FIP-2, an IkappaB-kinase-gamma-related protein,is associated with the Golgi apparatus and translocates to the marginal band during chicken erythroblast differentiation

Using an antiserum directed against marginal band associated proteins of chicken erythrocytes we isolated clones encoding the chicken homolog of 14.7K-interacting protein 2 (FIP-2), a protein potentially involved in tumor necrosis factor-alpha/nuclear factor-kappaB signaling, from a chicken erythroblast cDNA library. We found that chicken FIP-2 was expressed in a variety of tissues and cell types, but unlike its human counterpart, alternative splicing does not appear to take place. Analysis of intracellular localization revealed that FIP-2 was concentrated at the Golgi apparatus in most cells. Perturbation of the Golgi structure without loss of Golgi function (by treatment with nocodazole) resulted in a retention of FIP-2 at the dispersed Golgi fragments. In contrast, disruption of both Golgi structure and function (by brefeldin A) led to a loss of FIP-2 from Golgi membranes. Remarkably, during erythroblast differentiation FIP-2 was found to translocate from the Golgi to the marginal band. Our results support the hypothesis of a function of the Golgi apparatus in signal transduction. Moreover, our results raise the possibility that the marginal band of chicken erythrocytes, in addition to its role in morphogenesis, has a function in signal transduction and that FIP-2 is in some way involved in its formation.     

A novel Rho-like protein TbRHP is involved in spindle formation and mitosis in trypanosomes

Background

In animals and fungi Rho subfamily small GTPases are involved in signal transduction, cytoskeletal function and cellular proliferation. These organisms typically possess multiple Rho paralogues and numerous downstream effectors, consistent with the highly complex contributions of Rho proteins to cellular physiology. By contrast, trypanosomatids have a much simpler Rho-signaling system, and the Trypanosoma brucei genome contains only a single divergent Rho-related gene, TbRHP (Tb927.10.6240). Further, only a single RhoGAP-like protein (Tb09.160.4180) is annotated, contrasting with the >70 Rho GAP proteins from Homo sapiens. We wished to establish the function(s) of TbRHP and if Tb09.160.4180 is a potential GAP for this protein.

Methods/Findings

TbRHP represents an evolutionarily restricted member of the Rho GTPase clade and is likely trypanosomatid restricted. TbRHP is expressed in both mammalian and insect dwelling stages of T. brucei and presents with a diffuse cytoplasmic location and is excluded from the nucleus. RNAi ablation of TbRHP results in major cell cycle defects and accumulation of multi-nucleated cells, coinciding with a loss of detectable mitotic spindles. Using yeast two hybrid analysis we find that TbRHP interacts with both Tb11.01.3180 (TbRACK), a homolog of Rho-kinase, and the sole trypanosome RhoGAP protein Tb09.160.4180, which is related to human OCRL.

Conclusions

Despite minimization of the Rho pathway, TbRHP retains an important role in spindle formation, and hence mitosis, in trypanosomes. TbRHP is a partner for TbRACK and an OCRL-related trypanosome Rho-GAP.     

Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells.

Osmium impregnation was used to determine the number of Golgi apparatus in both interphase and mitotic HeLa cells. The number was found to increase substantially during mitosis to the point where random partitioning alone would explain the nearly equal numbers found in each daughter cell.     

Inositol phosphorylceramide synthase is located in the Golgi apparatus of Saccharomyces cerevisiae

The plasma membrane of eukaryotic cells differs in lipid composition from most of the internal organelles, presumably reflecting differences in many of its functions. In particular, the plasma membrane is rich in sphingolipids and sterols, one property of which is to decrease the permeability and increase the thickness of lipid bilayers. In this paper, we examine the length of transmembrane domains throughout the yeast secretory pathway. Although the transmembrane domains of cis and medial Golgi residents are similar to those of endoplasmic reticulum proteins, these domains lengthen substantially beyond the medial Golgi, suggesting a thickening of the bilayer. Yeast sphingolipids have particularly long acyl chains, and Aur1p, the inositol phosphorylceramide synthase that initiates yeast sphingolipid synthesis, was found to be located in the Golgi apparatus by both immunofluorescence and membrane fractionation, with its active site apparently in the Golgi lumen. Thus, it appears that sphingolipid synthesis in yeast takes place in the Golgi, separated from glycerophospholipid synthesis in the endoplasmic reticulum. A similar separation has been found in mammalian cells, and this conservation suggests that such an arrangement of enzymes within the secretory pathway could be important for the creation of bilayers of different thickness within the cell.     

A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this "internal" matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as "struts" stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.     

