L15: Cell migration part 2 Flashcards
pathogen migration
Listeria monocytogenes, a bacterial pathogen, can invade host cells and use the host’s actin polymerization machinery to propel itself from cell to cell.
The bacteria form actin “comets” behind them, where actin filaments polymerize at the rear of the bacterium and depolymerize at the front, creating a propulsive force.
The bacterium expresses ActA, a protein that mimics host proteins, including a sequence resembling vinculin, which is found in focal adhesions.
ActA contains proline-rich motifs that bind to vas (vasodilator-stimulated phosphoprotein), which in turn recruits profilin-actin complexes to the rear of the bacterium.
Profilin binds G-actin, promoting its polymerization into actin filaments, and this polymerization and depolymerization process drives the bacteria forward.
forcing driving migration?
De novo actin polymerization and hydrodynamic force propel the leading edge of the cell forward.
Actin filaments are indirectly connected to cell surface receptors at the front of the cell. These receptors interact with the extracellular matrix (ECM) and substratum.
The forces that drive the cell forward include:
Traction forces: Generated from the friction between the cell and the ECM (adhesion to the substratum).
Contractile forces: Produced by actin-myosin contraction within the cell.
The net effect of these forces is to push the actin filaments against the leading edge of the cell, which in turn drives the cell forward.
These processes happen synergistically, meaning they work together to create a coordinated movement of the cell’s leading edge, facilitating cell migration. The traction and contractile forces ensure that the actin network remains dynamic, allowing constant pushing at the front while the cell adheres to its surroundings.
rho gtpases effect different actin organisations
Major Regulatory Proteins and Their Effects on Actin Turnover:
Rho family GTPases (Rho, Rac, and Cdc42) are critical regulators of actin dynamics. They act in different parts of the cell, depending on which actin structure is needed or present at any given time.
Rho GTPase:
Regulates formation of stress fibers (contractile actin bundles), which are important for cell contraction and maintaining cell shape.
These stress fibers are found in the cell cortex and are typically contractile bundles.
Rac GTPase:
Regulates the formation of the lamellipodia, which is a gel-like network of actin filaments.
The transition from stress fibers to dendritic networks (in lamellipodia) is controlled by Rac.
Lamellipodia are important for cell spreading and migration, as they help the cell to extend forward.
Cdc42 GTPase:
Plays a key role in the formation of filopodia, which are finger-like projections.
These filopodia are involved in sensing environmental cues, such as chemicals and morphogens at the front of the cell.
Cdc42 also helps to form tight, parallel bundles in these filopodia, which help the cell navigate its environment and migrate.
Rho, Rac, and Cdc42 work synergistically:
Rho: Forms contractile bundles (stress fibers) in the cortex.
Rac: Promotes dendritic networks (in lamellipodia) for cell protrusion.
Cdc42: Drives filopodia formation for environmental sensing and cell guidance
rho family gtpase states?
Rho GTPases are bound to GDP (guanosine diphosphate) in the cytoplasm.
In the inactive state, they are sequestered by Rho-GDI (Guanine nucleotide Dissociation Inhibitor), which prevents the exchange of GDP for GTP and keeps the Rho GTPase inactive.
Active (On) State:
Rho-GDI helps deliver Rho-GDP to the plasma membrane (PM), where it interacts with guanine nucleotide exchange factors (GEFs).
GEFs promote the exchange of GDP for GTP, activating the Rho GTPase.
Once activated, the Rho GTPase binds to effector proteins and influences various cellular processes, including cytoskeletal dynamics, gene expression, and cell adhesion.
Rho GTPase: Drives the formation of stress fibers (contractile bundles).
Rac GTPase: Promotes the formation of lamellipodia (broad, sheet-like projections).
Cdc42 GTPase: Promotes the formation of filopodia (finger-like projections).
Inactivation of Rho GTPase:
Rho GTPase is inactivated by binding to a GTPase-activating protein (GAP).
GAPs accelerate the hydrolysis of GTP to GDP, turning the GTPase back into its inactive state.
Once inactive, Rho GTPase is sequestered again by Rho-GDI.
