Cytoskeleton Notes – Microtubules & Intermediate Filaments

Learning Objectives

• Describe the structure & function of:
  • Microtubules (MT)
  • Dynamic instability
  • Microtubule-organizing center (MTOC)
  • Microtubule-associated proteins (MAPs)
  • Cilia & flagella
  • Intermediate filaments (IF)
  • Participation in cell junctions
  • Mechanical stability
  • Pathological mutations
• Understand how drugs affect microtubule function; recall concrete examples.

Microtubules – Structure & Composition

• Built from polymers of tubulin
  • Tubulin subunit = heterodimer
  • $\alpha$-tubulin (≈ $445\text{–}450$ aa)
  • $\beta$-tubulin (≈ $445\text{–}450$ aa)
• Each monomer binds one GTP
  • $\alpha$-tubulin: GTP is permanently trapped (never hydrolyzed / exchanged)
  • $\beta$-tubulin: GTP hydrolyzed during polymerization → GDP; upon depolymerization GDP is exchanged back to GTP
• Heterodimers stack head-to-tail → protofilaments; $11\text{–}18$ protofilaments fold into a hollow cylinder
• Dimensions
  • Outer diameter: $24\text{–}25\,\text{nm}$
  • Lumen: $\approx15\,\text{nm}$

Dynamic Instability

• MTs alternate between growth (polymerization) & shrinkage (depolymerization)
  • Governed by the GTP-binding state of $\beta$-tubulin
• Mechanism
  • Addition of GTP-bound dimers to plus end → extension
  • If GTP hydrolysis rate > subunit addition rate → loss of GTP-cap → rapid catastrophe (shrinkage)
  • Rescue: re-establishment of a new GTP-cap lets MT grow again
• Key features
  • Provides structural plasticity
  • Enables rapid remodeling during mitosis, migration, organelle transport, etc.

Clinical & Biological Relevance of Dynamic Instability

• Cancer therapy
  • Drugs either stabilize or destabilize MT to arrest mitosis & trigger apoptosis
• Neurodegenerative disorders
  • Defects in MAPs (e.g., tau in Alzheimer’s) destabilize MT → axonal transport failure
• Cell migration & metastasis
  • MT reorganization directs polarity & motility of tumor and healing cells
• Developmental biology
  • Correct MT dynamics required for proliferation & differentiation
• Infection biology
  • Pathogens hijack MT for intracellular movement; targeting this pathway offers antimicrobial strategies

Microtubule-Organizing Center (MTOC)

• Site from which MTs nucleate, minus ends anchored, plus ends radiate
• Animal cells: centrosome is main MTOC
  • Composed of two perpendicular centrioles (small cylinders) + pericentriolar material
• During mitosis
  • Centrosomes duplicate → bipolar spindle
  • Astral MTs position spindle; kinetochore MTs segregate chromosomes
• After cytokinesis each daughter cell rebuilds interphase MT & actin arrays

Microtubule-Associated Proteins (MAPs)

• Motor MAPs — convert ATP into mechanical work along MT lattice
  • Kinesin family
  • Usually moves toward plus end
  • Two globular heads (ATPase) + coiled-coil stalk + cargo tail
  • Dynein family
  • Moves toward minus end
  • Two heavy-chain heads + intermediate/light chains for cargo/adaptor binding
• Non-motor MAPs — stabilize, bundle, or sever MTs; dictate polarity
• Functional consequences
  • Organelle/vesicle/mRNA transport
  • Establishment of cell polarity & asymmetric distribution of material
  • Melanosome transport in pigment cells (e.g., cephalopod camouflage)

Cilia & Flagella

• Highly specialized motile appendages with $9+2$ MT axoneme (nine outer doublets + central pair)
• Basal body = modified centriole anchoring axoneme to cell
• Dynein arms on doublets generate sliding forces; structural links convert sliding into bending → propulsion
• Biological roles
  • Cilia: move extracellular fluid (e.g., mucociliary clearance in airways)
  • Flagella: propel cells (e.g., spermatozoa)
• Prokaryotic flagella are not MT-based (composed of flagellin); similarity only at functional level

