Cytoskeletal Regulation by Rho GTPase and its Effectors

     
        Rho GTPases have emerged as key regulators of the actin cytoskeleton and, more recently, of the microtubule cytoskeleton. Activation of any Rho family protein results in cytoskeletal changes, but the exact changes depend on the family member. Rho and its relatives, Rac and Cdc42, are the founding members of the Rho subfamily signaling proteins that act as molecular switches.  Rho proteins cycle between an active GTP-bound state and an inactive GDP-bound state.  Their activation state is controlled by regulatory proteins such as guanine exchange factors (GEFs), which catalyze the exchange of GDP for GTP thereby activating Rho, guanine dissociation inhibitors (GDIs), which inhibit the release of GDP keeping Rho inactive, and GTPase activating proteins (GAPs), which increase the rate at which Rho hydrolyzes GTP and hence becomes inactivated.  While the overall sequence of events leading to activation of Rho family proteins by extracellular signals is known, gaps remain in the molecular details of these pathways and are areas of intensive study.

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.

Activated Rho GTPases interact with effector proteins (cellular target proteins) to drive a large variety of biological responses including reorganization of the actin cytoskeleton (affecting cell shape changes, cell polarity, cell movement, and cytokinesis), changes in gene transcription, chemotaxis, axonal guidance, cell cycle progression, cell adhesion, oncogenic transformation, and epithelial wound repair. Rho GTPases are also the targets of different classes of pathogens in disease-causing bacterial/viral infections. As with the upstream regulators of Rho function, details of the molecular pathways mediating these different Rho responses are mostly unknown.  Thus, a major question in the field is how these different responses are related.  Is the underlying mechanism for Rho's action in these diverse biological processes the same (e.g., actin regulation) or is Rho involved in a number of different molecular mechanisms (and how then are its different roles specified)?

Rho1 in Drosophila.

      Rho GTPases are required during early Drosophila development where they are essential in orchestrating the cell shape changes and movements that underlie morphogenesis. Several Rho family members have been identified molecularly in Drosophila including Rho1, Rac1, Rac2, Cdc42, RhoL, and Mtl.  We have been characterizing the Drosophila Rho1 mutation that provides in vivo developmental and organismal contexts in which to define the regulatory mechanism(s) underlying specific Rho-induced phenotypes.  Studies using ectopic expression of constitutively activated (ca) and dominant-negative (dn) forms of Rho1 indicate a role for this GTPase in dorsal closure by affecting JNK signaling, and during eye development where Rho1(dn) was shown to cause a collapse of the actin network important for proper ommatidial morphogenesis and planar polarity.

      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.

Egg chambers from females with reduced Rho1 function show a general disruption of the actin cytoskeleton, particularly in the outer ring canals and oocyte cortex.	Despite the disrupted actin cytoskeleton, oogenesis is able to proceed in these females leading to inviable embryos with patterning defects.

      Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure. Dorsal closure in Rho1 mutants occurs, but the process is not as well organized as in wildtype.  Leading edge cells in Rho1 mutants are pinched together or splayed out inappropriately rather than showing the well-organized, columnar organization typical of wildtype embryos.

Rho1 zygotic phenotypes (bottom) compared to wildtype (top).  Embryos undergoing dorsal closure stained with phalloidin to visualize actin.  Rho1 mutant embryos show increased actin staining in the head region where the abnormally shaped cells do not undergo the coordinated movements of head involution.  Higher magnification views show that while Rho1 mutant embryos do complete dorsal closure, actin organization at the dorsal midline is disrupted compared with wildtype.  The dorsal anterior hole in the larval cuticle is a result of failed internalization of head structures on the Rho1 embryo, resulting in the brain remaining on the anterior dorsal surface with the subsequent failure of this region to secrete cuticle.

We have generated a monoclonal antibody that specifically recognizes Rho1.  Rho1 protein is expressed ubiquitously, but accumulates at particular subcellular structures.  In the syncytial blastoderm Rho1 changes localization based on the phase of the cell cycle, localizing to the metaphase furrows during metaphase and surrounding the nuclei during interphase.  In the cellular blastoderm, Rho1 is enriched in the apical cytoplasm.  This apical accumulation is lost in embryos expressing a mutant form of Rho1 that lacks the isoprenylation site at its C-terminus (required for its insertion into the plasma membrane).  Rho1 also accumulates in the basal region of cells underlying the pole cells in the posterior of the embryo.

