8. GTPases + Migration - post

complete

What is cell migration?

Cells move from one location to another, often in response to chemical signals or environmental cues.

What role do neutrophils play in cell migration?

Neutrophils, a type of white blood cell, migrate toward sites of infection or injury to chase down and engulf bacteria or other pathogens.

A neutrophil detects chemical signals released by bacteria (chemotaxis) and moves directionally toward the higher concentration of these signals to reach and engulf the bacterium.

What are some examples of GTPases? (4)

The Rho family of GTPases, including Cdc42 and Rho.

Rabs GTPases - vesicle trafficking.

Arf GTPases - vesicle formation

Ran GTPases - nuclear import/export.

What are small GTPases, and what is their approximate size?

Signalling proteins that are typically around 21 kDa in size.

Which superfamily do small GTPases belong to, and what is their role?

Ras superfamily and play crucial roles in various cellular signalling pathways.

How do small GTPases function as molecular switches?

Change their conformation upon activation, enabling them to bind and activate downstream effectors that regulate specific cellular functions.

What is the primary function of Rab GTPases?

Endosomal trafficking, directing vesicles to their appropriate locations within the cell.

What cellular process is Arf GTPase primarily associated with?

Membrane budding, crucial for vesicle formation and trafficking.

What is the role of Ras GTPase in the cell, and why is it significant in cancer biology?

Regulates cell proliferation and can act as an oncogene, meaning mutations in Ras can lead to uncontrolled cell growth and cancer.

How does Rho GTPase contribute to cell migration?

Regulates the cytoskeleton, specifically controlling cell shape and migration by influencing actin filament organization.

What does GTPase signalling depend on?

The type of nucleotide bound to the GTPase, with GTP-bound GTPases being active and GDP-bound GTPases being inactive.

Why is nucleotide cycling essential for GTPase function?

Cycling between the GTP-bound (active) and GDP-bound (inactive) states allows GTPases to regulate signalling pathways accurately and enables controlled activation and inactivation.

What does it mean when Arf6 is "active"?

"Active" Arf6, in its GTP-bound form, can be inhibitory, showing that not all active GTPases promote downstream signalling.

Why is the distinction between "signalling active" and "hydrolysis active" important for GTPases?

"Signalling active" refers to GTPases that activate downstream pathways, while "hydrolysis active" refers to GTPases that efficiently convert GTP to GDP.

These functions may differ, as some GTPases need fast cycling, while others require prolonged activation.

GTPases can be signalling-active without immediately hydrolysing GTP, allowing sustained pathway activation, while fast hydrolysis allows rapid switching off, giving each GTPase flexibility in controlling cellular processes.

How does Arf6 CA (constitutively active) affect signalling?

(GTP-bound permanently) can act as a dominant negative (DN), interfering with normal cycling and potentially inhibiting proper signalling.

What is the significance of different GTP hydrolysis speeds among GTPases, such as Rac?

Allow GTPases to control their activity duration.

For example, Rac has a fast hydrolysis rate, enabling quick cycling and rapid response in processes like cell migration.

What is the role of a Guanine Nucleotide Exchange Factor (GEF) in cyclic GTPase regulation?

Promotes the exchange of GDP for GTP, activating the GTPase by accelerating this process up to 10^7 fold.

What is the role of GTPase-activating proteins (GAPs) in the GTPase cycle?

Accelerate the hydrolysis of GTP to GDP, inactivating the GTPase by releasing Pi (inorganic phosphate) and speeding up hydrolysis by 2000–10^5 fold.

Describe the role of Guanine Nucleotide Dissociation Inhibitors (GDIs) in the GTPase cycle.

Stabilize the GDP-bound (inactive) state of the GTPase, preventing spontaneous activation and sequestering the GTPase in its inactive form.

How does the cycle of activation and inactivation proceed in GTPases like Rac1?

Rac1 is activated by GEF, which exchanges GDP for GTP.

Rac1-GTP can then signal downstream (e.g., to PAK or Wave2).

GAP then inactivates Rac1 by hydrolysing GTP to GDP, and GDI can further stabilize Rac1 in its GDP-bound form.

In the cyclic regulation of Rac1, what downstream effectors are activated when Rac1 is in its GTP-bound form?

Can activate downstream effectors such as PAK (p21-activated kinase) and Wave2, which are involved in signalling pathways that regulate the cytoskeleton and cell migration.

