Cellular Dynamics and Polarity

• Cellular dynamics refers to cell migration.

Key Words from Last Lecture

• Cell polarity involves spatial differences in a cell's shape/structure, enabling unique functions.
• Apical-basal polarity is cell polarity vs planar cell polarity
• Par Complex, Crumbs, and Scribble are involved in the polarization of an epithelial cell.
• Cell-cell communication is neuronal polarization
• Axons vs dendrites involves MT organization.

Cell Polarization

• Cell polarity involves spatial differences in a cell's shape/structure, enabling unique functions.
• Apical-basal polarity is shown in epithelial cells via
  • The apical membrane faces the outside surface of a body.
  • The lumen of internal cavities and the basolateral membrane are oriented away from the lumen.
• Cell-cell communication is how neurons communicate in one direction
  • From dendritic terminals, through the soma and out through the axonal terminal.
• Cell migration needs a defined front and rear
  • The nucleus, Golgi apparatus, and microtubules guide cell migration.
  • Microtubules emanate asymmetrically from the centrosome to pull the nucleus.
  • Nuclear translocation and microtubule polarization define the direction of polarization in most mesenchymal cells.

Polarization and Migration

• Actin polymerization drives plasma membrane protrusion.
• Leading edge protrusion results from actin contraction at the rear and then pushes the plasma membrane forward.
• Cells can crawl across a solid substratum.
• This needs polarized organization of the actin and myosin cytoskeleton.
• Actin polymerization pushes the leading edge of the cell forward by creating a protrusion in the plasma membrane
• Myosin contraction is in the rear and then pushes the cell body forward.
• The actin cortex, focal adhesions, and lamelopodia are components of cells that can crawl across a solid substrate.

Cell Polarization and Migration types of protrusion

• Protrusions, or microspikes, include filopodia, lamellipodia, invadopodia, and blebbing.
• Filopodia are common in migrating growth cones of neurons and fibroblasts
  • They have one dimensional long bundles of parallel actin
• Lamellipodia are common in epithelial cells, fibroblasts, and neurons
  • They are 2-dimensional with a mesh-like network of branched actin.
• Invadopodia are often found in metastatic cancer
  • They are 3-dimensional actin-rich protrusions that penetrate tissue barriers.
• Blebbing is the protrusion of the membrane
  • It is due to the loss of the actin cortex and the formation of an immature spine.

Lamellipodia

• Cell movement starts with lamellipodia.
• Lamellipodia generate pushing forces that drive the cell forward.
• Actin filaments stretch out to the cell's periphery within lamellipodia.
• Microtubules do not penetrate this actin network.
• They direct cell motility by controlling cell adhesion and cell polarization.
• Cell polarization gives the cell its "internal compass".
• Also critical for wound healing in epithelial cells.

Lamellipodia contents and migration movement

• Actin filaments fill the lamellipodia and are responsible for the rapid movement.
• Microtubules and intermediate filaments are restricted to the area around the nucleus.
• Migration machinery depends on the ARP2/3 complex for nucleated branched actin.
• Actin, ARP2/3 complex-red for branched actin mesh.
• The plus ends of actin filaments orient towards the leading edge of the cell.
• Minus ends are anchored to other actin filaments by the ARP2/3 complex to create the actin mesh.
• Arp2/3 facilitates nucleation of new actin polymers at a 70 degree angle to the existing actin polymer.
• When ARP3 is missing there is still some movement governed by the filopodia and not lamellipodia

Actin dynamics of lamellipodia

• Cofilin disassembles actin filaments.
• Actin polymerization hydrolyzes ATP to ADP.
• ADP-Actin has a higher affinity for cofilin and is susceptible to cofilin depolymerization.
• Cofilin localizes to the rear of the leading edge ensuring actin polymerization is toward the leading edge only.
• Actin is at the leading edge and cofilin is at the back of the lamellipodia
  • Pulls actin monomers off via depolymerization at the rear side of the actin filaments.
  • Allows movement in one direction and ensures enough actin monomers are available to allow for growth.
• Constant polymerization of actin at the edge and depolymerization of actin at the rear pushes the cell forward

Filopodia

• They are thin, finger-like projections in contrast to the sheet-like lamellipodia.
• Filopodia are composed of tight parallel bundles of filamentous or "F"-actin.
• They act as cellular "antennae" probing the cell's microenvironment.
• They are involved in constructing cell-cell adhesions and guiding growing axons to chemoattractants.
• The MTs extend throughout the cytoplasm surrounding the nucleus.
• F-actin forms finger-like projections that contain receptors.

Growth Cone

• Wiring of complex patterns is achieved via actin and microtubule cytoskeleton controlling the growth cone
• The axon is steered by the growth cone's dynamics and is essential for directional migration.
• The lamellipodia extending the finger-like filopodia come off the growth cone of an axon.
• The MT is more stable than the actin filaments
  • Actin filaments constantly undergo growth and shrinking to allow exploration of the cell environment.

Neuronal Growth Cone dynamics

• Filopodia extend in the first step.
• The filopodia then contacts a cellular or ECM component and attaches.
• A vesicle fuses into the membrane at the leading edge of filopodia.
• Actin polymerization pushes filopodia forward.
• Microtubules then advance from the core.
• The cytoplasm then collapses to create a new segment of axon.
• Cytoplasm components, or organelles, enter attached to microtubules via motor proteins and the axon advances.

Neuronal Growth Cone steps

• Filopodium contacts an adhesive substance through receptors connected to the actin cytoskeleton.
• This includes integrins that link the actin cytoskeleton to the laminin or fibronectin of the ECM
• Allows for the formation of focal adhesions.
• Depolarization of the actin filament occurs at the minus end due to polymerization occurring at the plus end.
• The support growth at the plus end needs vesicle fusion at the PM so the actin filaments can polymerize for extension.
• Microtubules advance to the new position of the core structure after the filopodia has been extended
• The cytoplasm supporting the lamellipodia behind the growth cone collapses

Lamellopodia machinery

• Contains all machinery necessary for migration.
• In epithelial cells, lamellipodia are critical for wound healing
• They can close wounds by moving at 30um/min.

Invadopodia

• Invadopodia, or "invasive feet," are protrusions rich in actin
  • They extend into the extracellular matrix (ECM) in the cell membrane of some cells.
• Three main steps occur:
  • Initiation, assembly, and maturation.
• Cortactin is an actin-binding protein that regulates cell migration and invasion, helping remodel the cytoskeleton.
• Formins are a family of proteins that help elongate actin filaments.
• Localized relaxation leads to blebbing.

The 3 stages of Invadopodia Formation

• Initiation via
  • Growth factors and integrin-ECM interactions, activating Src kinase, PI3K, and Rho GTPases.
  • Cortactin phosphorylation and Arp2/3 activation promoting localized actin polymerization
  • Weakening of cortical actin network, and increased intracellular pressure.
• Assembly via
  • Arp2/3 complex, N-WASP, and cortactin to promote branched actin polymerization.
  • Tks5 scaffold protein binds to PI(3,4)P2 lipids and stabilizes the structure.
• Actin cytoskeleton continues to build.
• Maturation via
  • MMPs, especially MT1-MMP recruited to the invadopodia.
  • MMPs degrade collagen and ECM components to clear a path.
  • Invadopodia disassemble if ECM degradation is complete, or they persist for continued invasion.
• The final product is a protrusion, has stress fibers, ECM. focal adhesion complex etc.

Chemoinvasion Assay

• Tests how metastatic cells are.
• Grow cells in a well inset covered in Matrigel that is infused with GFP
• Invadopodia formation occurs and can be analyzed with confocal microscopy
• Cells are stained red to show actin
• Dark areas in Matrigel show where actin has pushed through

Cell Migration requirements

• Myosin contraction and cell adhesion allow cells to pull themselves forward.
• Needs de-adhesion at the rear of the cell.
• Myosin links the actin cytoskeleton to the substratum through integrin-mediated adhesion.
• actin polymerization with myosin contraction forms focal adhesions

Integrin complex

• includes active integrin, alpha and beta subunits, vinculin, talin and an actin filament.

Myosin Contraction and Cell Adhesion

• Myosin II links actin filaments at the rear of lamellipodia to pull them in a new position
• From perpendicular to the leading edge and then parallel.
• Pulls cell toward the leading edge, and leaves focal adhesion contacts intact
• A myosin contraction by cells is between actin filaments
• When myosin contracts, one begins to pull them together and pull contents forward
• The cell still maintains contact with the substratum via focal adhesions
• Drives the cell's physical movement, while cell adhesion ensures the cell stays anchored as it moves

Traction and adhesion

• Engagement of integrins with the ECM is needed to allow a contracting myosin to pull the actin filament forward
• Premature loss of focal adhesions allows actin filaments to slip back away from the leading edge

Cell Polarization and Migration requirements

• Controlled by the Rho protein family
• Cell must distinguish between leading edge and rear
  • requires polarization of the cytoskeleton by Rho protein family
  • includes Cdc42, Rac and Rho.

Filopodia Formation

• Cdc42 activation on the inner surface of the PM triggers actin polymerization and bundling.
• Cdc42 activates WASp protein family members, like N-WASP.
• Stabilizes open (active) N-WASp protein conformation - binds profilin in filopodia or ARP2/3 in lamellipodia.
• This promotes filamentous actin nucleation and some branched.
• Involves Cdc42 activation, filopodia formation, and stress fiber

Lamellipodia Formation

• Rac activation triggers actin polymerization in the cell periphery
  • The peripheral sheet-like lamellipodia forms
• RAC activates WASp
• Rac activates ARP2/3 complex and profilin (mostly ARP2/3).
• Activates Filamin
  • Which is an Actin crosslinking protein that stabilizes orthogonal actin networks in lamellipodia.
• Rac activates Rho
• This induces Myosin contraction in the region opposite to the leading edge
• Not until after full formation of the lamellipodia.
• Involves giant lamellipodia around the entire cell.

Contractile force

• Rac activates WASp - activating ARP2/3 gives branched actin.
• Pak activates filamin to stabilize the actin structure
• Pak inhibits MLCK so it does not phosphorylate and decreases the myosin activity.
• Rho activation promotes myosin activity by:
  • bundling actin filaments with myosin II forming stress fiber
  • clustering of integrins forming focal adhesions.
• Activates formin promoting actin bundling
• Inhibits cofilin blocking depolymerization via activating LIM kinase
• Promotes MLC phosphorylation promoting myosin contraction via inhibiting MLC phosphatase

Directionality of Migration

• The net force that leads to direction of migration involves three signalling cascades.

1. Filopodia formation - leading edge, includes F actin.
2. Lamellipodia formation - leading edge that is formed y branched actin
3. The focal adhesions attach the cell to the ECM using integrins.
4. Stress fibers and other focal adhesions are in the rear.

• Signals include:
  • Extracellular matrix adhesion
  • Cell surface adhesion
  • Fasciculation
  • Chemoattraction
  • Contact inhibition
  • Chemorepulsion

Signaling for Cellular Protrusion and Migration

• External signals dictate direction
• This is chemotaxis when signals help promote cell migration toward/away from signal.
• Chemoattractant binds to GCPR of migratory cell
• Activates PI3K activating RAC
• RAC activates ARP2/3 promoting lamellipodia formation
• PI3K is rapidly degraded - directionality in actin mesh
• GCPR activation activates Rho to promote myosin contraction
• RAC - leading edge, Rho - rear signals create polarity.
• GCPR binds chemoattractant to activate PI3K and PIP3 for Rac activation for branched actin and filopodia.
• At the same time, GCPR activates Rho for the formation of actin stress fibers.
• Rac and Rho inhibit each other creating cell polarity.
• PIP3/PI3K acts locally at the front of the cell, and doesn't diffuse.

The questions to consider:

• What letter (A to D) corresponds with the components of the diagram? The components are:
  • Rac-GTP
  • Rho-GTP
  • Lamellipodia formation and
  • Stress-fiber formation
• Explain the signalling events necessary for a bacterial peptide (chemoattractant) released from this bacterium that initiates the migration of a white blood cell
• Explain the dynamics of how a neuronal growth cone migrates towards its intended destination