Microtubules and Intermediate Filaments

• Microtubules and intermediate filaments are components of the cytoskeleton, which also includes microfilaments.

Microtubules

• Microtubules are polymers made of the protein tubulin.
• Tubulin subunits are heterodimers formed from two closely related globular proteins: alpha-tubulin and beta-tubulin, each comprised of 445-450 amino acids.
• Each tubulin has one GTP binding site.
• GTP bound to alpha-tubulin is trapped and not hydrolyzed or exchanged.
• GTP in beta-tubulin is hydrolyzed to GDP during polymerization (growth).
• GDP reconverts to GTP when the microtubule depolymerizes (shrinks), allowing beta-tubulin to polymerize again.
• Microtubules are hollow cylindrical structures built from heterodimeric protofilaments.
• Microtubules have alpha-beta tubulin heterodimers that stack head to tail and fold into a tube.
• Dynamic instability is how microtubules grow (polymerize) and shrink (depolymerize).
• Dynamic instability involves microtubules switching between growth (assembly) and shrinkage (disassembly) phases.
• Microtubules can dynamically switch between growing and shrinking phases.
• Structural plasticity/dynamics relies on the GTP-binding domain activity in beta-tubulin.
• Adding GTP-containing tubulin to a protofilament end causes the end to polymerize (grow).
• Microtubules depolymerize (shrink) if GTP hydrolysis happens faster than adding new subunits

Clinical Relevance of Microtubules Dynamic Instability

• Cancer therapy drugs target microtubule dynamics, either stabilizing or destabilizing microtubules to disrupt mitosis and induce cell death.
• Microtubule-associated protein dysfunction leads to instability occurs in neurodegenerative diseases, This contributes to neurodegeneration.
• Therapies that modulate dynamics of microtubules and allow control tumor cell movement are used in cell migration and metastasis, processes like wound healing and cancer metastasis are impacted.
• Microtubule dynamics are essential for cell division and differentiation during development with disruptions potentially leading to developmental disorders
• Some pathogens exploit microtubule dynamics for entry and movement within host cells.
• Understanding these interactions can lead to new therapeutic approaches against infections.
• Microtubules originate from the microtubule organizing center (MTOC).
• In animal cells, the centrosome is the primary MTOC.
• The centrosome includes 2 cylinders called centrioles.
• Centrioles are small cylindrical organelles near the nucleus in animal cells, occurring in pairs and involved in spindle fiber development.
• When a cell divides, MTs rearrange to form a bipolar mitotic spindle.
• Bipolar mitotic spindle aligns and segregates chromosomes.
• Astral microtubules occur during this process
• After division, both daughter cells reorganize their MT and actin filaments.

Microtubule-Associated Proteins (MAPs)

• MAPs move along microtubules, transporting vesicles to target organelles in the cell.
• Kinesin typically travels towards the (+) end of the microtubule.
• Dynein typically travels towards the (-) end of the microtubule.
• ATP hydrolysis in the globular head domains generates movement along the microtubule via the microtubule-binding domains for kinesin and dynein
• Dynein includes two identical heavy chains to make large globular head domains, and a variable number of intermediate and light chains.
• Dynein transports intracellular cargos toward the (-) end of the microtubule.
• Kinesin shares a similar structure to dynein.
• Kinesin transports diverse intracellular cargoes to the (+) end of the microtubule, including vesicles, organelles, protein complexes, and mRNAs.
• Selective stabilization of microtubules can polarize a cell (+ end or – end).
• MAPs can move organelles and vesicles within the cell.
• Cell polarity represents spatial differences in shape, structure, and function within a cell.
• MAPs transport vesicles with pigments (melanosomes) in the skin.

Cilia and Flagella

• Microtubules play a structural role in eukaryotic cilia and flagella.
• Cilia and flagella both have highly specialized and efficient motility structures.
• Cilia and flagella contain a basal body with a microtubule base of 9:2 structure.
• Dynein motor proteins move along MT strands in the cilium/flagellum, bending and generating the force for movement.
• Prokaryotes have tubulin-like proteins, however, prokaryotic flagella differ from eukaryotic flagella because they do not contain MT-based structures.
• Flagella move cells in aqueous environments (e.g., spermatozoids).
• Cilia move fluid around a cell (e.g., mucus upwards in the respiratory epithelium).

