Comprehensive Study Guide to Cytoskeleton, Cell Cycle, and Membrane Biology

Actin Filament Structure and Nucleation Kinetics

• Structural Properties of Actin Filaments:

  • Actin filaments are semi-flexible, asymmetric right-handed helical structures.
  • Unlike microtubules, which are hollow tubes consisting of $13$ protofilaments, actin filaments are solid strands.
  • They are polarized filaments, meaning they have distinct (+)-ends and (-)-ends, which distinguishes them from unpolarized tetrameric intermediate filaments.

• Transition from G-actin to F-actin:

  • The transition from globular (G-actin) to filamentous (F-actin) is primarily limited by the kinetics of the generation of stable dimers and trimers. This initial nucleation phase is the rate-limiting step of actin polymerization.

• Role of Profilin in Actin Dynamics:

  • Profilin regulates the pool of available G-actin by inhibiting spontaneous dimer and trimer formation.
  • It catalyzes the exchange of $ADP$ for $ATP$ on G-actin, ensuring a supply of $ATP$-bound monomers for polymerization.

• Arp2/3 Complex and Dendritic Networks:

  • The Arp2/3 complex is essential for creating branched (dendritic) actin networks.
  • Effective generation of these networks requires two primary components: an existing actin filament to act as a primer/template and a capping protein to limit the elongation of the branches, maintaining the branched structure.

Actin Disassembly and Regulatory Proteins

• ADF/Cofilin Activity:

  • ADF/cofilin induces the disassembly and fragmentation of actin filaments.
  • It preferentially binds to the $ADP$-bound (-)-end of the filament.
  • Tension Regulation: Excessive mechanical tension on a filament generally reduces the efficiency of ADF/cofilin fragmentation. This is because tension likely stabilizes the filament's helical conformation, making it more resistant to the localized mechanical stress or twisting induced by cofilin binding.

• Ena/VASP Proteins:

  • These proteins are critical for the formation of filopodia.
  • They promote prolonged actin elongation and actively prevent capping proteins from binding to the (+)-end, allowing the filaments to grow long enough to push against the membrane.

• Formins (e.g., mDia2):

  • Formins are used to construct actin-based sensory protrusions (filopodia) because they promote the persistent, uncapped elongation of parallel filament bundles. This provides the structural rigidity required to push narrow protrusions outward against membrane tension.
  • FH1 and FH2 Domains: The interaction between the FH1 domain of formins and profilin is essential for high rates of formin-dependent linear actin elongation.

Higher-Order Actin Structures and Motility

• Cdc42 and Filopodia: Constitutive activation of the small GTPase $Cdc42$ leads to the prominent formation of abundant filopodia.

• Retrograde Flow: The loss of depolymerization by ADF/cofilin at the rear of the lamellipodium can cause a sudden cessation of retrograde actin flow, as the recycling of monomers is halted.

• Actomyosin Contraction (In Vitro):

  • If actin filaments and myosin II are assembled in an assay without additional cross-linking proteins, the filaments will contract strongly and subsequently disassemble.

• Elastic and Viscous Properties:

  • Dendritic actin networks in the lamellipodium behave as an elastic solid on short time scales ($<1\,\text{min}$) but as a viscous fluid on longer time scales ($>10\,\text{min}$).
  • This transition is due to the binding and unbinding of cross-linkers and the continuous remodeling of the network by myosin activity.

Microtubules and Intermediate Filaments

• Microtubule Dynamic Instability:

  • This is defined as the stochastic switch between phases of rapid growth (polymerization) and rapid depolymerization (catastrophe) at the (+)-end.
  • Katanin: Katanin is a protein that severs microtubules. High expression in aggressive glioma cells may increase cytoskeletal flexibility and facilitate remodeling during invasion through narrow tissue spaces.
  • Force Generation: Depolymerizing microtubules can exert a pulling force on cargo via the lateral dissociation of protofilaments, which bend backward into ring-like shapes while remaining attached to the cargo.