Rho1 localizes to the metaphase furrows during metaphase (top) and surrounding the nuclei during interphase (bottom).	Rho1 protein is ubiquitously expressed, but concentrated apically, in blastoderm embryos (top).  Rho1 also accumulates basally in cells underlying the pole cells (arrow; bottom).	Higher magnification of a cellular blastoderm embryo highlighting the apical accumulation of Rho1 (top).  This apical accumulation is lost in embryos with a mutation in Rho1's isoprenylation site (Rho1E3.10).

      Recent studies suggest that Rho may act through multiple separable pathways to carry out its various functions.  While a handful of Rho targets have been identified in different systems to date, the molecular pathways in which each is involved largely remain to be elucidated.  We are currently investigating the molecular mechanisms associated with two specific dRho1 regulatory pathways mediated through its interaction with  alpha-catenin and p120-catenin, proteins involved in adherens junction formation, or mediated through its interaction with Cappuccino, a formin homology protein important for actin and microtubule cytoskeleton integrity.

Rho1 and the coordination of microtubule/microfilament dynamics.

We have found that Drosophila Rho1 is required for maintenance of proper microfilament and microtubule architecture during oogenesis.  We have also found that the de novo actin nucleation factors Cappuccino (Capu; a formin-homology (FH) protein) and Spire (a WH2 domain protein) act downstream of Rho1 to regulate the onset of ooplasmic streaming. We find that double heterozygosity for LOF alleles of the small GTPase Rho1 and capu or spire results in 100% maternal effect lethality and abnormal microfilament architecture at the oocyte cortex. This maternal lethality in Rho1-capu or Rho1-spire trans-heterozygotes is due to premature ooplasmic streaming similar to that described for homozygous capu or spire mutants, suggesting that Rho1, Capu, and Spire share a common pathway.

Rho1-capu doubly heterozygous oocytes undergo premature ooplasmic streaming.  (top right corner) Schematic of a stage 10 egg chamber.  Boxed area indicates region of egg chamber shown in other panels. Still confocal micrographs (left column) or 6-frame confocal temporal projections of time-lapse movies (middle column) of wildtype or Rho-capu trans-heterozygous oocytes stained with trypan blue to visualize dynamic yolk granule movement. (right column) Confocal micrographs of stage 7 oocytes from wildtype or Rho1-capu trans-heterozygous females stained with alpha-tubulin to visualize dynamic microtubules. Wildtype stage 10 oocyte (top row) during cytoplasmic streaming.  Unlike wildtype stage 7 oocytes (middle row), stage 7 oocytes from Rho1-capu trans-heterozygous mothers undergo premature ooplasmic streaming, as indicated by the spiral patterns of fluorescence seen in the temporal projections.

      In Drosophila, capu and spire are both required for the proper timing of ooplasmic streaming.  In wildtype oocytes, vigorous ooplasmic streaming is associated with rapid growth during stages 10b-13, and is never observed prior to this stage.  Homozygous capu or spire mutants exhibit premature ooplasmic streaming, beginning at stage 7/8 and continuing through stage 13. This premature streaming interferes with transport mechanisms required for the localization of early polarity markers, resulting in disruption of dorsal-ventral and anterior-posterior body axes.  We have also observed this premature ooplasmic streaming phenotype in oocytes from mothers with reduced maternal Rho1 activity, and from mothers trans-heterozygous for Rho1-capu, Rho1-spire and capu-spire.  Both the wildtype streaming event and the premature streaming in homozygous capu or spire mutants are microtubule-based. Thus, it was somewhat paradoxical that Spire and Capu nucleate actin, but are not known to affect microtubule architecture or dynamics.

      We addressed the role of actin nucleation in preventing premature ooplasmic streaming, by examining the nucleation properties of CapuFH2 proteins harboring point mutations corresponding to the strong allele capu^RK12 and weak allele capu^2F, both of which cause premature streaming.  The capu^2F (P597T) mutation does not substantially affect the nucleation activity of CapuFH2, indicating that nucleation is not solely responsible for the premature streaming phenotype. The capu^RK12 (L768H) mutation results in complete loss of nucleation activity in vitro, yet CapuFH2^RK12 (L768H) is still able to dimerize, suggesting that this mutation creates a nucleation-dead FH2 domain.

