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.

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. Mutation of cbe 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, also known as Sauron) 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.

Cytokinesis in Eukaryotic Cells: The Furrow Complexity at a Glance.

The duplication cycle is the fascinating process that, starting from a cell, results in the formation of two daughter cells and it is essential for life. Cytokinesis is the final step of the cell cycle, it is a very complex phase, and is a concert of forces, remodeling, trafficking, and cell signaling. All of the steps of cell division must be properly coordinated with each other to faithfully segregate the genetic material and this task is fundamental for generating viable cells. Given the importance of this process, molecular pathways and proteins that are involved in cytokinesis are conserved from yeast to humans. In this review, we describe symmetric and asymmetric cell division in animal cell and in a model organism, budding yeast. In addition, we illustrate the surveillance mechanisms that ensure a proper cell division and discuss the connections with normal cell proliferation and organs development and with the occurrence of human diseases.

Spindle pole power in health and disease.

Saccharomyces cerevisiae has been widely used as a model system for the study of basic biological processes which are usually evolutionarily conserved from yeasts to multicellular eukaryotes. These studies are very important because they shed light on mechanisms that are altered in human diseases and help the development of new biomarkers and therapies. The mitotic spindle is a conserved apparatus that governs chromosome segregation during mitosis. Given its crucial role for genome stability and, therefore, for cell viability, its structure and function are strictly regulated. Recent findings reveal new levels of regulation in mitotic spindle dynamics and link spindle pole diversification with cell fate determination, health, disease and aging.

Swe1 and Mih1 regulate mitotic spindle dynamics in budding yeast via Bik1.

The mitotic spindle is a very dynamic structure that is built de novo and destroyed at each round of cell division. In order to perform its fundamental function during chromosome segregation, mitotic spindle dynamics must be tightly coordinated with other cell cycle events. These changes are driven by several protein kinases, phosphatases and microtubule-associated proteins. In budding yeast, the kinase Swe1 and the phosphatase Mih1 act in concert in controlling the phosphorylation state of Cdc28, the catalytic subunit of Cdk1, the major regulator of the cell cycle. In this study we show that Swe1 and Mih1 are also involved in the control of mitotic spindle dynamics. Our data indicate that Swe1 and the Polo-like kinase Cdc5 control the balance between phosphorylated and unphosphorylated forms of Mih1, which is, in turn, important for mitotic spindle elongation. Moreover, we show that the microtubule-associated protein Bik1 is a phosphoprotein, and that Swe1 and Mih1 are both involved in controlling phosphorylation of Bik1. These results uncover new players and provide insights into the complex regulation of mitotic spindle dynamics.

Snf1/AMPK is involved in the mitotic spindle alignment in Saccharomyces cerevisiae.

Before anaphase onset, budding yeast cells must align the mitotic spindle parallel to the mother-bud axis to ensure proper chromosome segregation. The protein kinase Snf1/AMPK is a highly conserved energy sensor, essential for adaptation to glucose limitation and in response to cellular stresses. However, recent findings indicate that it plays important functions also in non-limiting glucose conditions. Here we report a novel role of Snf1/AMPK in the progression through mitosis in glucose-repressing condition. We show that active Snf1 is localized to the bud neck from bud emergence to cytokinesis in a septin-dependent manner. In addition, loss of Snf1 induces a delay of the metaphase to anaphase transition that is due to a defect in the correct alignment of the mitotic spindle. In particular, genetic data indicate that Snf1 promotes spindle orientation acting in parallel with Dyn1 and in concert with Kar9. Altogether this study describes a new role for Snf1 in mitosis and connects cellular metabolism to mitosis progression.

Divide Precisely and Proliferate Safely: Lessons From Budding Yeast.

A faithful cell division is essential for proper cellular proliferation of all eukaryotic cells; indeed the correct segregation of the genetic material allows daughter cells to proceed into the cell cycle safely. Conversely, errors during chromosome partition generate aneuploid cells that have been associated to several human pathological conditions, including cancer. Given the importance of this issue, all the steps that lead to cell separation are finely regulated. The budding yeast Saccharomyces cerevisiae is a unicellular eukaryotic organism that divides asymmetrically and it is a suitable model system to study the regulation of cell division. Humans and budding yeast are distant 1 billion years of evolution, nonetheless several essential pathways, proteins, and cellular structures are conserved. Among these, the mitotic spindle is a key player in chromosome segregation and its correct morphogenesis and functioning is essential for genomic stability. In this review we will focus on molecular pathways and proteins involved in the control mitotic spindle morphogenesis and function that are conserved from yeast to humans and whose impairment is connected with the development of human diseases.

COG7 deficiency in Drosophila generates multifaceted developmental, behavioral and protein glycosylation phenotypes.