A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus

SAR1, a gene that has been isolated as a multicopy suppressor of the yeast ER-Golgi transport mutant sec12, encodes a novel GTP-binding protein. Its nucleotide sequence predicts a 21-kD polypeptide that contains amino acid sequences highly homologous to GTP-binding domains of many ras-related proteins. Gene disruption experiments show that SAR1 is essential for cell growth. To test its function further, SAR1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SAR1 copy disrupted. This mutant grows normally in galactose medium but arrests growth 12-15 h after transfer to glucose medium. At the same time, mutant cells accumulate ER precursor forms of a secretory pheromone, alpha-mating factor, and a vacuolar enzyme, carboxypeptidase Y. We propose that Sec12p and Sarlp collaborate in directing ER-Golgi protein transport.     

The abnormal spindle protein is required for germ cell mitosis and oocyte differentiation during Drosophila oogenesis

In this study, we present evidence that the asp function is required in oogenesis for germline cell divisions as well as for cyst polarity and oocyte differentiation. Consistent with previously described roles in spindle organization during Drosophila meiosis and mitosis, asp mutation leads to severe defects in spindle microtubule organization within the germarium. The mitotic spindles of the mutant cystocytes are composed by wavy microtubules and have abnormal poles that often lack gamma-tubulin. The fusome structure is also compromised. In the absence of asp function, the cystocyte divisions fail resulting in egg chamber with fewer than 16 germ cells. Moreover, the microtubule network within the developing germline cysts may assemble incorrectly in turn affecting the microtubule based transport of the specific determinants that is required during mid-oogenesis for the oocyte differentiation program.     

The cilia protein IFT88 is required for spindle orientation in mitosis

Cilia dysfunction has long been associated with cyst formation and ciliopathies. More recently, misoriented cell division has been observed in cystic kidneys, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells. Several IFT proteins also localize to spindle poles in mitosis, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737(orpk) and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies.     

RAF1-activated MEK1 is found on the Golgi apparatus in late prophase and is required for Golgi complex fragmentation in mitosis

Amitotically activated mitogen-activated protein kinase 1 (MEK1) fragments the pericentriolar Golgi stacks in mammalian cells. We show that activated MEK1 is found on the Golgi apparatus in late prophase. The fragmented and dispersed Golgi membranes in prometaphase and later stages of mitosis do not contain activated MEK1. MEK1-dependent Golgi complex fragmentation is through activation by RAF1 and not MEK1 kinase 1. We propose that a RAF1-dependent activation of MEK1 and its presence on the Golgi apparatus in late prophase is required for Golgi complex fragmentation.     

Newly synthesized acetylcholine receptors are located in the Golgi apparatus

Chick skeletal muscle cells in tissue culture were fixed and treated with saponin to allow [125I]alpha-bungarotoxin access into the cells while preserving ultrastructure. The kinetics of binding of iodinated alpha-bungarotoxin to intracellular acetylcholine (ACh) receptors and to surface A Ch receptors were comparable. About half of the intracellular ACh receptors are newly synthesized and in the pathway leading to incorporation into the plasma membrane. Correlated electron microscope autoradiographic and kinetic studies of this receptor population suggest that a substantial fraction of the newly synthesized ACh receptors are located in the Golgi apparatus, where they reside for approx. 2 h.     

Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis

The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.     

The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus.

The formation and maintenance of the bipolar mitotic spindle apparatus require a complex and balanced interplay of several mechanisms, including the stabilization and separation of polar microtubules and the action of various microtubule motors. Nonmicrotubule elements are also present throughout the spindle apparatus and have been proposed to provide a structural support for the spindle. The Nuclear-Mitotic Apparatus protein (NuMA) is an abundant 240 kD protein that is present in the nucleus of interphase cells and concentrates in the polar regions of the spindle apparatus during mitosis. Sequence analysis indicates that NuMA possesses an unusually long alpha-helical central region characteristic of many filament forming proteins. In this report we demonstrate that microinjection of anti-NuMA antibodies into interphase and prophase cells results in a failure to form a mitotic spindle apparatus. Furthermore, injection of metaphase cells results in the collapse of the spindle apparatus into a monopolar microtubule array. These results identify for the first time a nontubulin component important for both the establishment and stabilization of the mitotic spindle apparatus in multicellular organisms. We suggest that nonmicrotubule structural components may be important for these processes.     

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.     

Skeletor, a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix

A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.     

A novel 58-kDa protein associates with the Golgi apparatus and microtubules

With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo function of 58K is to provide an anchorage site for microtubules on the outer surface of the Golgi.     

Identification of a novel protein (G210) specific to the Golgi apparatus

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.     

Synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of Golgi apparatus

Rat liver membranes were subjected to centrifugation in a sucrose density gradient in which the Golgi apparatus was separated into several subfractions. Two enzymes involved in the synthesis of the phosphorylated recognition marker in lysosomal enzymes, UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase fractionated with alpha-1,2-mannosidase, a marker enzyme of cis Golgi membranes and differently from galactosyltransferase, a marker enzyme of trans Golgi membranes.