Cytoskeletal Regulation by Rho1 GTPase and its Effectors

Rho GT pase

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.

Model for Rho-mediated signaling
An upstream signal leads to the conversion of inactive GDP-bound Rho to active GTP-bound Rho. In the example shown here, an extracellular signal activates a heterotrimeric G-protein coupled receptor. Activated Rho is then proposed to interact with a number of downstream targets (Effectors) leading to a variety of biological responses.

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.

Egg chambers from female Drosophila with reduced Rho1 function
Egg chambers from female Drosophila with reduced Rho1 function show a general disruption of the actin cytoskeleton, particularly in the outer ring canals and oocyte cortex.

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.

Microtubule (MT) and microfilament bundling/crosslinking assays
Microtubule (MT) and microfilament bundling/crosslinking assays showing that Wash's bundling and crosslinking activities are disrupted by the addition of SpireD. This inhibition is relieved by the addition of activated Rho1.

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.

Model for the regulation of microtubule/microfilament crosslinking and ooplasmic streaming by Rho1, Wash, and SpireD
(left) Wildtype oocyte prior to the onset of ooplasmic streaming (stage 7). MTs are shown in orange and cortical microfilaments in green. Active (GTP-bound) Rho1 promotes MT-microfilament crosslinking by sequestering SpireD, thereby preventing it from binding to Wash, SpireC, and Capu. MT arrays are restricted to the oocyte cortex. (right) Wildtype oocyte during ooplasmic streaming (stage 10b). Upstream signaling events result in GTP hydrolysis by Rho1, allowing Spire D to bind to Wash, SpireC, and Capu. This blocks MT-microfilament crosslinking, resulting in ooplasmic streaming.

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.

Schematic of Drosophila hemocyte developmental migrations in stage 10-16 embryos
Schematic of Drosophila hemocyte developmental migrations in stage 10-16 embryos. The major events indicated are : 1) transmigration of hemocytes from the head to the tail, 2) posterior migration of head hemocytes along ventral midline, 3) anterior migration of tail hemocytes along ventral midline, 4) lateral migration of hemocytes from ventral midline to form three parallel lines. Lateral or ventral perspectives are indicated. A=anterior, P=posterior, D=dorsal, V=ventral.
Time-lapse series of surface projections (ventral view) of migrating hemocytes expressing GFP in control
Time-lapse series of surface projections (ventral view) of migrating hemocytes expressing GFP in control, Rho1 mutant, and Wash knock-down embryos. Head and Tail are indicated. Hemocytes that began their developmental migrations in the tail were tracked for 180°. Note: all of the tail hemocytes present in Wash knockdown embryos arrived from the anterior.
Cellular protrusions are severely reduced in Rho1 > Wash > Arp2/3, but not Wash Regulatory Comples (Strumpelin, CCDC53), posterior hemocytes
Cellular protrusions are severely reduced in Rho1 > Wash > Arp2/3, but not Wash Regulatory Comples (Strumpelin, CCDC53), posterior 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.