Drugs Targeting Microtubules

• Stabilizers
  • Paclitaxel (Taxol) — binds MT, prevents depolymerization → freezes spindle; cytostatic against breast & ovarian cancer; isolated from Pacific yew
• Destabilizers / Polymerization inhibitors
  • Vinca alkaloids (vincristine, vinblastine) — bind tubulin, block spindle formation → metaphase arrest
  • Colchicine — binds tubulin, prevents polymerization; uses
  • Treats gout & familial Shar-pei fever by inhibiting secretion of serum amyloid A (SAA) & leukocyte motility
  • Colcemid & nocodazole — research tools & antimitotics
• Comprehensive comparison (from Table 16-2)
  • Actin-specific: phalloidin (stabilizes), cytochalasin (caps + end), swinholide (severs), latrunculin (sequesters G-actin)
  • MT-specific: Taxol (stabilizes), colchicine/colcemid, vinblastine/vincristine, nocodazole (all prevent polymerization)

Intermediate Filaments – General Features

• Provide mechanical strength & integrity; resist shear stress
• No intrinsic polarity (no +/– ends)
• Subunits bind neither ATP nor GTP; not tracks for motor proteins
• Not involved in motile force generation

Molecular Architecture

• Central building block = coiled-coil dimer (two $\alpha$-helical monomers, hydrophobic interface)
• Two dimers stagger in antiparallel fashion → tetramer (non-polar)
• $8$ tetramers laterally associate → rope-like IF filament (final filament ≈ $10\,\text{nm}$ diameter; overall $16$ dimers = $32$ monomers)

Major IF Classes in Vertebrates (Table 16-1)

• Nuclear lamins (A, B, C) — nuclear lamina
• Vimentin-like family
  • Vimentin (mesenchyme), desmin (muscle), GFAP (astrocytes), peripherin (neurons)
• Epithelial keratins
  • Type I (acidic), Type II (basic) — epithelia, hair, nails
• Axonal neurofilament proteins (NF-L, NF-M, NF-H) — neurons

Keratin Filaments

• Most diverse IF family; produced by keratinocytes
• Constitute horns, nails, hair, scales
• Link epithelia to basal lamina & neighbors via desmosomes/hemidesmosomes → tissue cohesion
• Clinical utility
  • Specific keratin expression profiles identify carcinoma origin, guiding therapy
  • Mutations → epidermolysis bullosa simplex (EBS) & other blistering diseases
  • Example variants: KRT5 (Cardigan Welsh Corgi), PLEC nonsense (Eurasier dogs)

Other IFs & Functions

• Neurofilaments
  • Abundant in axons; regulate axon caliber & conduction velocity; confer tensile strength
• Desmin
  • Links Z-disks & organelles → structural scaffold in skeletal/cardiac muscle
• Lamins
  • Support nuclear envelope; organize chromatin; disassemble/reassemble during mitosis

Comparative Cytoskeletal Overview (summary)

• Microtubules
  • Structure: hollow tubes, $24\text{–}25\,\text{nm}$ diameter, $11\text{–}18$ protofilaments
  • Subunit: $\alpha/\beta$-tubulin dimers
  • Functions: cell shape, organelle transport, chromosome segregation, motility
  • Localization: originate at MTOC, radiate toward periphery
• Intermediate filaments
  • Structure: rope-like polymers, $8\text{–}12\,\text{nm}$ diameter
  • Subunit: diverse IF proteins
  • Functions: mechanical resilience, nuclear lamina, tissue integrity
  • Localization: cytoplasmic network + nuclear lining
• Microfilaments (Actin)
  • Structure: two intertwined actin strands, $7\text{–}8\,\text{nm}$ diameter
  • Subunit: G-actin
  • Functions: cortex tension, cell motility, muscle contraction
  • Localization: cell periphery, stress fibers, lamellipodia

Ethical & Practical Considerations

• Anti-MT chemotherapeutics prolong survival but cause neuropathy (due to MT’s role in axonal transport)
• IF gene testing enables early diagnosis & breeding decisions in veterinary medicine (e.g., EBS in dogs)
• Environmental sustainability of drug sources (e.g., Pacific yew harvesting for Taxol) led to synthetic & semi-synthetic production methods

Key Numerical / Statistical References

• Tubulin monomer length: $\approx445\text{–}450$ aa
• MT outer diameter: $24\text{–}25\,\text{nm}$; lumen: $15\,\text{nm}$
• IF diameter: $8\text{–}12\,\text{nm}$
• Actin filament diameter: $7\text{–}8\,\text{nm}$
• MT protofilaments per tube: $11\text{–}18$ (often $13$)

Connections to Previous / Foundational Concepts

• Actin & MT drugs illustrate specificity of small molecules for distinct cytoskeletal elements → paradigm for targeted therapy
• Polarity concept contrasts among cytoskeletal systems: MT & actin polarized (support motor directionality); IF non-polar
• Energy dependency: MT & actin dynamics require nucleotide (GTP/ATP) hydrolysis, IF assembly does not