Intrinsic GTP Hydrolysis:
Rho GTPases have intrinsic GTPase activity, meaning they can hydrolyze GTP to GDP on their own, but this process is often slow. The activity of GAPs accelerates this conversion, speeding up the inactivation process.
rho family gtpase signalling pathways?
The downstream effects of these GTPases are complex and involve a wide range of cellular processes, including:
Membrane trafficking and vesicle movement.
Cytokinesis (the final stage of cell division).
Cell cycle progression.
Microtubule stability.
Linking the actin cytoskeleton to the cell membrane.
Myosin phosphorylation and activation, influencing contractility.
Reactive oxygen species (ROS) production, involved in signaling and defense mechanisms.
Cell proliferation.
Actin polymerization and dynamics.
Cell adhesion.
Gene expression regulation: Particularly AP1-dependent (AP1 is a transcription factor).
Focal adhesion formation and cell-matrix interactions.
Cdc42 is involved in orienting the Golgi apparatus in relation to the microtubule organizing center (MTOC). This is crucial for proper intracellular organization, especially during cell division and secretion.
Upstream Regulation of Rho, Rac, and Cdc42:
The activity of Rho family GTPases is regulated by various upstream signals, including:
GEFs (Guanine nucleotide Exchange Factors): These proteins activate Rho GTPases by promoting the exchange of GDP for GTP.
GAPs (GTPase-activating Proteins): These inactivate Rho GTPases by accelerating the hydrolysis of GTP to GDP.
Rho GTPase activation can be influenced by several factors:
Integrin adhesion to the ECM (extracellular matrix): Cell-matrix interactions are critical for activating Rho family GTPases.
Tyrosine kinase receptors: These are involved in cell signaling and can activate GEFs.
G protein-coupled receptors (GPCRs): These receptors also regulate Rho GTPase signaling through GEFs.
Cadherins: Involved in cell-cell adhesion and regulation of Rho GTPase activity.
Adhesion via Ig superfamily receptors: These receptors also affect Rho GTPase signaling.
Mechanical stress: Physical forces on the cell can regulate Rho GTPase activity, particularly during cell migration or tension.
rho family gtpase activity?
RhoA and Actin Contractility:
RhoA activates ROCK (Rho-associated protein kinase), which then phosphorylates MLC (myosin light chain). This results in increased actin-myosin contraction.
RhoA → ROCK → Phosphorylation of MLC → Increased contraction.
LIM Kinase:
LIMK (LIM domain kinase) phosphorylates cofilin, inhibiting its activity. This stabilization of F-actin (filamentous actin) helps prevent depolymerization at the pointed end of actin filaments.
This is important for actin filament stabilization and reducing excessive depolymerization.
mDia Activation:
RhoA also activates mDia (formin), which binds to the barbed end of existing actin filaments.
mDia recruits profilin-G-actin complexes to add new actin monomers at the growing barbed end, promoting actin polymerization.
Rac1 and Actin Polymerization:
Rac1 activates the WAVE complex, which in turn activates the Arp2/3 complex.
The Arp2/3 complex promotes the nucleation of new actin filaments that grow at a 70-degree angle to existing filaments, leading to the formation of the branched network characteristic of lamellipodia.
PI(4,5)P2 (Phosphatidylinositol 4,5-bisphosphate):
Rac1 activates PI 4-5 kinase, producing PIP2, which is involved in actin polymerization.
PIP2 binds to proteins that mediate actin polymerization and stabilize the link between focal adhesions and F-actin.
Rac1 and p21-activated kinase (PAK):
Rac1 activates PAK, which in turn phosphorylates LIMK (LIM kinase). This promotes actin stabilization by inhibiting cofilin (which depolymerizes actin).
PAK can also phosphorylate MLCK (myosin light chain kinase), reducing its ability to phosphorylate MLC, thus decreasing actin-myosin contraction.
Cdc42 and Actin Dynamics:
Cdc42 activates N-WASP (neural Wiskott-Aldrich Syndrome protein), which in turn activates the Arp2/3 complex to promote actin polymerization. This helps form filopodia and other actin-driven protrusions.
Cdc42 and Golgi Orientation:
Cdc42 activates PAR6, which is involved in Golgi orientation relative to the microtubule organizing center (MTOC).
This helps direct the movement of vesicles from the Golgi towards the plasma membrane (PM) in a specific direction, which is essential for the polarization of the cell.