Congenital disorders of glycosylation (CDG) comprise a family of human multisystemic diseases caused by recessive mutations in genes required for protein N-glycosylation. More than 100 distinct forms of CDGs have been identified and most of them cause severe neurological impairment. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2 and COG4-COG8 cause monogenic forms of inherited, autosomal recessive CDGs. We have generated a Drosophila COG7-CDG model that closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects associated with altered N-glycome profiles. Consistent with these alterations, larval neuromuscular junctions of Cog7 mutants exhibit a significant reduction in bouton numbers. We demonstrate that the COG complex cooperates with Rab1 and Golgi phosphoprotein 3 to regulate Golgi trafficking and that overexpression of Rab1 can rescue the cytokinesis and locomotor defects associated with loss of Cog7. Our results suggest that the Drosophila COG7-CDG model can be used to test novel potential therapeutic strategies by modulating trafficking pathways.

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.

Factors that Control Mitotic Spindle Dynamics.

Mitosis is the last phase of the cell cycle and it leads to the formation of two daughter cells with the same genetic information. This process must occurr in a very precise way and this task is essential to preserve genetic stability and to maintain cell viability. Accurate chromosome segregation during mitosis is brought about by an important cellular organelle: the mitotic spindle. This structure is made of microtubules, polymers of alpha and beta tubulin, and it is highly dynamic during the cell cycle: it emanates from two microtubules organizing centers (Spindle Pole Bodies, SPBs, in yeast) that are essential to build a short bipolar spindle, and it undergoes two steps of elongation during anaphase A and anaphase B in order to separate sister chromatids. Several proteins are involved in the control of mitotic spindle dynamics and their activity is tightly coordinated with other cell cycle events and with cell cycle progression.

Analysis of the Functionality of the Mitotic Checkpoints.

During cell division the main goal of the cell is to produce two daughter cells with the same genome as the mother, i.e., maintain its genetic stability. Since this issue is essential to preserve the cell ability to proliferate properly, all eukaryotic cells have developed several pathways, called mitotic checkpoints, that regulate mitotic entry, progression, and exit in response to different cellular signals. Given the evolutive conservation of mechanisms and proteins involved in the cell cycle control from yeast to humans, the budding yeast S. cerevisiae has been very helpful to gain insight in these complex regulations. Here, we describe how the checkpoint can be activated and which cellular phenotypes can be used as markers of checkpoint activation.

Budding yeast Swe1 is involved in the control of mitotic spindle elongation and is regulated by Cdc14 phosphatase during mitosis.

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr(19)). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G2/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.

Vhs2 is a novel regulator of septin dynamics in budding yeast.

In budding yeast, septins are assembled into structures that undergo dramatic changes during the cell cycle. The molecular mechanisms that drive these remodelings are not fully uncovered. In this study, we describe a characterization of Vhs2, a nonessential protein that revealed to be a new player in septin dynamics. In particular, we report that Vhs2 is important to maintain the stability of the double septin ring structure until telophase. In addition, we show that Vhs2 undergoes multiple phosphorylations during the cell cycle, being phosphorylated during S phase until nuclear division and dephosphorylated just before cell division. Importantly we report that cyclin-dependent protein kinase Cdk1 and protein phosphatase Cdc14 control these Vhs2 post-translational modifications. These results reveal that Vhs2 is a novel Cdc14 substrate that is involved in the control of septin organization.

Saccharomyces cerevisiae Dma proteins participate in cytokinesis by controlling two different pathways.

Cytokinesis completion in the budding yeast S. cerevisiae is driven by tightly regulated pathways, leading to actomyosin ring contraction coupled to plasma membrane constriction and to centripetal growth of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. Here we show that both the lack of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild-type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of the Cyk3-positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis.

Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels and localization.

Timely down-regulation of the evolutionarily conserved protein kinase Swe1 plays an important role in cell cycle control, as Swe1 can block nuclear division through inhibitory phosphorylation of the catalytic subunit of cyclin-dependent kinase. In particular, Swe1 degradation is important for budding yeast cell survival in case of DNA replication stress, whereas it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septin structure. We show that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Moreover, Swe1 is stabilized, restrained at the bud neck, and hyperphosphorylated in dma1Δ dma2Δ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Taken together, the data highlight a previously unknown role of these proteins in the complex regulation of Swe1 and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation.

The spindle position checkpoint: how to deal with spindle misalignment during asymmetric cell division in budding yeast.

During asymmetric cell division, spindle positioning is critical to ensure the unequal segregation of polarity factors and generate daughter cells with different sizes or fates. In budding yeast the boundary between mother and daughter cell resides at the bud neck, where cytokinesis takes place at the end of the cell cycle. Since budding and bud neck formation occur much earlier than bipolar spindle formation, spindle positioning is a finely regulated process. A surveillance device called the SPOC (spindle position checkpoint) oversees this process and delays mitotic exit and cytokinesis until the spindle is properly oriented along the division axis, thus ensuring genome stability.

The spindle position checkpoint in budding yeast: the motherly care of MEN.

Mitotic exit and cytokinesis must be tightly coupled to nuclear division both in time and space in order to preserve genome stability and to ensure that daughter cells inherit the right set of chromosomes after cell division. This is achieved in budding yeast through control over a signal transduction cascade, the mitotic exit network (MEN), which is required for mitotic CDK inactivation in telophase and for cytokinesis. Current models of MEN activation emphasize on the bud as the place where most control is exerted. This review focuses on recent data that instead point to the mother cell as being the residence of key regulators of late mitotic events.