Capu and Spire affect actin dynamics.  (a) Pyrene-actin polymerization assays were conducted with varying concentrations of CapuFH2 (in µM).  (b) Capu actin nucleation activity is not subject to auto-inhibition. CapuN1 alone has no affect on actin dynamics.  (c) The capu2F (P597T) mutation does not substantially affect actin nucleation, whereas the capuRK12 (L768H) mutation abolished nucleation activity.

      The prevailing model for DRF proteins is that their actin nucleation activity is regulated through an inhibitory intra-molecular interaction between the conserved N-terminal DID and C-terminal DAD domains that is relieved by Rho GTPase binding to the N-terminal GBD domain. We find that Capu and the three Spire isoforms bind directly to Rho1 in GST-pulldown assays showing a preference for Rho in its GTP-bound active state. We mapped the binding sites for Rho1 on SpireD, SpireC, and Capu to broad regions using smaller protein constructs (see figure below).  Although Capu does not have a DAD domain by sequence homology, because of its physical interaction with Rho1 we thought it possible that Capu activity is regulated by an auto-inhibitory mechanism. While CapuN1 and CapuFH2 associate in vitro, we do not observe any auto-inhibitory effect of CapuN1 on actin nucleation by CapuFH2, suggesting that this self-association may either not take place or not be important in the context of Drosophila oocyte development.

Diagram of a canonical Diaphanous-related formin (DRF) protein and of the Capu protein (left), and of the Spire-A, -C, and -D protein isoforms (right).  The smallest Capu and Spire protein fragments that bind to Rho1 are indicated by thicker lines. While mapping to a similar region of the protein, Capu's Rho1-binding domain (GBD) does not contain sequence homology to Rho binding motifs in other DRF proteins.

      We made antibodies and GFP-fusion constructs of Rho1, Capu and the Spire C and D isoforms and examined their expression during oogenesis.  We find that all of these proteins are enriched at the cortex of both nurse cells and the oocyte, and are diffusely distributed throughout the cytoplasm of these cells.  SpireC exhibits punctate expression throughout the cytoplasm.  Based on the co-localization of Rho1, Capu, and Spire at the oocyte cortex, the apparent coordination of microtubules (MTs) and microfilaments required to regulate the onset of ooplasmic streaming, and the lack of phenotype correlation with actin nucleation activity, we hypothesized that these proteins may directly coordinate MTs and actin, rather than affecting MT dynamics indirectly. Using in vitro MT/microfilament crosslinking assays with purified recombinant proteins, we found that the C-terminal fragment of Capu (CapuFH2) exhibits potent novel F-actin and MT bundling and cross-linking activity.  In addition, an N-terminal portion of Capu (CapuN1) exhibits novel F-actin bundling activity.  We also tested the crosslinking activity of CapuFH2^RK12 (L768H) and CapuFH2^2F (P597T), and found that the L768H mutation, which results in a more penetrant phenotype in vivo, also abrogates crosslinking activity, whereas CapuFH2^2F (P597T) only attenuates cross-linking activity, suggesting that Capu-mediated actin nucleation is not sufficient to prevent premature ooplasmic streaming.

MT and microfilament bundling and crosslinking properties of wildtype and mutant forms of Capu.  Stabilized MTs (middle row) and F-actin (top row) were incubated with protein(s) indicated.

      We have also examined SpireA, -C and -D for crosslinking and bundling activities. We find that SpireC, which lacks the four tandem WH2-domains that are sufficient for actin nucleation in vitro, has actin bundling and actin/MT crosslinking activity similar to that of CapuFH2. However, the actin nucleating isoform SpireD has no crosslinking or bundling activity, suggesting that the C and D isoforms of Spire regulate distinct aspects of cytoskeletal architecture. Interestingly, SpireA bundles MTs, but not actin. Importantly, we find that when added at equimolar ratios with Capu, SpireD blocks crosslinking and MT bundling whereas SpireC has no effect. Notably, we find that addition of activated Rho1 (Rho1^GTP), but not inactive Rho1 (Rho1^GDP), restores crosslinking and MT bundling by CapuFH2 in the presence of SpireD. These novel bundling and crosslinking activities suggest the possibility that Capu and Spire regulate the onset of ooplasmic streaming by directly mediating coordination of actin assembly and MT architecture. Moreover, our finding that Rho1 has no effect on actin nucleation, but can relieve the inhibition of CapuFH2-mediated crosslinking by SpireD, provides a mechanistic basis for the genetic interaction of Rho1, capu, and spire.

(Left) MT and microfilament bundling/crosslinking properties of Spire isoforms. (Right) SpireD interferes with Capu bundling/crosslinking activity and this inhibition is restored by the presence of active Rho1.

      Our results suggest that Rho1 regulates the timing of ooplasmic streaming by regulating the MT/microfilament crosslinking that occurs at the oocyte cortex. In this model, crosslinking antagonizes the formation of the dynamic subcortical microtubule arrays that are required for ooplasm streaming.  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, Capu, and Spire isoforms C and D. (a) Diagram of wildtype oocyte prior to the onset of ooplasmic streaming.  MTs are shown in orange and cortical microfilaments in green. (inset) Close-up view of the oocyte cortex.  Active (GTP-bound) Rho1 promotes MT-microfilament crosslinking by sequestering SpireD, thereby preventing it from binding to SpireC and Capu.  MT arrays are restricted to the oocyte cortex.  (b) Diagram of wildtype oocyte during ooplasmic streaming.  (inset) Close-up view of the oocyte cortex.  Upstream signaling events result in GTP hydrolysis by Rho1, allowing SpireD to bind to SpireC and Capu.  This blocks MT-microfilament crosslinking, resulting in ooplasmic streaming.

     Further support for our model comes from a recent study from the Reichmann lab demonstrating that Chickadee, encoding fly profilin, is required for the formation of cortical actin bundles in the oocyte, and that Capu and Spire anchor the minus ends of microtubules to a scaffold made from these cortical actin bundles. Together, these results suggest dual or multifaceted biochemical roles for these proteins in regulating developmental processes. Consistent with this, non-actin nucleating roles for other formins (i.e., actin severing/depolymerization, microtubule stabilization, signaling, and transcriptional regulation) are beginning to be reported.

      Thus to date, we have identified Capu and Spire as effectors of Rho1 activity required for regulating the proper timing of ooplasmic streaming.  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 Spire and Capu is regulated to coordinate the ooplasmic streaming event in vivo.  As so little is 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 and catenins.

      Rho GTPases have recently been recognized as 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 genetically and physically with the adherens junction components  α-catenin and p120^ctn.  While Rho1 protein is present throughout the cell, it accumulates apically, particularly at sites of cadherin-based adherens junctions.  Cadherin and catenin localization is disrupted in Rho1 mutants, implicating Rho1 in their regulation.

Adherens junction protein expression in wildtype and Rho1 mutant embryos. DE-cadherin expression in stage 15 wildtype (top left) or Rho1 mutant (bottom left) embryos.  Note the disruption of DE-Cadherin localization near the leading edge, but not in more lateral regions.  No difference in Neurexin expression (labeling septate junctions) is observed in Rho1 mutant embryos (bottom right) compared to wildtype (top right).

     Rho1 binds directly to  α-catenin and p120^ctn in vitro.  p120^ctn interacts preferentially with Rho-GDP, consistent with its ability to negatively regulate Rho by keeping it in a GDP-bound state when present in the cytoplasm, while the interaction with  α-catenin is GTP-independent.  These interactions map to distinct surface-exposed regions of the protein not previously assigned functions.  Consistent with a direct interaction, both  α-catenin and p120^ctn co-immunoprecipitate with Rho1-containing complexes from embryo lysates. 

Diagram of the Rho1 protein with the major protein domains indicated (top).  Computer models of GDP-bound RhoA crystal structure (bottom).  The residues required for  α-catenin binding are highlighted in yellow and those required for p120ctn binding are highlighted in red.  For reference the effector domain is highlighted in green.

      p120^ctn has recently been suggested to inhibit Rho activity through an unknown mechanism. Since no mutations have been reported for either p120^ctn or  α-catenin, we have used RNA interference (RNAi) to disrupt their function.  Loss of function for either catenin results in severe morphogenetic defects and aberrant Rho1 localization, as well as an accumulation of Rho1 in response to lowered p120^ctn activity. Our observations suggest that  α-catenin and p120^ctn are key players in a mechanism of recruiting Rho1 to its sites of action.

Adherens junction formation and maturation.  The extracellular domains of cadherin molecules bind weakly to each other.  These initially weak interactions are strengthened by the clustering of multiple cadherin complexes and the release of IQGAP from the cadherin complexes, allowing  α-catenin to associate with the cadherin complexes and link them to the actin cytoskeleton.  JMD: juxtamembrane domain;  CBD: catenin-binding domain,  PM: plasma membrane;   α:  α-catenin;   β:  β-catenin.

Rho1 and endocytosis.

Maternal Rho1 mutants fail to maintain segment polarity gene expression.  Embryos with reduced maternal Rho1 exhibit a segmentation phenotype due in part to improper maintenance of segment polarity gene expression.  In particular, expression of the segment polarity genes Engrailed (En), Wingless (Wg), and Hedgehog, while initiated normally, are not maintained properly, leading to the fusion or absence of stripes. We found that this phenotype is the result Rho1 affecting the Wg signal transduction pathway through its effects on general cellular processes including regulation of the cytoskeleton required for proper endocytosis. Actin cytoskeletal regulation has been linked to regulation of both the endocytic and secretory pathways. Maternal Rho1 mutants also exhibit defective secretion of Wg.

Z-series projections of wildtype, shibire, and maternal Rho1 mutant stage 9 embryos stained with antibodies against Wingless (Wg, green) and Discontinuous Actin Hexagon (Dah, red) to outline cell boundaries.  The boxed regions of embryos are shown at higher magnification to the right.  Z-series cross-sections noted by dashed lines are projected in ant, mid, post.  Note the punctate accumulations of Wg protein spreading out from the Wg-expressing cells in wildtype that are reduced in shibire and maternal.		A ventral view of 3 stripes of extracellular Wg staining in wildtype, shibire, and maternal Rho1 mutant embryos.  Higher magnification views of one of the stripes is shown to the right.

     Similar to embryos, fly S2R+ cells treated with Rho1 dsRNA cannot respond properly to Wg signaling. Treatment of S2R+ cells with Rho1 dsRNA has been shown to result in a multinucleate phenotype due to defects in cytokinesis, mimicking defects in cellularization exhibited by maternal Rho1 mutants. Adding fluorescently labeled dextran to the Wg-conditioned medium prior to treating the cells allowed us to directly assess their endocytic activity.  Wildtype cells show an internalization of both Wg and dextran, with Wg-containing endosomes comprising a subset of those present within the cell, whereas these Wg-containing endosomes are smaller and reduced in number in cells treated with Rho1 dsRNA.

(Left) S2R+ cells treated with Rho1 dsRNA do not respond properly to Wg signaling.  Wildtype and Rho1 dsRNA treated S2R+ cells without or with Wg treatment (as indicated).  Accumulation of Arm (left column) and DE-Cad (middle column) in response to Wg signaling is lost with Rho1 dsRNA treatment.  Upon treatment with Wg-conditioned medium, Wg-positive punctate structures can be seen in wildtype cells, but not those treated with Rho1 dsRNA.  (Right) Wildtype and Rho1 dsRNA-treated cells that have internalized Texas-Red-conjugated 70kD dextran molecules (Dx; red) stained with antibodies to Wg (green).  Note the punctate yellow accumulations (arrows), indicating co-localization of Wg and Dx in wildtype cells.  Cells treated with Rho1 dsRNA fail to properly internalize Wg.

     We also examined the activity of the other signaling pathways known to require endocytosis for their proper function. EGFR signaling is under feedback control whereby activation of signaling leads to endocytosis of active receptor and attenuation of the signal. Another signal transduction pathway whose activity is regulated by endocytosis is that of the receptor tyrosine kinase Torso, which is involved in the specification of terminal pattern in Drosophila.  Signaling from both the EGFR and Torso pathways were disrupted in Rho1 mutants compared to wildtype suggesting that maternal Rho1 mutants are generally compromised in endocytosis and secretion.  Thus, our analysis of the embryonic phenotype associated with reduced Rho1 activity is consistent with a role for Rho1 in regulating signaling events governing proper developmental patterning.

Reagents Available

   The Rho1 loss-of-function mutant (Rho1^1B) is available from the Bloomington Drosophila Stock Center - stock #9477.

   Transgenic lines expressing GFP-Rho1 under the control of the endogenous Rho1 promoter are available from the Bloomington Drosophila Stock Center - stocks #9527, 9528, 24762.

   The Rho1 monoclonal antibody (P1D9) is available from the Developmental Studies Hybridoma Bank.

Last updated 10/28/08