What is the relationship between GEFs and GAPs in the regulation of GTPase activity?

GEFs activate GTPases by facilitating the exchange of GDP for GTP, while GAPs inactivate GTPases by promoting the hydrolysis of GTP to GDP.

What is the role of the P-loop in GTPases?

Coordinates the phosphate groups of GTP, stabilising the nucleotide binding and helping facilitate GTPase activity.

Why is Mg²⁺ essential for GTPase function?

Crucial for nucleotide binding, as it stabilizes the interaction between GTP and the GTPase, allowing proper positioning for catalytic activity.

What is the function of Switch 1 (residues 25–39) in GTPases?

Changes conformation upon GTP binding and is involved in binding downstream effector proteins, enabling signalling.

Describe the role of Switch 2 (residues 57–75) in GTPases.

Undergoes conformational changes that help regulate effector binding and contain the catalytic residue, glutamine-61, which is essential for GTP hydrolysis.

How do Switch regions contribute to GTPase signalling?

The conformational changes in Switch 1 and Switch 2 upon GTP binding create specific binding surfaces for effectors - activate downstream effectors, facilitating signal transduction.

Why are the nucleotide and effector-binding sites important for GTPase activity?

These sites control the binding of GTP and effector proteins, ensuring that GTPases can cycle between active and inactive states, which is essential for precise cellular signalling.

Why is phosphorylation not typically used to measure GTPase activity directly?

GTPase activity involves GTP binding and hydrolysis rather than phosphorylation. GTPases do not generally get phosphorylated as part of their activation cycle; instead, they switch between active (GTP-bound) and inactive (GDP-bound) states.

How can antibodies be used to measure GTPase activity?

Specific antibodies can be used to detect the conformational changes in Switch 1 and Switch 2 that occur when GTPases bind GTP.

These antibodies are often conformation-sensitive and can distinguish between the active (GTP-bound) and inactive (GDP-bound) forms of the GTPase.

What role does effector binding play in measuring GTPase activity?

Effector binding assays can measure GTPase activity by assessing the ability of the GTPase to bind downstream effectors.

Since only the GTP-bound (active) form of the GTPase binds effectors, binding indicates that the GTPase is in its active state.

What is the role of the catalytic glutamine in GTP hydrolysis?

Helps in the positioning of the attacking water molecule, facilitating the hydrolysis of GTP.

Name the 2 active mutants affecting GTPase activity and their effects.

Q61L mutation - catalytic mutant, disrupts the positioning of the attacking water molecule, impairing GTP hydrolysis.

G12V mutation - pushes the Q61 out of position and disturbs the P-loop, which disrupts GTP hydrolysis.

What is the role of the P-loop in GTPase catalysis?

The P-loop (12GxxGKT17) forms hydrogen bonds and interacts with lysine residues, which counteracts the negative charge at the phosphates, helping in GTP hydrolysis.

What is the role of GTPase Activating Proteins (GAPs) in GTPase signalling?

Accelerate GTP hydrolysis, helping to turn off GTPase signalling by converting GTP to GDP, thereby inactivating the GTPase.

In the context of Rac1, what specific residue does GAP target to stabilize for hydrolysis?

Gln 61 and Thr 35

What are the roles of each of the following in the GAP hydrolysis mechanism?

• Arg 85

• Mg²⁺

• Positioning of water molecules

• Arg 85 aligns with the phosphate group, helping to stabilize the transition state for hydrolysis.

• Mg²⁺ is positioned to coordinate with the phosphate groups, helping to stabilize the molecule and assist in catalysis.

• Water molecules are positioned optimally by GAP to enable a nucleophilic attack on the phosphate group, aiding in the breakdown of GTP.

How does GAP reduce the entropy barrier in GTP hydrolysis?

Restricts the freedom of movement of GTP, reducing the entropy barrier and making the hydrolysis reaction more efficient.

What is the significance of stabilizing Thr 17 and Thr 35 in Rac1 during GAP-mediated hydrolysis?

Aids in properly aligning the molecule for hydrolysis, ensuring effective inactivation of Rac1.

What is the role of Guanine Nucleotide Exchange Factors (GEFs) in GTPase regulation?

Accelerate the exchange of GDP for GTP, activating the GTPase.

How do GEFs stabilize the GTPase during nucleotide exchange?

Stabilise the nucleotide-free, Mg²⁺-free GTPase, promoting the exchange of GDP for GTP.

What is the effect of the T17N mutation on GTPase activity?

Dominant negative mutation that prevents the binding of Mg²⁺, effectively "mopping up" GEFs and blocking the activation of the GTPase.

What is the function of the Dbl-homology domain in GEFs?

Involved in the activation of GTPases by facilitating the exchange of GDP for GTP.

What is the role of the DOCK-family in GEFs?

Helps activate GTPases, though their specific mechanisms and structures differ from other GEFs.

How does the Sec7 domain contribute to the function of GEFs?

Involved in the activation of small GTPases, specifically in the exchange of GDP for GTP.

How many members are in the Dbl family of GEFs?

Over 70

What is the significance of Tiam1 in GEF specificity?

It’s a GEF.

Has 9 residues from the body and Switch 2 that form a contiguous (common border) contact, contributing to its specificity in activating certain GTPases.

How does the W56F mutation in Rac affect its response to GEFs?

Makes it insensitive to Tiam1 but sensitive to ITSN (Intersectin), showing how GEF specificity can differ for the same GTPase.

What is actin-based motility?

A complex process that involves the assembly of actin filaments to generate force and enable cells to move.

Assemble into different structures to promote protrusion and retraction.

What role does RhoA play in actin-based motility?

Regulates actin contraction and the formation of stress fibres, which are essential for cell motility.

What are stress fibres and which GTPase is involved in their formation?

Bundles of actin filaments that provide structural support for cells, and their formation is regulated by RhoA.

What is the role of Cdc42 in actin-based motility?

Regulates the formation of filopodia, which are thin, protrusive structures involved in cell movement and sensing the environment.

What is the role of Rac1 in actin-based motility?

Regulates the formation of lamellipodia, which are broad, sheet-like extensions that push the cell membrane forward during migration.

What is the role of adhesion in actin-based motility?

Adhesion molecules help anchor the cell to the substrate, allowing the cell to move forward by providing stable points for the lamellipodia and filopodia to push against.

What is the signalling pathway downstream of GTP-RhoA?

GTP-RhoA activates Rho kinase, which then phosphorylates Myosin Light Chain, leading to actomyosin contraction, which helps generate force for processes like cell migration.

What is the role of each of the following in the downstream signalling of GTPases (GTP-RhoA)?

• Rho kinase

• Myosin Light Chain

• Rho kinase phosphorylates Myosin Light Chain, which activates the actomyosin contraction mechanism.

• The phosphorylation of Myosin Light Chain by Rho kinase activates myosin motor proteins, leading to actomyosin contraction.

What type of signals are Cdc42 and Rac in actin-based motility?

Protrusive signals that promote the extension of cellular structures like filopodia and lamellipodia.

What type of signal is RhoA in actin-based motility?

Contractile signal that promotes actomyosin contraction and the formation of stress fibres.

How do Cdc42/Rac and RhoA function in relation to each other?

Cdc42 and Rac promote protrusion (extension of cell structures), while RhoA promotes contraction, making these signals antagonistic in regulating cell movement.

How does cell migration differ in a 2D environment?

Cell migration is random, flat, and fast.

How does cell migration differ in a 3D environment?

Cell migration is directional, integrated, and often slower compared to 2D migration.

What is the role of the cell-derived matrix in the in vivo environment?

Provides structural support and facilitates cellular migration.

Long matrix strands provide a scaffold or "train tracks" that guide and direct the movement of cells during processes like wound healing.

How does the tissue environment support cell migration in vivo?

Offers biochemical and physical cues, including matrix components, that help guide cells during migration, particularly in processes such as wound healing.

Does increasing Rac1 activity lead to faster migration?

Doesn’t necessarily lead to faster, similar or slower migration, not as simple as more active protein = faster migration.

The effect of increasing Rac1 activity on migration depends on the context: in cells with properly localized signals, increased Rac1 activity accelerates migration, but in cells with dysregulated Rac1 activity, it can impair directional movement and make migration slower or more erratic.

How does active Rac1 localization affect cell migration in wild-type (WT) cells?

Active Rac1 is localized at the front of the cell, enabling efficient, directed migration along fibrous matrices, allowing cells to move in a straight line and reach their destination quickly.

What happens to cell migration in SDC4 -/- (mutant) cells that have dysregulated Rac1 activity?

Active Rac1 is not localized and is spread across the entire cell, leading to random, wandering movement despite the presence of guidance cues (matrix "train tracks"). This results in a lack of directionality and inefficient migration.