3/23 Lecture

Overview: Focus on receptor tyrosine kinases using VEGF (Vascular Endothelial Growth Factor) receptors as an example.
Context: Discussion centers on downstream signaling, particularly regarding the regulation of the actin cytoskeleton and cellular migration.

Actin Filaments: Crucial for organizing into different arrangements under signaling responses.
Signaling Cascades: Various downstream signaling components activated by the VEGF receptor and other signaling pathways.
Function of the Actin Cytoskeleton: Vital for cell migration; structure depends on the direction of the incoming signals.

Leading Edge: Forms lamellipodia, which consist of actin organized by the $ ext{Arp2/3}$ complex with a 70-degree branching pattern.
- Pushing Mechanism: Actin polymerization pushes the front of the cell forward.
- Anchor Points: Anchored to the substrate via focal adhesions (integrin-based junctions).
Back End Motion: Pulls the cell forward using contractile bundles of actin, often referred to as stress fibers.

Filopodia: Protrusions that act as sensors, enriched with receptors, containing fimbrin to organize tight, parallel bundles of actin.
Dendritic Network: Formed by the $ ext{Arp2/3}$ complex that helps establish branched structures.
Contractile Bundles: Contain alpha-actinin and interactions with active myosin motors that facilitate contraction.

CDC42 Activation: Promotes the formation of filopodia when activated, causing a uniform extension in all directions.
RAC Protein: Activation leads to the formation of lamellipodia around the entire cell surface, hindering the cell's directional migration.
RHO Protein: Activation results in contractile bundles (stress fibers) formation, enabling the structural integrity necessary for directional movement.

RAC Pathway:
- Activates the WASP protein family, essential for $ ext{Arp2/3}$ activation, facilitating branched actin webs formation.
- Activates PAK (p21-activated kinase) that targets filament proteins and inhibits myosin light chain kinase (MLCK), reducing myosin activity and stress fiber formation.
RHO Pathway:
- Activates Rho-dependent kinase (ROCK), leading to stress fiber stabilization by preserving long actin filaments and phosphorylating myosin light chain, increasing myosin activity.
- Induces formin activation for actin filament growth.

Opposing Actions: RHO induces stress fiber formation while inhibiting lamellipodia. In contrast, RAC promotes lamellipodia formation and inhibits stress fiber development.
Cellular Context:
- Both pathways can be active simultaneously within a single cell, allowing for spatial organization of different actin configurations.

Example of Pathway Integration:
- A G protein-coupled receptor (GPCR) example shows a MAP kinase signaling cascade activated by the VEGF receptor or other GPCR.
Chemoattractant Response: Cells move towards a signal (chemoattractant) due to spatial activation of pathways creating asymmetric signaling responses in different parts of the cell.

Integrin Activation: Signaling pathways must activate integrins for adhesion to the extracellular matrix leading to focal adhesion formation, essential for migration.
RAF-1 Protein: A significant activator for integrin recruitment and adhesion stabilization.

Slow Response: Involves altering gene expression through new protein synthesis (e.g., cyclins required for cell cycle progression).
Fast Response: Rapid response utilizing existing proteins and altering their activities without requiring new protein synthesis.
- Rapid reactions can include phosphorylation and recruitment of pre-existing proteins.

Example of Notch Signaling: Distinguishes responses among vascular endothelial cells exposed to the same VEGF signal, leading to tip cells and tube-forming cells.
Mechanism: The Notch pathway employs contact-dependent signaling to regulate gene expression and response to nearby cells, allowing for differentiation in function.
Calcium Waves: Initial uniform response of cells to the VEGF signal marked by varying calcium wave frequency, determining which cell becomes the leading (tip) cell.

Delta Ligand and Notch Activation:
- Delta from one cell activates Notch on neighboring cells, inhibiting those cells from expressing delta.
- Activation results in differential responses to VEGF, leading to some cells proliferating while others migrate.
- Notch intracellular domain enters the nucleus to modulate gene expression, influencing the levels of VEGF receptor and downstream effects.

Mutations in Notch Signaling: Can disrupt the migration and differentiation of cells, leading to vascular malformations due to all cells acting as potential tip cells when they should not.

Exam Preparation: Focus on the intricate details of each pathway discussed, their interactions, and the functional implications of signaling disturbances. The session will culminate with an exploration of cell signaling components in cell cycle phases during the subsequent lecture.