These vesicles contain new integrins, which are incorporated into the cell surface, stabilizing cell polarity and ensuring directed migration or specific cellular functions.
cancer metastasis?
- Cancer cells move from a primary tumour site to a secondary site
elsewhere in the organism in a process termed metastasis. - This process involves many changes in cell adhesion and migration.
Cancer Cell Migration and Metastasis:
Detachment from Primary Tumor:
Cancer cells in the primary tumor detach from the local cell mass and migrate towards blood vessels formed through angiogenesis (the growth of new blood vessels).
Intravasation:
Migratory cancer cells enter the bloodstream through a process called intravasation. Once in the blood, these cells are transported to distant parts of the body.
Extravasation and Secondary Tumor Formation:
Cancer cells extravasate (exit the bloodstream) and infiltrate surrounding tissues, where they can form secondary tumors. This process is known as metastasis.
Changes in Adhesion and Actin Remodeling:
Metastasis is driven by changes in cell-cell adhesion (e.g., cadherins) and cell-matrix adhesion (e.g., integrins). These changes enable actin remodeling, allowing cancer cells to move and invade new tissues.
Defects in Cell Signaling for Tumor Growth:
Uncontrolled Signaling:
Tumor growth requires defects in cell signaling, such as uncontrolled mitogenic (cell growth), motogenic (cell movement), and survival signaling.
This can result from constitutive activation of integrins (which don’t require binding to the matrix) or activated receptor tyrosine kinases (RTKs) that do not need their ligand for activation.
These abnormalities activate various tyrosine kinases, like focal adhesion kinases and Src family kinases, leading to uncontrolled signaling pathways that promote tumor growth and invasion.
Alpha 6 Beta 4 Integrin and Alternative Pathways:
Integrins such as alpha 6 beta 4 (important in hemidesmosomes) can activate alternative pathways, leading to PI3K activation, which consolidates uncontrolled signaling and tumor progression.
Loss of Cell-Cell Adhesion:
Downregulation of Cadherins:
For tumor cells to migrate away from the primary site, they need to lose or downregulate cadherin-based cell-cell adhesions (particularly E-cadherin).
Integrins or receptor tyrosine kinases (RTKs) can activate signaling pathways (like integrin-linked kinase and focal adhesion kinase or Src family kinases) that activate transcription repressors such as Snail and Slug.
Snail and Slug:
These transcription repressors move to the nucleus and suppress the expression of E-cadherin, leading to a decrease in E-cadherin levels on the cell surface.
In parallel, Src family kinases can activate proteins like HAX-1, which can bind to the cytoplasmic tail of E-cadherin, leading to endocytosis of E-cadherin and disrupting cell-cell adhesion.
Extracellular Matrix (ECM) Degradation:
Mesenchymal Migration:
In the early stages of metastasis, tumor cells often use mesenchymal modes of migration, characterized by the ability to degrade the ECM to create a path for movement.
Matrix Metalloproteinases (MMPs):
MMP2 (matrix metalloproteinase 2) is an enzyme that degrades the ECM, facilitating cell migration.
uPAR (urokinase plasminogen activator receptor) binds to uPA, leading to the conversion of plasminogen into plasmin, which is a protease that also degrades the ECM, further enabling cell migration.
Role of Rho GTPases in Metastasis:
Rho Family GTPases:
The tight regulation of Rho family GTPases (Rho, Rac, and Cdc42) plays a critical role in coordinating actin remodeling during cell migration.
These GTPases drive the formation of protrusions (e.g., lamellipodia and filopodia) and regulate the contractile forces necessary for cell movement through the matrix.
Metastasis:
Downstream signaling of Rho GTPases is involved in several stages of metastasis, including cell adhesion, motility, and extravasation. The regulation of actin polymerization by these GTPases helps the cancer cell move through tissues, enter the bloodstream (intravasate), and ultimately extravasate to form a secondary tumor.
Cadherin Upregulation in Metastatic Cells:
During metastasis, cancer cells often upregulate cadherins on their surface to facilitate the formation of secondary tumors. This upregulation helps in adhesion to the extracellular matrix and other cells, aiding in tumor formation at the secondary site.