• Intermediate Filament Construction:

  • The primary building block of cytoplasmic intermediate filaments is the unpolarized tetramer.
  • These tetramers are formed by antiparallel coiled-coil dimers.
  • Plectin: A critical protein that provides mechanical linkage between intermediate filaments, actin filaments, microtubules, and desmosomes. Defective plectin results in severe mechanical instability across cytoskeletal networks.

Cell Mechanics: Blebbing and Cortex

• Cell Blebbing Initiation: The most direct cause of membrane protrusion during blebbing is local cortex tearing or the loss of cortex-membrane adhesion, coupled with internal hydrostatic pressure.
• Bleb Size: The maximal size a bleb can reach is dictated by the initial growth rate balanced against the time required for the actin cortex to re-polymerize and the resistance offered by membrane unfolding.
• Actin Cortex Definition: The cortex is not an "elastic, static shell." It is accurately characterized as undergoing rapid, continuous turnover driven by cross-linker kinetics, myosin contraction, and cofilin severing, displaying glassy, visco-elastic properties.
• Deformation Resistance: For rapid, small-scale cellular deformations (e.g., 5\text{%} stretch over $1\,\text{second}$), cross-linked actin networks dominate the response because they are stiffer at low strains. Intermediate filaments are more flexible and only harden at high strains.

The Cell Cycle: Phases and Regulation

• Stages of the Cycle:

  • G1 Phase: Typically the longest stage of the cell cycle.
  • G0 Phase: A permanent dormant phase entered by cells that do not normally divide, such as neurons and cardiac muscle cells.
  • G1 Restriction Point: The most critical milestone where the cell commits to division based on growth factors and nutrients; after this point, extracellular growth factors are no longer required to proceed.

• Cyclins and CDKs:

  • Cyclins: Regulatory proteins whose concentrations fluctuate in a coordinated cyclical pattern of synthesis and proteasome-mediated degradation. Their primary role is to bind and activate Cyclin-Dependent Kinases ($CDKs$).
  • CDKs: Their concentration remains relatively constant throughout the cycle.
  • M Phase Activity: The Cyclin B-CDK1 complex is most active during the M phase. It facilitates events such as the breakdown of the nuclear envelope by phosphorylating/breaking down lamins.
  • S Phase Licensing: Origin of replication firing happens only once because key licensing factors (like $Cdc6$ and $Cdt1$) are phosphorylated and removed by Cyclin E-CDK2. Hyperactive Cyclin E-CDK2 leads to premature replication and genomic instability.

• Anaphase-Promoting Complex (APC):

  • The APC is responsible for the polyubiquitination and degradation of Cyclin B. Inactivation of the APC results in mitotic arrest because the cell cannot exit the M phase.

Tumor Suppressor Proteins and Genes

• p53 ("Guardian of the Genome"):

  • In the event of DNA damage, $p53$ acts as a transcription factor for p21.
  • If $p53$ is lost (loss-of-function mutation), cells will continue to divide despite severe DNA damage.

• p21:

  • A tumor suppressor that binds to and inhibits $CDK$ activity, causing cell cycle arrest.
  • P21 level is an indicator of cell cycle arrest, not a definitive indicator of apoptosis.

• Rb (Retinoblastoma) Protein:

  • Hypophosphorylated (Active) State: Rb binds and sequesters the transcription factor E2F, preventing the transcription of S-phase genes.
  • Prevention of Rb phosphorylation arrests cells at the G1/S boundary.

