary Ciliary Dyskinesia (Ciliary Dynein)
Infertility (Male: Sperm/ Female: Fallopian tubes)
Respiratory/Sinus Infections
Situs Inversus (organ positions inverted)

33. Intermediate Filaments Functions
Mechanical Strength
Cell and tissue integrity
Cell Adhesion
Cell-cell adhesion (Desmosome)
Cell-matrix adhesion (Hemidesmosome)
Axon Diameter and Strength (Neurofilaments)
Amyotropic Lateral Sclerosis
Nuclear Lamins
Nucleus integrity
Laminopathies
Epidermolysis
Bullosa
Simplex
ALS: Neurofilament accumulations found in neurons

34. Intermediate Filament Structure and Assembly
Rope-Like
High Physical Strength
- Non-Polar
Soluble Subunit
A tetramer (two coiled-coil dimers) is the soluble subunit for Intermediate Filaments.

35. Intermediate Filament Cross-Linking
C-terminal domains binds neighboring filaments
Bundled cytokeratins are called tonofilaments
Accessory proteins
Filaggrin (Filament Aggregation)
Eczema and/or Ichthyosis Vulgaris
Plectin (Vimentin, Actin, Microtubules)
Muscular Dystrophy with Epidermolysis Bullosa Simplex

36. TYPES OF INTERMEDIATE FILAMENTS COMPONENT POLYPEPTIDES
Diversity of Intermediate Filament Proteins
Large gene family
Tissue specific expression
Useful for tumor diagnosis
TYPES OF INTERMEDIATE FILAMENTS
COMPONENT POLYPEPTIDES
CELLULAR LOCATION
Nuclear
Lamins A, B, and C
Defect: Progeria
Nuclear Lamina
(Inner lining of nuclear envelope)
Vimentin-like
Vimentin
Desmin
Glial fibrillary acidic protein
Peripherin
Many cells of mesenchymal origin; Muscle
Glial cells (Astrocytes, Schwann cells)
Some neurons
Epithelial
Keratins, type I (acidic) (~10 genes)
Keratins, type II (basic) (~10 genes)
Hair Keratins (~10 genes)
Epithelial cells
Hair and Nails (Epithelial Appendages)
Axonal
Neurofilament proteins
(NF-L, NF-M, and NF-H)
Neurons