The Fog signaling pathway: Insights into signaling in morphogenesis

Epithelia form the building blocks of many tissue and organ types. Epithelial cells often form a contiguous 2-dimensional sheet that is held together by strong adhesions. The mechanical properties conferred by these adhesions allow the cells to undergo dramatic three-dimensional morphogenetic movements while maintaining cell–cell contacts during embryogenesis and post-embryonic development. The Drosophila Folded gastrulation pathway triggers epithelial cell shape changes that drive gastrulation and tissue folding and is one of the most extensively studied examples of epithelial morphogenesis. This pathway has yielded key insights into the signaling mechanisms and cellular machinery involved in epithelial remodeling. In this review, we discuss principles of morphogenesis and signaling that have been discovered through genetic and cell biological examination of this pathway. We also consider various regulatory mechanisms and the system?s relevance to mammalian development. We propose future directions that will continue to broaden our knowledge of morphogenesis across taxa.     

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.     

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and ¹H,¹³C,¹⁵N-based NMR spectroscopy. The two domains assume a “chair” shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.     

Phosphorylation of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) at Threonine 544 by Aurora B Kinase Promotes the Binding of Polo-like Kinase 1 to MyoGEF

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.     

Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs.     

Effect of the Rho kinase inhibitor Y-27632 on the proteome of hearts with ischemia-reperfusion injury

Growing attention has been given to the role of the Rho kinase pathway in the development of heart disease and ischemia/reperfusion (I/R) injury. Y‐27632 is a Rho kinase inhibitor demonstrated to protect against I/R injury, but the exact mechanism by which it does so remains to be elucidated. The goal of this project was to determine new targets by which Y‐27632 can protect the heart against I/R injury. Isolated rat hearts were perfused under aerobic conditions or subjected to I/R in the presence or absence of Y‐27632. Administration of Y‐27632 (1 μM) before ischemia and during the first 10 min of reperfusion resulted in complete recovery of cardiac function. 2‐D electrophoresis followed by MS identified four proteins whose levels were affected by Y‐27632 treatment. Lactate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase were significantly increased in the Y‐27632 treated group, while creatine kinase was normalized to control levels. In addition, we found increased level of two different molecular fragments of ATP synthase, which were normalized by Y‐27632. This increase suggests that during ischemia ATP synthase is subjected to degradation. The changes in metabolic enzymes' levels and their regulation by Y‐27632 suggest that the cardioprotective effect of Y‐27632 involves increased energy production.     

Measles virus contact with T cells impedes cytoskeletal remodeling associated with spreading, polarization, and CD3 clustering

CD3/CD28-induced activation of the PI3/Akt kinase pathway and proliferation is impaired in T cells after contact with the measles virus (MV) glycoprotein (gp) complex. We now show that this signal also impairs actin cytoskeletal remodeling in T cells, which loose their ability to adhere and to promote microvilli formation. MV exposure results in an almost complete collapse of membrane protrusions associated with reduced phosphorylation levels of cofilin and ezrin/radixin/moesin (ERM) proteins. Consistent with their inability to activate Cdc42 and Rac1 in response to the ligation of CD3/CD28, T cells exposed to MV fail to acquire a morphology consistent with spreading and lamellopodia formation. In spite of these impairments of cytoskeleton-driven morphological alterations, these cells are recruited into conjugates with dendritic cells as efficiently as control T cells. The signal elicited by MV, however, prevents T cells to polarize as documented by a failure to redistribute the microtubule organizing center toward the synapse. Moreover, CD3 cannot be efficiently clustered and redistributed to the central region of the immunological synapse. Thus, by inducing microvillar collapse and interfering with cytoskeletal remodeling, MV signaling disturbs the ability of T cells to adhere, spread, and cluster receptors essential for sustained T-cell activation.     

An essential role of Cdc42-like GTPases in mitosis of HeLa cells

Here we used RNA interference and examined possible redundancy amongst Rho GTPases in their mitotic role. Chromosome misalignment is induced significantly in HeLa cells by Cdc42 depletion and not by depletion of either one or all of the other four Cdc42-like GTPases (TC10, TCL, Wrch1 or Wrch2), four Rac-like GTPases or three Rho-like GTPases. Notably, combined depletion of Cdc42 and all of the other four Cdc42-like GTPases significantly enhances chromosomal misalignment. These observations suggest that Cdc42 is the primary GTPase functioning during mitosis but that the other four Cdc42-like GTPases can also assume the mitotic role in its absence.     

The diaphanous-related formin DAAM1 collaborates with the Rho GTPases RhoA and Cdc42, CIP4 and Src in regulating cell morphogenesis and actin dynamics

Binding partners for the Cdc42 effector CIP4 were identified by the yeast two-hybrid system, as well as by testing potential CIP4-binding proteins in coimmunoprecipitation experiments. One of the CIP4-binding proteins, DAAM1, was characterised in more detail. DAAM1 is a ubiquitously expressed member of the mammalian diaphanous-related formins, which include proteins such as mDia1 and mDia2. DAAM1 was shown to bind to the SH3 domain of CIP4 in vivo. Ectopically expressed DAAM1 localised in dotted pattern at the dorsal side of transfected cells and the protein was accumulated in the proximity to the microtubule organising centre. Moreover, ectopic expression of DAAM1 induced a marked alteration of the cell morphology, seen as rounding up of the cells, the formation of branched protrusions as well as a reduction of stress-fibres in the transfected cells. Coimmunoprecipitation experiments demonstrated that DAAM1 bound to RhoA and Cdc42 in a GTP-dependent manner. Moreover, DAAM1 was found to interact and collaborate with the non-receptor tyrosine kinase Src in the formation of branched protrusions. Taken together, our data indicate that DAAM1 communicates with Rho GTPases, CIP4 and Src in the regulation of the signalling pathways that co-ordinate the dynamics of the actin filament system.