L15: better phrasing for an essay Flashcards
what is cell migration?
Cell migration is the directed movement of cells from one location to another, often through tissues or along surfaces. Many cells use crawling to migrate, driven by changes in the cytoskeleton and interactions with the extracellular environment. During embryogenesis, gastrulation involves coordinated cell movements over the surface of the blastocyst to form the three germ layers. Neural crest cells migrate long distances from the neural tube to various regions in the developing embryo, where they contribute to multiple tissues. Macrophages and neutrophils exit blood vessels and migrate into surrounding tissues in response to chemokines released at sites of infection or injury. Growth cones at the tips of neuronal axons extend and migrate through the organism to establish proper neural connections. Fibroblasts move through connective tissue and remodel the extracellular matrix as part of the wound healing process. In cancer, metastatic cells leave the primary tumour site, invade surrounding tissue, enter the bloodstream or lymphatic system, and colonise distant organs to form secondary tumours.
modes of migration
There are three main modes of cell migration: mesenchymal, collective, and amoeboid.
Mesenchymal migration involves cells moving through collagen-rich extracellular matrices using integrin-mediated adhesion. Cells attach to the ECM via integrins, secrete matrix-degrading proteases such as matrix metalloproteinases (MMPs), and then advance through the tissue. This mode is commonly used by fibroblasts during wound healing, where they remodel the ECM as they migrate. Fibroblast migration is guided by chemotaxis, where fibroblasts detect and move toward a source of chemoattractant. In response to the chemical gradient, the fibroblast polarises, orienting itself toward the source. At the front of the cell, it extends a lamellipodium, a thin, sheet-like protrusion driven by actin polymerisation. This lamellipodium advances in the direction of the chemoattractant. At the rear of the cell, focal adhesions that anchor it to the extracellular matrix are disassembled. This release, combined with actomyosin contraction, generates a propulsive force that moves the entire cell forward. The process is tightly coordinated, involving cytoskeletal rearrangements and adhesion turnover to ensure efficient directed migration.
Collective migration occurs when groups of cells move together while maintaining stable cell-cell junctions. The cells at the leading edge form integrin-based adhesions with the ECM and guide the direction of movement. The trailing cells stay connected through cadherins or tight junctions, ensuring coordinated migration. This is seen during gastrulation, where embryonic cells move over the blastocyst surface, and in neural crest migration, where cells travel from the neural tube to various parts of the developing embryo.
Amoeboid migration is characterised by cells squeezing through gaps in the ECM without degrading it or forming strong adhesions. This mode does not rely on integrins. Instead, movement is driven by cytoskeletal contractions and dynamic shape changes. Neutrophils use amoeboid migration to rapidly respond to infection. They detect bacterial signals such as formyl-methionine-leucine-phenylalanine (fMLP), follow the chemotactic gradient, and migrate toward the source, where they engulf bacteria via phagocytosis.
2D VS 3D migration
Although most in vitro studies use 2D systems, cell migration in vivo typically occurs in 3D environments. In 2D migration, such as on collagen-coated glass, fibroblasts form a triangular, flattened shape with broad lamellipodia containing criss-crossed F-actin. Cells move along the surface, and focal adhesions are largely restricted to the x–y plane where the cell contacts the substrate. The matrix is stiff, and the underlying glass surface offers no deformability or mechanical feedback. There is no natural soluble gradient unless introduced artificially, such as via a microneedle. The result is a “fried egg” morphology where spreading is unconstrained.
In contrast, 3D migration occurs within a collagen matrix, where the environment is softer and more complex. Cells adopt a more rounded shape, often with pseudopods or finger-like projections extending into the matrix to probe directionality. These projections interact with discrete collagen fibrils, which can be pulled on to generate traction. Soluble gradients of ECM breakdown products and signalling molecules are often present, guiding migration. Focal adhesions form in all directions, not just in the x–y plane, allowing cells to sense and respond to mechanical cues from multiple angles. However, spreading and movement are physically constrained by the surrounding collagen fibres, making migration more dynamic and adaptive than in 2D systems.
The role of actin
Cell migration relies on the spatial and temporal regulation of both actin polymerisation and actomyosin-based contraction. Different actin structures are formed in distinct regions of the cell, depending on specific regulatory signals. These signals control when and where actin filaments are assembled or disassembled, coordinating cell polarity, protrusion, and retraction. The dynamic regulation of actin allows the formation of lamellipodia or filopodia at the leading edge, while actomyosin contraction at the rear helps retract the trailing edge, together driving forward movement.
Migration cycle?
Cell migration is a cyclic and tightly regulated process driven by actin dynamics and actomyosin contraction. At the leading edge, actin polymerisation pushes the plasma membrane forward to form protrusions such as lamellipodia or filopodia. This is facilitated by the addition of actin monomers at the barbed ends of filaments near the membrane. The membrane itself is thought to vibrate slightly, creating transient gaps that allow monomers to insert and extend the filament, preventing membrane retraction.
At the rear of the cell, actomyosin contraction generates a hydrodynamic pressure that contributes to overall cell propulsion. This pressure not only helps push the cell body forward but also assists in transporting G-actin monomers, produced by depolymerisation at the rear, toward the front to fuel further polymerisation. This movement is driven, not passive, and relies on cytosolic flow rather than diffusion.
Simultaneously, new focal adhesions are formed at the front of the cell to anchor protrusions to the substrate. As the cell advances, old adhesions at the rear are actively disassembled, reducing resistance and enabling retraction. These steps—protrusion, adhesion, traction, and retraction—are spatially and temporally coordinated, ensuring efficient forward movement.
Stress fibres, composed of actin and myosin, span the cell and link front and back. Their contraction adds additional force to propel the cell. Friction between focal adhesions and the substrate provides resistance to balance this force, maintaining controlled, directed migration.
actin turnover regulation
Actin turnover is tightly regulated by various actin-binding proteins that control filament nucleation, growth, stabilisation, and disassembly.
Together, these proteins coordinate actin dynamics to allow controlled filament growth, turnover, and structural organisation during processes like cell migration.
Nucleation and Formins: Both the ARP2/3 complex and formin proteins play critical roles in actin filament nucleation. The ARP2/3 complex promotes the formation of branched filaments by initiating new filaments from pre-existing ones, while formin proteins drive the elongation of linear filaments by adding actin monomers at the barbed (+) end.
Polymerization at the Barbed End: At the barbed (+) end, formin and profilin work together to drive actin polymerization. Profilin binds to G-actin (globular actin) in its ATP-bound state, facilitating its addition to the growing filament. This ATP-G-actin is incorporated at the barbed end, supporting the extension of the filament.
Instability of Actin Filaments: Actin filaments are inherently unstable and need stabilization to maintain structure. Capping proteins bind to the barbed (+) end, preventing further polymerization or depolymerization. Additionally, tropomyosin stabilizes the filament by binding along its sides, protecting it from severing by proteins like gelsolin and inhibiting depolymerization by cofilin.
Tropomodulin and Tropomyosin: Tropomodulin binds to the pointed (-) end of actin filaments, preventing disassembly by inhibiting depolymerization. Tropomyosin, which stabilizes the filament by binding along its length, is necessary to prevent severing by gelsolin and depolymerization by cofilin. For gelsolin to sever filaments or cofilin to depolymerize them, tropomyosin must be displaced.
Profilin and Thymosin: Thymosin-β4 sequesters G-actin in its ADP-bound state, preventing it from polymerizing. In contrast, profilin promotes polymerization by exchanging ADP for ATP on G-actin, converting it into the ATP-bound form, which is primed for incorporation into the growing filament at the barbed end. This exchange mechanism is vital for maintaining a ready supply of ATP-actin for filament growth.
Gelsolin Severing Actin Filaments: Gelsolin severs actin filaments by creating two new barbed (+) ends at the sites of severing. This increases the number of polymerization sites, which speeds up actin turnover. By promoting the formation of new barbed (+) ends, gelsolin enhances the recycling of G-actin and accelerates both polymerization and depolymerization, facilitating rapid cytoskeletal remodeling.
G-Actin Pool and Polymerization: The severing action of gelsolin increases the number of barbed (+) ends available for polymerization and pointed (-) ends for depolymerization, thus accelerating overall actin turnover. Continuous recycling of G-actin ensures a dynamic actin cytoskeleton that can rapidly reorganize in response to cellular needs.
