Development of conformational BRET biosensors that monitor ezrin, radixin and moesin activation in real time.

Ezrin, radixin and moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape, as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel bioluminescence resonance energy transfer (BRET)-based conformational biosensors, compatible with high-throughput screening, that monitor individual ezrin, radixin or moesin activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.

Moesin activation controls bone resorption and tunneling nanotube-dependent osteoclast fusion.

Osteoclasts are multinucleated cells unique in their ability to resorb bone. Osteoclastogenesis involves several steps of actin-driven rearrangements that participate not only in the cell-cell fusion process, but also in the formation of the sealing zone, the adhesive structure determining the resorption area. Despite the importance of these actin cytoskeleton-based processes, their precise mechanisms of regulation are still poorly characterized. Here, we found that moesin, a member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role for both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin is negatively regulated to potentiate their ability to fuse and degrade bone. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances formation of tunneling nanotubes (TNTs), F-actin-containing intercellular bridges that we revealed to trigger osteoclast fusion. In addition, via a ß3-integrin/RhoA/SLK pathway and independently of its role in fusion, moesin regulates the number and organization of sealing zones in mature osteoclast, and thus participates in the control of bone resorption. Supporting these findings, we found that moesin-deficient mice are osteopenic with a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of osteoclast biology, and open new opportunities to specifically target osteoclast activity in bone disease therapy.

Differential roles of PtdIns(4,5)P2 and phosphorylation in moesin activation during Drosophila development.

The ezrin, radixin and moesin (ERM) proteins regulate cell membrane architecture in several cellular contexts. Current models propose that ERM activation requires a PtdIns(4,5)P(2)-induced conformational change, followed by phosphorylation of a conserved threonine. However, how these inputs contribute in vivo to orchestrate ERM activation is poorly understood. We addressed this issue by evaluating the contribution of PtdIns(4,5)P(2) and phosphorylation to the regulation of moesin during Drosophila development. Unexpectedly, we found that a form of moesin that cannot be phosphorylated displayed significant activity and could substitute for the endogenous product during wing morphogenesis. By contrast, we also show that PtdIns(4,5)P(2) binding is essential for moesin recruitment to the membrane and for its subsequent phosphorylation. Our data indicate that PtdIns(4,5)P(2) acts as a dosing mechanism that locally regulates ERM membrane recruitment and activation, whereas cycles of phosphorylation and dephosphorylation further control their activity once they have reached the cell cortex.

Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.

At mitotic entry, reorganization of the actomyosin cortex prompts cells to round-up. Proteins of the ezrin, radixin, and moesin family (ERM) play essential roles in this process by linking actomyosin forces to the plasma membrane. Yet, the cell-cycle signal that activates ERMs at mitotic entry is unknown. By screening a compound library using newly developed biosensors, we discovered that drugs that disassemble microtubules promote ERM activation. We further demonstrated that disassembly of interphase microtubules at mitotic entry directs ERM activation and metaphase cell rounding through GEF-H1, a Rho-GEF inhibited by microtubule binding, RhoA, and its kinase effector SLK. We finally demonstrated that GEF-H1 and Ect2, another Rho-GEF previously identified to control actomyosin forces, act together to drive activation of ERMs and cell rounding in metaphase. In summary, we report microtubule disassembly as a cell-cycle signal that controls a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.

Penile Cancer-Derived Cells Molecularly Characterized as Models to Guide Targeted Therapies.

Penile cancer (PeCa) is a common disease in poor and developing countries, showing high morbidity rates. Despite the recent progress in understanding the molecular events involved in PeCa, the lack of well-characterized in vitro models precludes new advances in anticancer drug development. Here we describe the establishment of five human primary penile cancer-derived cell cultures, including two epithelial and three cancer-associated fibroblast (CAF) cells. Using high-throughput genomic approaches, we found that the epithelial PeCa derived- cells recapitulate the molecular alterations of their primary tumors and present the same deregulated signaling pathways. The differentially expressed genes and proteins identified are components of key oncogenic pathways, including EGFR and PI3K/AKT/mTOR. We showed that epithelial PeCa derived cells presented a good response to cisplatin, a common therapeutic approach used in PeCa patients. The growth of a PeCa-derived cell overexpressing EGFR was inhibited by EGFR inhibitors (cetuximab, gefitinib, and erlotinib). We also identified CAF signature markers in three PeCa-derived cells with fibroblast-like morphology, indicating that those cells are suitable models for PeCa microenvironment studies. We thus demonstrate the utility of PeCa cell models to dissect mechanisms that promote penile carcinogenesis, which are useful models to evaluate therapeutic approaches for the disease.

NF45 and NF90 Regulate Mitotic Gene Expression by Competing with Staufen-Mediated mRNA Decay.

In human cells, the expression of ∼1,000 genes is modulated throughout the cell cycle. Although some of these genes are controlled by specific transcriptional programs, very little is known about their post-transcriptional regulation. Here, we analyze the expression signature associated with all 687 RNA-binding proteins (RBPs) and identify 39 that significantly correlate with cell cycle mRNAs. We find that NF45 and NF90 play essential roles in mitosis, and transcriptome analysis reveals that they are necessary for the expression of a subset of mitotic mRNAs. Using proteomics, we identify protein clusters associated with the NF45-NF90 complex, including components of Staufen-mediated mRNA decay (SMD). We show that depletion of SMD components increases the binding of mitotic mRNAs to the NF45-NF90 complex and rescues cells from mitotic defects. Together, our results indicate that the NF45-NF90 complex plays essential roles in mitosis by competing with the SMD machinery for a common set of mRNAs.

STRIPAK regulates Slik localization to control mitotic morphogenesis and epithelial integrity.

Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. We previously reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. We also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, we conducted a genome-wide RNAi screen, looking for new regulators of moesin activity. We identified that Slik is a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). We discovered that the phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. Our results implicate the Slik-STRIPAK complex in the control of multiple morphogenetic processes.

Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network.

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.

Misshapen coordinates protrusion restriction and actomyosin contractility during collective cell migration.

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces.

IPIP27 Coordinates PtdIns(4,5)P2 Homeostasis for Successful Cytokinesis.

During cytokinesis, an actomyosin contractile ring drives the separation of the two daughter cells. A key molecule in this process is the inositol lipid PtdIns(4,5)P2, which recruits numerous factors to the equatorial region for contractile ring assembly. Despite the importance of PtdIns(4,5)P2 in cytokinesis, the regulation of this lipid in cell division remains poorly understood. Here, we identify a role for IPIP27 in mediating cellular PtdIns(4,5)P2 homeostasis. IPIP27 scaffolds the inositol phosphatase oculocerebrorenal syndrome of Lowe (OCRL) by coupling it to endocytic BAR domain proteins. Loss of IPIP27 causes accumulation of PtdIns(4,5)P2 on aberrant endomembrane vacuoles, mislocalization of the cytokinetic machinery, and extensive cortical membrane blebbing. This phenotype is observed in Drosophila and human cells and can result in cytokinesis failure. We have therefore identified IPIP27 as a key modulator of cellular PtdIns(4,5)P2 homeostasis required for normal cytokinesis. The results indicate that scaffolding of inositol phosphatase activity is critical for maintaining PtdIns(4,5)P2 homeostasis and highlight a critical role for this process in cell division.

PTEN reduces endosomal PtdIns(4,5)P2 in a phosphatase-independent manner via a PLC pathway.

The tumor suppressor PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 Here, we make the unexpected discovery that in Drosophila melanogaster PTEN reduces PtdIns(4,5)P2 levels on endosomes, independently of its phosphatase activity. This new PTEN function requires the enzymatic action of dPLCXD, an atypical phospholipase C. Importantly, we discovered that this novel PTEN/dPLCXD pathway can compensate for depletion of dOCRL, a PtdIns(4,5)P2 phosphatase. Mutation of OCRL1, the human orthologue of dOCRL, causes oculocerebrorenal Lowe syndrome, a rare multisystemic genetic disease. Both OCRL1 and dOCRL loss have been shown to promote accumulation of PtdIns(4,5)P2 on endosomes and cytokinesis defects. Here, we show that PTEN or dPLCXD overexpression prevents these defects. In addition, we found that chemical activation of this pathway restores normal cytokinesis in human Lowe syndrome cells and rescues OCRL phenotypes in a zebrafish Lowe syndrome model. Our findings identify a novel PTEN/dPLCXD pathway that controls PtdIns(4,5)P2 levels on endosomes. They also point to a potential new strategy for the treatment of Lowe syndrome.

RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery.

Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.

Proteomics Screen Identifies Class I Rab11 Family Interacting Proteins as Key Regulators of Cytokinesis.

The 14-3-3 protein family orchestrates a complex network of molecular interactions that regulates various biological processes. Owing to their role in regulating the cell cycle and protein trafficking, 14-3-3 proteins are prevalent in human diseases such as cancer, diabetes, and neurodegeneration. 14-3-3 proteins are expressed in all eukaryotic cells, suggesting that they mediate their biological functions through evolutionarily conserved protein interactions. To identify these core 14-3-3 client proteins, we used an affinity-based proteomics approach to characterize and compare the human and Drosophila 14-3-3 interactomes. Using this approach, we identified a group of Rab11 effector proteins, termed class I Rab11 family interacting proteins (Rab11-FIPs), or Rip11 in Drosophila We found that 14-3-3 binds to Rip11 in a phospho-dependent manner to ensure its proper subcellular distribution during cell division. Our results indicate that Rip11 plays an essential role in the regulation of cytokinesis and that this function requires its association with 14-3-3 but not with Rab11. Together, our results suggest an evolutionarily conserved role for 14-3-3 in controlling Rip11-dependent protein transport during cytokinesis.

DHTP is an allosteric inhibitor of the kinesin-13 family of microtubule depolymerases.

The kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells.

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex.

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM's role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM-microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility.

The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasion-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative-feedback loop that restricts Gab2-dependent epithelial cell motility. On the basis of the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems.

The unexpected role of Drosophila OCRL during cytokinesis.

Inositides are intrinsic components of cell membranes that regulate a wide variety of cellular functions. PtdIns(4,5)P(2,) one of the most abundant phosphoinositides, is restricted at the plasma membrane where it regulates numerous functions including cell division. We have recently established that the Drosophila inositol 5-phosphatase, dOCRL, is essential for cytokinesis, the last step of cell division (Ben El Kadhi et al. 2011).(8) We demonstrated that dOCRL is required for the dephosphorylation of PtdIns(4,5)P(2) at the surface of endosomes, resulting in the restriction of this phosphoinositide to the cell cortex during cytokinesis. dOCRL is the Drosophila ortholog of human OCRL1, a PtdIns(4,5)P(2) phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome. Here, we discuss the relevance of our findings with reference to the role of human OCRL1 in non-pathological and pathological conditions.

The inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis and is necessary for cytokinesis.

During cytokinesis, constriction of an equatorial actomyosin ring physically separates the two daughter cells. At the cleavage furrow, the phosphoinositide PI(4,5)P2 plays an important role by recruiting and regulating essential proteins of the cytokinesis machinery [1]. Accordingly, perturbation of PI(4,5)P2 regulation leads to abortive furrowing and binucleation [2-4]. To determine how PI(4,5)P2 is regulated during cytokinesis, we individually knocked down each of the enzymes controlling the phosphoinositide (PIP) cycle in Drosophila. We show that depletion of the Drosophila ortholog of human oculocerebrorenal syndrome of Lowe 1 (OCRL1), an inositol 5-phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome, triggers a high rate of cytokinesis failure. In absence of dOCRL, several essential components of the cleavage furrow were found to be incorrectly localized on giant cytoplasmic vacuoles rich in PI(4,5)P2 and in endocytic markers. We demonstrate that dOCRL is associated with endosomes and that it dephosphorylates PI(4,5)P2 on internal membranes to restrict this phosphoinositide at the plasma membrane and thereby regulates cleavage furrow formation and ingression. Identification of dOCRL as essential for cell division may be important to understand the molecular basis of the phenotypic manifestations of Lowe syndrome.