A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila.

In animal cell cytokinesis interaction of non-muscle Myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. cbe mutation impairs binding of Zipper protein to the regulatory light chain Spaghetti Squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the Phosphatidylinositol 4-phosphate [PI(4)P] binding protein Golgi phosphoprotein 3 (GOLPH3) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly

The Dynamic Cell The roles of the oncoprotein GOLPH3 in contractile ring assembly and membrane trafficking during cytokinesis

Abstract Cytokinesis is an intricate process that requires an intimate interplay between actomyosin ring constriction and plasma membrane remodelling at the cleavage furrow. However, the molecular mechanisms involved in coupling the cytoskeleton dynamics with vesicle trafficking during cytokinesis are poorly understood. The highly conserved Golgi phosphoprotein 3 (GOLPH3), functions as a phosphatidylinositol 4-phosphate (PI4P) effector at the Golgi. Recent studies have suggested that GOLPH3 is up-regulated in several cancers and is associated with poor prognosis and more aggressive tumours. In Drosophila melanogaster, GOLPH3 localizes at the cleavage furrow of dividing cells, is required for successful cytokinesis and acts as a key molecule in coupling phosphoinositide (PI) signalling with actomyosin ring dynamics. Because cytokinesis failures have been linked with pre-malignant disease and cancer, the novel connection between GOLPH3 and cytokinesis imposes new fields of investigation in cancer biology and therapy

GOLPH3 protein controls organ growth by interacting with TOR signaling proteins in Drosophila

The oncoprotein GOLPH3 (Golgi phosphoprotein 3) is an evolutionarily conserved phosphatidylinositol 4-phosphate effector, mainly localized to the Golgi apparatus, where it supports organelle architecture and vesicular trafficking. Overexpression of human GOLPH3 correlates with poor prognosis in several cancer types and is associated with enhanced signaling downstream of mTOR (mechanistic target of rapamycin). However, the molecular link between GOLPH3 and mTOR remains elusive. Studies in Drosophila melanogaster have shown that Translationally controlled tumor protein (Tctp) and 14-3-3 proteins are required for organ growth by supporting the function of the small GTPase Ras homolog enriched in the brain (Rheb) during mTORC1 (mTOR complex 1) signaling. Here we demonstrate that Drosophila GOLPH3 (dGOLPH3) physically interacts with Tctp and 14-3-3ζ. RNAi-mediated knockdown of dGOLPH3 reduces wing and eye size and enhances the phenotypes of Tctp RNAi. This phenotype is partially rescued by overexpression of Tctp, 14-3-3ζ, or Rheb. We also show that the Golgi localization of Rheb in Drosophila cells depends on dGOLPH3. Consistent with dGOLPH3 involvement in Rheb-mediated mTORC1 activation, depletion of dGOLPH3 also reduces levels of phosphorylated ribosomal S6 kinase, a downstream target of mTORC1. Finally, the autophagy flux and the expression of autophagic transcription factors of the TFEB family, which anti correlates with mTOR signaling, are compromised upon reduction of dGOLPH3. Overall, our data provide the first in vivo demonstration that GOLPH3 regulates organ growth by directly associating with mTOR signaling proteins

Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis

The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle–dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction

Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. Here we provide evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. We demonstrate that the gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. We show that Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. We further demonstrate that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. We propose that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis

Identification of GOLPH3 Partners in <i>Drosophila</i> Unveils Potential Novel Roles in Tumorigenesis and Neural Disorders.

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases

Identification of GOLPH3 Partners in Drosophila Unveils Potential Novel Roles in Tumorigenesis and Neural Disorders.

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases

Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. Here we provide evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. We demonstrate that the gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. We show that Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. We further demonstrate that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. We propose that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis

Modeling Congenital Disorders of N-Linked Glycoprotein Glycosylation in Drosophila melanogaster

Protein glycosylation, the enzymatic addition of N-linked or O-linked glycans to proteins, serves crucial functions in animal cells and requires the action of glycosyltransferases, glycosidases and nucleotide-sugar transporters, localized in the endoplasmic reticulum and Golgi apparatus. Congenital Disorders of Glycosylation (CDGs) comprise a family of multisystemic diseases caused by mutations in genes encoding proteins involved in glycosylation pathways. CDGs are classified into two large groups. Type I CDGs affect the synthesis of the dolichol-linked Glc3Man9GlcNac2 precursor of N-linked glycosylation or its transfer to acceptor proteins. Type II CDG (CDG-II) diseases impair either the trimming of the N-linked oligosaccharide, the addition of terminal glycans or the biosynthesis of O-linked oligosaccharides, which occur in the Golgi apparatus. So far, over 100 distinct forms of CDGs are known, with the majority of them characterized by neurological defects including mental retardation, seizures and hypotonia. Yet, it is unclear how defective glycosylation causes the pathology of CDGs. This issue can be only addressed by developing animal models of specific CDGs. Drosophila melanogaster is emerging as a highly suitable organism for analyzing glycan-dependent functions in the central nervous system (CNS) and the involvement of N-glycosylation in neuropathologies. In this review we illustrate recent work that highlights the genetic and neurobiologic advantages offered by D. melanogaster for dissecting glycosylation pathways and modeling CDG pathophysiology

Identification of Drosophila Mitotic Genes by Combining Co-Expression Analysis and RNA Interference

RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression–based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression–based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins