BMP3 is a novel locus involved in the causality of ocular coloboma.

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, these data suggest that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

MS/MS-based strategies for proteomic profiling of invasive cell structures.

Acquired capacity of cancer cells to penetrate through the extracellular matrix of surrounding tissues is a prerequisite for tumour metastatic spread - the main source of cancer-associated mortality. Through combined efforts of many research groups, we are beginning to understand that the ability of cells to invade through the extracellular matrix is a multi-faceted phenomenon supported by variety of specialised protrusive cellular structures, primarily pseudopodia, invadopodia and podosomes. Additionally, secreted extracellular vesicles are being increasingly recognised as important mediators of invasive cell phenotypes and therefore may be considered bona fide invasive cell structures. Dissection of the molecular makings underlying biogenesis and function of all of these structures is crucial to identify novel targets for specific anti-metastatic therapies. Rapid advances and growing accessibility of MS/MS-based protein identification made this family of techniques a suitable and appropriate choice for proteomic profiling of invasive cell structures. In this review, we provide a summary of current progress in the characterisation of protein composition and topology of protein interaction networks of pseudopodia, invadopodia, podosomes and extracellular vesicles, as well as outline challenges and perspectives of the field.

Met receptor tyrosine kinase signals through a cortactin-Gab1 scaffold complex, to mediate invadopodia.

Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure-function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1-cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.

Emerging roles of Ruk/CIN85 in vesicle-mediated transport, adhesion, migration and malignancy.

Ruk/CIN85 is an adaptor protein. Similar to many other proteins of this type, Ruk/CIN85 is known to take part in multiple cellular processes including signal transduction, vesicle-mediated transport, cytoskeleton remodelling, programmed cell death and viral infection. Recent studies have also revealed the potential importance of Ruk/CIN85 in cancer cell invasiveness. In this review we summarize the various roles of this protein as well as the potential contribution of Ruk/CIN85 to malignancy and the invasiveness of cancer cells. In the last section of the paper we also speculate on the utility of Ruk/CIN85 as a target for novel anti-cancer therapies.

Dock7: a GEF for Rho-family GTPases and a novel myosin VI-binding partner in neuronal PC12 cells.

Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.

The Axenfeld-Rieger Syndrome Gene FOXC1 Contributes to Left-Right Patterning.

Precise spatiotemporal expression of the Nodal-Lefty-Pitx2 cascade in the lateral plate mesoderm establishes the left-right axis, which provides vital cues for correct organ formation and function. Mutations of one cascade constituent PITX2 and, separately, the Forkhead transcription factor FOXC1 independently cause a multi-system disorder known as Axenfeld-Rieger syndrome (ARS). Since cardiac involvement is an established ARS phenotype and because disrupted left-right patterning can cause congenital heart defects, we investigated in zebrafish whether foxc1 contributes to organ laterality or situs. We demonstrate that CRISPR/Cas9-generated foxc1a and foxc1b mutants exhibit abnormal cardiac looping and that the prevalence of cardiac situs defects is increased in foxc1a-/-; foxc1b-/- homozygotes. Similarly, double homozygotes exhibit isomerism of the liver and pancreas, which are key features of abnormal gut situs. Placement of the asymmetric visceral organs relative to the midline was also perturbed by mRNA overexpression of foxc1a and foxc1b. In addition, an analysis of the left-right patterning components, identified in the lateral plate mesoderm of foxc1 mutants, reduced or abolished the expression of the NODAL antagonist lefty2. Together, these data reveal a novel contribution from foxc1 to left-right patterning, demonstrating that this role is sensitive to foxc1 gene dosage, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld-Rieger syndrome patients.

Adaptor protein Ruk/CIN85 is associated with a subset of COPI-coated membranes of the Golgi complex.

Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.

Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS.

BACKGROUND: Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins. RESULTS: In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division. CONCLUSION: Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division.

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins.

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

Simultaneous Targeting of Two Distinct Epitopes on MET Effectively Inhibits MET- and HGF-Driven Tumor Growth by Multiple Mechanisms.

Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro The importance of these effector functions was confirmed in vivo using an Fc-effector function-attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780-91. ©2017 AACR.

5'-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion.

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease.