Rho family GTPases (Rho, Rac, and Cdc42) are molecular switch proteins that play a central role in diverse biological processes such as actin cytoskeleton organization (affecting cell shape changes, cell polarity, cell movement, and cytokinesis), microtubule dynamics, changes in gene transcription, chemotaxis, axonal guidance, cell cycle progression, cell adhesion, oncogenic transformation, and wound repair. Rho GTPases are also the targets of different classes of pathogens in disease-causing bacterial/viral infections.
Activated Rho GTPases interact with effector proteins (cellular target proteins) to drive this large variety of biological responses. While a handful of Rho targets have been identified in different systems to date, the majority of molecular pathways in which each is involved remain to be elucidated. We are currently investigating the molecular mechanisms associated with Rho1 and three of its downstream effectors: the de novo linear actin nucleation factors Capu (a formin-homology protein) and Spire (a WH2 domain protein), which act downstream of Rho1 to regulate the onset of ooplasmic streaming during oogenesis, and Wash, a new subfamily of Wiskott-Aldrich Syndrome family proteins, that activates the Arp2/3 complex to nucleate branched actin filaments and functions to remodel actin structures and elicit changes in cell shape and movement.
Rho1 in Drosophila.
Loss of Rho1 function results in both maternal and zygotic defects that are NOT identical to those reported from ectopic expression studies using a dominant-negative form of Rho1. Null clones in the germline cannot be recovered, indicating that Rho1 is required for cell viability or proliferation. Reduction of maternal Rho1 activity (using wimp) results in two phenotypes: the ovarian actin cytoskeleton is disrupted, particularly in the outer ring canals and oocyte cortex, and embryos resulting from these females display segmentation defects. Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure.
Rho1 and the coordination of microtubule/microfilament dynamics.
Rho GTPases are important regulators of the actin cytoskeleton and have been shown to affect microtubule stability. We find that Drosophila Rho1, through its downstream effectors Wash, Capu, and Spire, is required for maintenance of proper microfilament and microtubule architecture to regulate the onset of ooplasmic streaming during oogenesis. While this streaming event is microtubule-based, actin assembly is required for its timing. In addition to their actin nucleation activity, we find that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC, and SpireD). SpireD was shown to nucleate actin, but the activity of the other isoforms had not been addressed. We find that SpireD does not have crosslinking activity, while SpireC is a potent crosslinker. We have found that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition.
Our results suggest that Rho1 regulates the timing of ooplasmic streaming by regulating the microtubule/microfilament crosslinking that occurs at the oocyte cortex: crosslinking antagonizes the formation of the dynamic subcortical microtubule arrays that are required for ooplasmic streaming. Rho1, Capu and Spire appear to be elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments. We have proposed that activated Rho1 transduces a signal during stages 8-10a that promotes the crosslinking activity of Capu and SpireC by preventing binding of SpireD to both Capu and SpireC. Rho1 then becomes inactivated at stage10b, presumably by a signaling event, allowing SpireD to bind to Capu and SpireC, thereby inhibiting MT/microfilament crosslinking.
Thus, our work establishes Rho1 as a direct regulator of a broader group of actin nucleating proteins, and is the first evidence for how the activity of Wash, as well as the linear actin nucleators Spire and Capu, is regulated to coordinate ooplasmic streaming in vivo. As little is yet known about coordination of the actin and MT cytoskeletons, we are using this as a model system to reveal general mechanisms underlying MT/microfilament cross-talk and as a readout for key cell biological steps in cell motility, polarity, morphology, and division.
Rho1, Wash, and immune cell developmental migrations.
When not responding to immune crises, Drosophila immune cells (hemocytes) undergo a stereotyped developmental program in the embryo wherein hemocytes initially found in the head region are distributed throughout the embryo in a series of invasive and non-invasive migrations.
We find that Rho1 is required for the third hemocyte developmental migration in which hemocytes in the posterior move anteriorly along the ventral midline. This migration requires the interaction of Rho1 with its downstream effector the Wiskott Aldrich Syndrome family protein Wash.
Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same migratory defects as Rho1 knockdown. This phenotype is further recapitulated in hemocytes with impaired Arp2/3 function, suggesting that Wash’s actin nucleation activity is required. Wash activity is, however, independent of the multi-subunit WASH regulatory complex as knockdown of the Drosophila ortholog for Strumpellin, SWIP, or CCDC53 results in no migratory defect.
Our results suggest a WASH complex-independent signaling pathway in which Rho1 interacts with the Arp2/3 activator Wash to regulate the cytoskeleton dynamics in hemocytes.
Rho1 and catenins.
Rho GTPases are crucial regulators of cadherin-mediated adhesion where they have been proposed to play a role in assembly or disassembly of adherens junctions. Rho activity is thought to be required early in the process for cadherin clustering at sites of cell-cell contact. We find that Rho1 interacts physically with alpha-catenin and p120ctn. For the catenin proteins these interactions map to distinct surface-exposed regions of the Rho protein not previously assigned functions. Cadherin and catenin localization is disrupted in Rho1 mutants, implicating Rho1 in their regulation. In addition, we find that Rho1 accumulates in response to lowered p120ctn activity. We find that p120ctn interacts genetically with Rho1 and that loss of p120ctn enhances the Rho1 phenotype. Our results suggest that, consistent with work done in mammalian systems, p120ctn is an important, if not essential, component of adherens junction integrity, and that p120ctn/Rho complexes are present in a tightly controlled equilibrium between the cytoplasm and the membrane.