Lecture 7: Migration

To gain an understanding of the mechanisms of cytoskeletal regulation underpinning migration guidance.

Learning Objectives

●     Discussion of the molecular basis of migration regulation

●     Introduction to Rho family small GTPases

●     Understand the molecular basis of GTPase regulation

●      Understand how GTPase activity directs cytoskeletal reorganisation

●      Consider how migration guidance is achieved

●      Recognise the in vivo consequences of regulated migration

Overview of Small GTPases and the Rho Family

• Rho GTPases are small proteins, typically around $21\,kDa$, that serve as critical molecular switches in cell signaling.

• They belong to the Ras superfamily, which represents one of the largest groups of signaling proteins in biology.

• The functional state of these proteins is determined by the nucleotide bound to them; they undergo a conformational change upon activation that allows them to bind and activate various downstream effectors.

• Specific members of the small GTPase families and their roles include:

  • Ras: Primarily involved in cell proliferation and acts as a major oncogene when mutated.

  • Ran: Facilitates nuclear import and the organization of the mitotic spindle during cell division.

  • Arf: Involved in membrane budding and vesicular transport processes.

  • Rab: Regulates endosomal trafficking and membrane fusion.

  • Rho: Controls the cytoskeleton and is essential for cell migration.

Physiological Roles and Subject Mapping for Small GTPases

• GTPases permeate nearly every aspect of cellular life, mapped across various biological topics and specific lecturers:

  • Cell Communication (Zeidler) and Channels/Receptors (Gravells): Relate to G-protein coupled receptors ($GPCRs$).

  • Migration (Bass) and Mechanobiology (King): Driven by the Rho family of GTPases.

  • Cell Polarity (Smythe): Influenced by $Cdc42$.

  • Membrane Fusion (Peden) and Vesicular Trafficking (Smythe): Controlled by Rabs and Arfs.

  • DNA and Cell Division (Gravells): Controlled by the Ran GTPase.

  • Autophagy (King): Mediated by Rab GTPases.

  • Metabolism (Gokhale) and Immunology (Zeidler): Further specific biological functions involving GTPase signaling.

Biochemical Cycling: Signalling and Hydrolysis States

• The activity of a GTPase depends on whether it is bound to Guanosine Triphosphate ($GTP$) or Guanosine Diphosphate ($GDP$).

• Cycling between these two states is essential for proper signaling. The "active" state is typically the $GTP$-bound state, which connects to downstream effectors, while the $GDP$-bound state is inactive.

• It is critical to distinguish between "signaling active" (ability to bind effectors) and "hydrolysis active" (the enzymatic process of breaking down $GTP$).

• In some specific cases, such as $Arf6$, the "active" bound state can actually be inhibitory.

Structural Biology and Nucleotide Binding of GTPases

• The structure of a GTPase includes several key functional domains identified via crystal structure analysis:

  • P-Loop (Phosphate-binding loop): Located near residues $12GxxGKT17$, it coordinates the phosphate groups and carries a large amount of charge.

  • Switch 1 (Residues $25-39$) and Switch 2 (Residues $57-75$): These are the primary regions that change conformation to bind downstream effectors upon $GTP$ loading.

  • Magnesium Ion ($Mg^{2+}$): This ion is essential for nucleotide binding and is coordinated by the P-loop and the hydroxyl group of $Thr17$.

  • Catalytic Glutamine-61 ($Q61$): This residue is central to the hydrolysis reaction.

• Measuring Activity: The best way to detect GTPase action is through effector-binding assays rather than looking for phosphorylation or using simple antibodies.

Molecular Mechanisms of GTP Hydrolysis and Catalytic Mutations

• The catalysis of $GTP$ hydrolysis involves two primary steps:

  1. Positioning of the attacking water molecule: This is mediated by the catalytic residue $Q61$.

  2. Counteracting the negative charge of the phosphates: This is achieved via the P-loop ($12GxxGKT17$), which utilizes hydrogen bonds and a lysine residue to stabilize the transition state.

• Specific mutations can lock the GTPase in an active state:

  • $Q61L$: A catalytic mutant that prevents hydrolysis.

  • $G12V$: This mutation pushes the $Q61$ residue out of its optimal position and disturbs the P-loop, thereby inhibiting hydrolysis and keeping the protein signaling-active.

Regulatory Proteins: GAPs, GEFs, and GDIs

• GTPase Activating Proteins ($GAPs$): These proteins turn off signaling by accelerating the intrinsic rate of $GTP$ hydrolysis by $2000$ to $10^{5}$ fold.

  • $GAPs$ work by stabilizing the transition state and restricting the freedom of relevant molecules, which reduces the entropy barrier and positions water optimally for nucleophilic attack.

  • Example: The protein $p50\,RacGAP$ provides an "Arginine finger" ($Arg85$) to neutralize negative charges during the hydrolysis of $GTP$ in $Rac1$.

• Guanine Nucleotide Exchange Factors ($GEFs$): These proteins activate signaling by accelerating the exchange of $GDP$ for $GTP$ by $10$ to $10^{7}$ fold.

  • Families include the Dbl-homology domain family ($DH$), DOCK-family, and Sec7 domain proteins.

  • $GEFs$ work by stabilizing the nucleotide-free and $Mg^{2+}$-free state of the GTPase to allow a new $GTP$ molecule to enter.

  • $T17N$ is a dominant-negative mutant that prevents efficient exchange.

• Guanine Nucleotide Dissociation Inhibitors ($GDIs$): These act to prevent the release of $GDP$, thereby keeping the GTPase in an inactive, sequestered state.

GEF Specificity and Structural Interactions

• There are over 70 members of the Dbl family of $GEFs$, demonstrating high specificity for their target GTPases.

• $Tiam1$ specifically recognizes its targets using nine residues from the GTPase body and Switch 2 that form a contiguous contact surface.

• Mutation-based specificity: For example, the $Rac\,W56F$ mutant is insensitive to the $GEF\,Tiam1$ but remains sensitive to the $GEF\,ITSN$.

Rho GTPases in Cell Migration and Actin Dynamics

• Migration is a complex movement built around the coordinated action of small GTPases:

  • $Cdc42$: Located at the leading edge, it regulates Directional sensing and the formation of Filopodia (actin-rich spikes).

  • $Rac1$: Responsible for the formation of Lamellipodia (broad actin sheets) and focal adhesions to provide traction.

  • $RhoA$: Controls the formation of stress fibers and the retraction of the cell tail via actomyosin contraction.

• Example: A neutrophil chasing a bacterium (as observed by David Rogers, Vanderbilt) exemplifies the rapid cytoskeletal reorganization driven by these molecules.

Downstream Signaling and the Mechanics of Contraction

• Actively bound $RhoA$ triggers a signaling cascade to induce contraction:

  1. $GTP-RhoA$ activates its downstream effector, $Rho\,Kinase$.

  2. $Rho\,Kinase$ phosphorylates the Myosin Light Chain.

  3. This leads to the activation of actomyosin contraction, where myosin motors "walk" along actin filaments, pulling them together to generate force.

Coordination and Spatial Localization in Migration

• Antagonistic Signaling: $Cdc42$ and $Rac1$ provide protrusive signals, while $RhoA$ provides contractile signals. For maximum protrusion to be reached, $RhoA$ must be suppressed at the leading edge where $Rac$ activity is high.

• Migration Guidance and Environments:

  • 2D Migration: Occurs on flat surfaces; it is generally fast but can be random in direction.

  • 3D Migration: Occurs within complex tissues or cell-derived matrices; it is typically more integrated and directional, though often slower than 2D movement.

• Localized Signals: For a cell to move effectively, signals must be localized.

  • In wild-type cells, localized signals ensure a single direction of movement.

  • Mutant cells with globally elevated $Rac1$ activity (e.g., $SDC4-$) may form protrusions in multiple directions, leading to a loss of directionality and potentially slower overall migration depending on how much $Rho$ is present and where it is localized.