Arp2/3
Actin-Related Protein 2/3 (ARP2/3) Complex:
The ARP2/3 complex consists of seven subunits, including ARP2 and ARP3, which are essential for actin nucleation. The complex is typically inactive until it is activated by a factor that induces a conformational change in ARP2 and ARP3. This change enables the complex to bind to G-actin monomers.
When activated, the ARP2/3 complex binds to G-actin through its pointed (-) end, where it caps the end of the actin filament, preventing its disassembly. Filament growth then proceeds at the barbed (+) end of the newly formed actin filament. The ARP2/3 complex remains attached at the pointed (-) end, and the new filament grows at an angle of approximately 70 degrees relative to the original actin filament.
This mechanism results in the formation of a dendritic network, which is crucial for processes like cell motility and actin cytoskeleton remodeling, as it facilitates the branching of actin filaments.
Actin polymerization is a key mechanism driving cell movement, occurring primarily at the leading edge of the cell. Here, the ARP2/3 complex nucleates new actin filaments, promoting their growth at the barbed (+) end. This polymerization at the leading edge generates a propulsive force, pushing the plasma membrane (PM) forward, enabling cell movement.
Cofilin plays a crucial role in the disassembly of actin filaments, but it acts further from the leading edge, where it binds to actin filaments and accelerates their depolymerization. This process helps to clear space for the continuous polymerization of new filaments at the front of the cell.
Thymosin β4 sequesters G-actin (globular actin), preventing its premature polymerization. This sequestration pushes G-actin toward the front of the cell, where it is bound by Profilin. Profilin exchanges ADP for ATP on the G-actin, preparing it for polymerization.
Profilin then facilitates the addition of ATP-bound G-actin to the growing barbed end of actin filaments, contributing to the formation of new filaments at the leading edge. As the filaments elongate, the ARP2/3 complex dissociates from the growing filament and is recycled to the front of the cell, where it continues to nucleate new actin filaments, sustaining the directional movement of the cell.
pathogen migration?
Listeria monocytogenes, a bacterial pathogen, exploits the host cell’s actin polymerization machinery to propel itself from one cell to another. The bacteria form actin “comets” behind them, where actin filaments polymerize at the rear of the bacterium and depolymerize at the front. This dynamic actin turnover generates a propulsive force, enabling the bacteria to move within the host cell.
The bacteria express ActA, a protein that mimics host cell proteins, particularly those involved in focal adhesions, such as vinculin. ActA contains proline-rich motifs that bind to vasodilator-stimulated phosphoprotein (VASP), a key protein involved in actin polymerization.
VASP recruits profilin-actin complexes to the rear of the bacterium. Profilin binds to G-actin, exchanging ADP for ATP, and promotes the polymerization of G-actin into actin filaments. As these filaments polymerize at the rear and depolymerize at the front, the actin polymerization cycle propels the bacterium forward, facilitating its movement through and between host cells.
forces driving cell migration
Cell migration is driven by de novo actin polymerization and hydrodynamic forces at the leading edge. Actin filaments at the front of the cell are indirectly connected to cell surface receptors, which interact with the extracellular matrix (ECM) and substratum, helping to anchor the cell.
The forces driving the cell forward are:
Traction Forces: These forces are generated from the friction between the cell and the ECM, particularly through adhesion to the substratum.
Contractile Forces: Produced by actin-myosin contraction within the cell, these forces help to generate movement and shape the cell.
Together, these forces push the actin filaments against the cell’s leading edge, driving the cell forward. The synergy between traction and contractile forces ensures a dynamic, coordinated movement, allowing the cell to maintain forward progression while adhering to its environment. This integrated movement is critical for effective cell migration. The downstream effects of these GTPases are complex and involve a wide range of cellular processes, including:
rho family gtpases
Major Regulatory Proteins and Their Effects on Actin Turnover
The Rho family GTPases—Rho, Rac, and Cdc42—are important regulators of actin dynamics. These proteins control different aspects of the actin cytoskeleton, depending on the cell’s needs. Each one has specific functions that contribute to cell movement and shape.
Rho GTPase:
Key Role: Regulates the formation of stress fibers, which are contractile actin bundles. These are important for cell contraction and maintaining the cell shape.
Location: Found in the cell cortex (the outer part of the cell), where they form bundles of actin filaments.
Function: Stress fibers help to generate force for cell contraction and play a role in adhesion to the extracellular matrix (ECM).
Rac GTPase:
Key Role: Regulates the formation of lamellipodia, which are sheet-like projections made up of a gel-like network of actin filaments.
Function: Lamellipodia are critical for cell spreading and migration, as they help extend the cell’s leading edge forward.
Transition: Rac controls the transition from stress fibers to dendritic networks in the lamellipodia, helping cells to form these branched structures that drive movement.
Cdc42 GTPase:
Key Role: Promotes the formation of filopodia, which are finger-like projections of the cell.
Function: Filopodia help the cell sense its environment by detecting chemical signals or other cues at the front of the cell.
Structure: Cdc42 promotes the formation of tight, parallel bundles of actin filaments in the filopodia, which help the cell navigate and migrate.
Synergistic Action of Rho, Rac, and Cdc42:
Rho: Forms stress fibers (contractile actin bundles) in the cortex, important for cell shape and contraction.
Rac: Promotes dendritic networks (in lamellipodia) that help push the cell forward for migration.
Cdc42: Drives filopodia formation to guide the cell’s direction by sensing environmental cues.
Together, these three GTPases work in a coordinated way to control the dynamic actin cytoskeleton, which is essential for processes like cell movement, shape maintenance, and response to external signals.
Inactive State (Rho GTPase Bound to GDP):
Rho GTPases are in their inactive state when bound to GDP (guanosine diphosphate) in the cytoplasm.
In this state, Rho GTPases are sequestered by Rho-GDI (Guanine nucleotide Dissociation Inhibitor), which prevents the exchange of GDP for GTP and keeps the Rho GTPase inactive.
Active State (GDP to GTP Exchange):
Rho-GDI helps deliver Rho-GDP to the plasma membrane (PM), where the Rho GTPase interacts with guanine nucleotide exchange factors (GEFs).
GEFs promote the exchange of GDP for GTP, activating the Rho GTPase.
Once activated, Rho GTPase binds to effector proteins, influencing a variety of cellular processes such as:
Cytoskeletal dynamics (affecting actin polymerization),
Gene expression,
Cell adhesion.
Each Rho GTPase has a specific function:
Rho: Drives the formation of stress fibers (contractile actin bundles),
Rac: Promotes the formation of lamellipodia (broad, sheet-like projections),
Cdc42: Promotes the formation of filopodia (finger-like projections).
Inactivation of Rho GTPase:
Rho GTPase is inactivated by GTPase-activating proteins (GAPs), which accelerate the hydrolysis of GTP to GDP, turning the GTPase back into its inactive state.
Once inactivated, Rho GTPase is again sequestered by Rho-GDI to remain inactive.
Intrinsic GTP Hydrolysis:
Rho GTPases possess intrinsic GTPase activity, meaning they can hydrolyze GTP to GDP on their own, but the process is slow.
The activity of GAPs accelerates this hydrolysis, speeding up the transition from the active (GTP-bound) state to the inactive (GDP-bound) state.
This regulatory cycle allows Rho GTPases to control various cellular processes like actin turnover, cell migration, and cell shape maintenance.
rho signalling
The downstream effects of Rho, Rac, and Cdc42 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, where 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), which is crucial for proper intracellular organization, especially during cell division and secretion. The activity of Rho family GTPases is regulated by various upstream signals, including guanine nucleotide exchange factors (GEFs) that activate Rho GTPases by promoting the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) that inactivate Rho GTPases by accelerating the hydrolysis of GTP to GDP. Rho GTPase activation can be influenced by several factors such as integrin adhesion to the extracellular matrix (ECM), which is critical for activating Rho family GTPases, tyrosine kinase receptors involved in cell signaling, and G protein-coupled receptors (GPCRs), which also regulate Rho GTPase signaling through GEFs. Cadherins, involved in cell-cell adhesion, are also important in regulating Rho GTPase activity, along with adhesion via Ig superfamily receptors. Additionally, mechanical stress, such as physical forces on the cell during migration or tension, can also regulate Rho GTPase activity.
rho fam 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.