Cell Death: Apoptosis vs. Necrosis

• Apoptosis ("Falling Off"):

  • Morphological Hallmarks: Formation of apoptotic bodies, cell shrinkage, and maintenance of membrane integrity (though the membrane is altered).
  • Intrinsic Pathway: Centrally involves the mitochondrion and the release of Cytochrome C.
  • Executor Caspase: Caspase-3 is the ultimate executor of apoptosis, irreversibly cleaving DNA and lamins.
  • Bcl-2 Family: Pro-apoptotic members (Bax/Bak) and anti-apoptotic members (Bcl-2) compete. Bcl-2 maintains mitochondrial integrity by preventing Bax/Bak from forming pores.
  • Extrinsic Pathway: Involves diagnostic markers like Caspase-8.
  • C8+ T-cell Induction: Granzymes enter target cells through perforin pores and activate pro-apoptotic regulators like Bid.
  • Developmental Role: Essential for removing tissue between developing digits (failure leads to syndactyly).

• Necrosis:

  • Pathological death caused by extreme injury or toxins (e.g., rapid depletion of all intracellular $ATP$).
  • Hallmarks: Cell swelling, loss of membrane integrity, and leakage of cytoplasmic contents.
  • Physiological Response: The release of intracellular enzymes/contents provokes a massive inflammatory response.

Biological Membranes: Structure and Lipids

• The Plasma Membrane:

  • The main permeability barrier is the hydrophobic core of the lipid bilayer.
  • Membranes are best described as fluid, mosaic, and asymmetric.
  • They behave as two-dimensional fluids.

• Membrane Lipids:

  • Phospholipids: Most abundant membrane lipids; they are amphiphilic (containing a hydrophilic head and hydrophobic fatty acid tails). They form bilayers spontaneously to minimize water contact with the hydrophobic tails.
  • Cholesterol: A sterol that orients itself with its $-OH$ group near the phospholipid heads. It modulates membrane fluidity.
  • Fluidity Factors: Fluidity increases with higher temperatures and an increase in unsaturated fatty acids.
  • Lipid Rafts: Dynamic microdomains enriched in cholesterol and sphingolipids.
  • Lipid Droplets: Originate from the ER membrane, store neutral lipids, and are surrounded by a monolayer (not a bilayer).

• Membrane Asymmetry:

  • Glycolipids: Primarily found in the outer leaflet and form the glycocalyx (carbohydrate coat for protection).
  • Phosphatidylserine (PS): Normally located in the inner (cytosolic) leaflet. Externalization of PS to the outer leaflet is a definitive indicator of apoptosis.
  • Protein Kinase C (PKC): Binds preferentially to the PS-rich cytosolic leaflet.

Membrane Proteins and Transport

• Types of Associations:

  • Transmembrane Proteins: Typically amphiphilic, featuring $\alpha-helical$ domains or $\beta-barrel$ structures (common in outer bacterial membranes).
  • Lipid Anchors: Includes Myristoylation (fatty acid added to N-terminus), Palmitoylation (on cysteine residues), and GPI anchors (attach proteins to the outer leaflet).
  • Peripheral Proteins: Attach via noncovalent interactions and can be released by high salt concentrations or pH changes.

• Protein Environment and Modification:

  • The cytosolic environment is reducing.
  • Disulfide bonds form mainly in the noncytosolic (extracellular/lumenal) domains because the environment there is oxidizing.
  • Glycosylation: Occurs in the ER and Golgi.

• Experimental Techniques:

  • FRAP (Fluorescence Recovery After Photobleaching): Measures the lateral diffusion of membrane molecules.
  • Heterokaryon Experiments: Demonstrated the mobility of membrane proteins.
  • Detergents: Solubilize membranes by disrupting the bilayer. At the CMC (Critical Micelle Concentration), they form micelles. Nonionic detergents are preferred for preserving protein structure.
  • Nanodiscs: Used to stabilize a membrane protein within a small patch of bilayer for study.

• Structural Support and Curvature:

  • Spectrin: Provides mechanical support to the red blood cell membrane; loss of spectrin results in fragile, abnormally shaped cells.
  • Tight Junctions: Restrict the lateral diffusion of membrane proteins.
  • Curvature Induction: Can be achieved by membrane-bending proteins or by a protein inserting into only one leaflet of the bilayer.