Cell Biology: Microtubules and Cytoskeleton

Chapter 3: Cytoskeleton

• The cytoskeleton is a network of filamentous proteins within the cytoplasm
• It provides mechanical support, maintains cell shape, and enables cell motility
• It facilitates chromosome migration during mitosis, organelle movement, and biochemical reactions
• The cytoskeleton includes actin microfilaments (AF), intermediate filaments (IF), and microtubules (MT)

Cytoplasm Composition

• Cytoplasm (cytosol, hyaloplasm) is a jelly-like fluid with
• Water (70%)
• Small molecules (low MW): ions, gases, glucose, amino acids, fatty acids, urea, glycerol
• Large molecules (high MW): proteins, lipids, carbohydrates, nucleic acids (RNA)
• Inert inclusions (non-living)
• Proteins (proteasome)
• Lipids (droplets)
• Carbohydrates (rosettes, little clouds)

Cytoskeletal Components

• Actin Microfilaments (AF): 5-8 nm diameter
• Intermediate Filaments (IF) : 8-10 nm diameter
• Microtubules (MT): 20-30 nm diameter

Actin Microfilaments (AMF) Structure and Nucleation

• Monomer: G-actin (globular protein) with 1 ATP groove
• Polymer: Several G-actins bind together to form F-actin. ATP grooves on same side. Involves ARP2/3 (a complex) forming 2 twisted chains.

Actin Polymerization/Depolymerization

• ATP hydrolysis drives polymerization and depolymerization
• Polymerization occurs at the plus end, while depolymerization occurs at the minus end
• Factors like ARP2/3, Profilin, and CapZ regulate polymerization
• The polymerization/depolymerization rate depends on G-actin/ATP concentration
• The plus end is referred to as a barbed end and the minus end is a pointed end

AMF Roles

• Maintain cell shape using cellular cortex (actin layer)
• Enable cytoplasmic currents (movement of molecules and organelles)
• Support structures like microvilli (intestinal and kidney cells)
• Facilitate vesicle exchanges (endocytosis/exocytosis)
• Essential during cell division: forms a contractile ring
• Enables diapedesis (movement of white blood cells out of vessels)
• Enables cell migration by the polymerization and depolymerization of AMFs

Microtubules (MT)

• Organize the positions of organelles
• Direct intracellular transport
• Composed of tubulin dimers (α-tubulin and β-tubulin) arranged in a hollow tube structure with 13 protofilaments per circumference.

MT Structure and Assembly

• Monomer: α-tubulin and β-tubulin
• Polymerization: Tubulin dimers bind to each other using GTP, forming protofilaments. 13 protofilaments combine to form a microtubule
• The γ-TuRC complex is crucial for MT nucleation (initiating assembly). It's considered a strong nucleator.
• MTs are stabilized by accessory proteins. MAP2 is a common example.

MT Polymerization

• Depends on equilibrium between free and bound tubulins. Free GTP form leads to polymerization; otherwise depolymerization.
• Imbalance in the GTP-bound to GDP-bound tubulin ratio can cause instability and collapse. This is called a disaster phenomenon.

MT Associated Proteins

• γ-TuRC: MT nucleation
• MAP2: MT stability
• Kinetochore: Attaches chromosomes to mitotic spindle during mitosis
• Motor proteins (Kinesin and Dynein): convert chemical energy into mechanical energy for transport of cargo along microtubules. Kinesin moves towards the + end, Dynein towards the minus end.

Intermediate Filaments (IF)

• Stability: Never polymerize or depolymerize
• Cytoskeleton Role: Maintain cell shape
• Nuclear Lamina: Form a nuclear lamina in the nucleus (3 types of lamins: A, B, and C). Reinforces the nuclear envelope. They are crucial against the pressure of the nucleus.
• Diagnostics: Used in immunohistochemistry (IHC) to determine the cancer type
• Families:
  • Keratins (epithelial cells) - crucial in diagnosing carcinomas
  • Desmin (muscle cells, myoblastomas)
  • Vimentin (connective tissue, sarcomas)
  • Neurofilaments (neurons, neuroblastomas)
  • GFAP (glial cells, gliomas)

IF Structure and Assembly

• Composed of filamentous monomers with two heads
• Monomers form dimers, followed by tetramers, then a rope-like structure. The specific tetramer arrangements within the rope will depend on the type of intermediate filament.

IF Functions

• Morphogenesis: Forms a scaffold for maintaining cell shape.
• Muscles: In muscle cells, IFs orient actin and myosin to maintain organization, and thus contraction.
• Resistance: IFs link desmosomes and hemidesmosomes to each other for cell resistance. Disruptions in this can cause diseases like epidermolysis bullosa.

Cilia and Flagella

• Microtubules control their beating
• Cilia move fluids (e.g., mucus in the throat)
• Flagella propel cells (e.g., sperm)

Axonal Transport

• Kinesin and Dynein move cargo along microtubules. Kinesin moves towards the + end and Dynein moves towards the - end.
• Cargo can be transported in either direction.

Centrosomes and Centrioles

• Centrosomes have a pair of centrioles at its core
• Centrioles are made of microtubules arranged in a specific configuration that differ in their longitudinal and cross sections.

Pharmacological Effects on MTs

• Certain drugs (e.g., colchicine, taxol, vincristin, podophyllotoxin) interfere with MTs, impacting polymerization, depolymerization, and overall microtubule function. This is a major target in chemotherapy and cancer treatment.