Microtubule Specific Drugs

• Paclitaxel (Taxol): Prevents depolymerization of MT, decreasing dynamic nature of cytoskeletal structures
  • Taxol is an anticancer chemotherapy drug
• Taxol is a cytostatic drug used in cancer therapy for breast/ovarian cancer
• Taxol inhibits mitotic cell progression to G1 phase via interference with spindle formation without affecting other microtubule function during anaphase/telophase
• Vinca Alkaloids (e.g., Vinblastin, Vincristine, Vindesin):
  • Vincristine binds irreversibly to microtubules and spindle proteins
    • This interferes with formation of the mitotic spindle, arresting tumor cells in metaphase
  • Vinca alkaloids are found in periwinkles (Vinca rosae, Vinca minor)
  • Binds free tubulin and prevents MT polymerization and cell division
    • Used as cytostatic in treatments for Leukemia, Lymphoma, breast cancer
• Colchicine from alters tubulin in alkaloids the autumn crocus (Colchicum autumnale)
  • Colchicine as a result, prevents MT polymerisation and cell division, demonstrating anti-inflammatory effects.
• Colchicine can aid in familial Shar-pei fever treatment
  • Colchicine Prevents MT polymerization and consequently secretion of SAA (serum amyloid A – reactive amyloidosis) thus resulting in Disruption of moment of leukocytes and other inflammatory cells

Intermediate Filaments

• No polarity (+) or (-) end exists on Intermediate Filaments

• Subunits don't contain ATP or GTP

• Intermediate filaments not involved in cell movement

• No motor proteins associate

• Intermediate filaments associate with cell-cell junctions strengthening cells and epithelia, tissue mechanical stability

• It lines the inner face of the nuclear envelope, forming a protective cage for the cell's DNA

• In the cytosol, they are twisted into strong cables

• Bundles hold epithelial cells sheets together

• Nerve cells extend long and robust axons

• Hair and fingernails contain Intermediate Filaments

• The central building block of Intermediate Filaments:

  • two intertwined proteins = 'coiled-coil' dimer
  • The building block are all bound together by hydrophobic interactions

• In a final IF there are 16 dimers of IF monomers (=32 coiled coils)

• Major Types of Intermediate Filament Proteins in Vertebrate Cells

  • Nuclear :
    • lamins A, B, and C- which helps to shape the nuclear lamina (inner lining of nuclear envelope)
  • Vimentin-like- vimentin, which helps in:
    • cells of mesenchymal origin to function,
    • desmin to build muscle,
    • glial fibrillary acidic protein to help glial cells (astrocytes and some Schwann cells) -peripherin also works in glial cells and some neurons
  • Epithelial:
    • type I keratins (acidic)
    • type II keratins (basic) give rise to epithelial cells and their derivatives (e.g., hair and nails)
  • Axonal:
    • neurofilament proteins help neurons to operate.

• Epithelial Keratin Filaments:

  • Includes the most diverse intermediate filament family

  • Produced by keratinocytes in the epidermis

  • Help in the Formation of horns, nails, hair, scales

  • Anchoring of epithelial cells occurs via desmosomes/hemidesmosomes

• The diversity of keratins is clinically useful in diagnosis of

  • Epithelial cancers (carcinoma)
  • Particular set of keratin filaments expressed indicates epithelial tissue in which the cancer originated and thus guides the choice of treatment
    • Ex Mutation in keratin genes causes genetic diseases of affecting various tissues-liver, intestines, skin

• Gene Mutation causes various effects such as - Epidermolysis bullosa simplex (EBS), the final protein results in disruption of the keratin network in basal cells of the skin

  • As a result, the epidermis detaches (blistering)
    • EBS may arise from KRT5 missense variant in Cardigan Welsh Corgi or PLECnonsense variant.

• Vertebrate neurons make the neurofilament, helping the neurons to function- Participates(axonal growth) Participates in axonal growth (length and diameter)

  • Neurofilaments give strength and stability to the axon for the neuron

Cytoskeleton - Summary

• Microfilaments
  • Structure: Two helical crossed actin strands
  • Diameter: 7-8 nm
  • Subunit: Actin
  • Functions: Cell shape maintenance, shape changes, muscle contraction, cell movement
  • Localization: Periphery of the cell, sometimes running parallel
• Microtubules
  • Structure: Tubes of 11-18 strands per MT
  • Diameter: 24-25 nm with 15 nm Lumen
  • Subunit: Tubulin (α- and β-Tubulin)
  • Functions: Cell shape maintenance, chromosome movement during cell division, organelle movement, cell movement
  • Localization: Coming from an organizing center (e.g., centrosomes) and spreading towards the cell periphery
• Intermediate Filaments
  • Structure: Long molecule polymers
  • Diameter: 8-12 nm
  • Subunit: Keratin, Lamin, Vimentin
  • Functions: Cell shape maintenance, mechanical strength, formation of nuclear lamina
  • Localization: Distributed along the whole cytoplasm of the cell, building a network of filaments in the cytosol