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At least 1,063 records · Page 59Linked to original sources

PubMed2005

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs.

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.

Animals↗

PubMed2004

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus.

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).

Animals↗

PubMed2002

Isolation and characterization of Golgi apparatus-specific GODZ with the DHHC zinc finger domain.

We identified a novel Golgi apparatus-specific protein with the DHHC zinc finger domain and four putative transmembrane regions, designated as GODZ. The amino acid sequences were highly conserved among mouse and human GODZs and homologous proteins in human, mouse, rat, Drosophila melanogaster, and Caenorhabditis elegans, implying a functional significance of the GODZ protein family. Overexpression of mouse GODZ in COS7 cells suppressed the sorting of the glutamate receptor GluRalpha1 from the Golgi apparatus. These results suggest that GODZ plays a role in the membrane protein trafficking.

Amino Acid Sequence↗

PubMed2003

The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference.

The secretory-pathway Ca(2+)-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca(2+) pump found mostly in the Golgi compartment. We have explored the contribution of this Ca(2+) pump to cytosolic Ca(2+) signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca(2+) concentration in the lumen of the Golgi apparatus by specifically targeting the Ca(2+)-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca(2+) uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca(2+) spiking when stimulated with histamine, indicating that the SPCA1-containing Ca(2+) store was not absolutely needed to set up these oscillations. However, baseline Ca(2+) oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca(2+) signal in HeLa cells.

Calcium↗

PubMed2003

Targeting of the AE2 anion exchanger to the Golgi apparatus is cell type-dependent and correlates with the expression of Ank(195), a Golgi membrane skeletal protein.

Sodium-independent anion exchangers (AE1-4) show remarkable variability in their tissue-specific expression and subcellular localization. Currently, isoform-specific targeting mechanisms are considered to be responsible for this variable localization. Here, we report that targeting can also be cell type-specific. We show that the full-length AE2 protein and its green fluorescent protein- or DsRed-tagged variants localize predominantly either to the Golgi apparatus in COS-7 cells, or to the plasma membrane in HeLa cells. This alternative targeting did not seem to result from either translational or post-translational differences, but rather from differential expression of at least one of the Golgi membrane skeletal proteins, ankyrin(195) (Ank(195)), between the two cell types. Comparative studies with several different cell lines revealed that the Golgi localization of the AE2 protein correlated strictly with the expression of Ank(195) in the cells. The two Golgi-associated proteins also co-localized well and similarly resisted detergent extraction in the cold, whereas the plasma membrane-localized AE2 in Ank(195)-deficient cells was mostly detergent-soluble. Collectively, our results suggest that Ank(195) expression is a key determinant for the variable and cell type-dependent localization of the AE2 protein in the Golgi apparatus in mammalian cells.

Animals↗

PubMed2004

The AE2 anion exchanger is necessary for the structural integrity of the Golgi apparatus in mammalian cells.

The structural integrity of the Golgi apparatus is known to be dependent on multiple factors, including the organizational status of microtubules, actin and the ankyrin/spectrin-based Golgi membrane skeleton, as well as vesicular trafficking and pH homeostasis. In this respect, our recently identified Golgi-associated anion exchanger, AE2, may also be of importance, since it potentially acts as a Golgi pH regulator and as a novel membrane anchor for the spectrin-based Golgi membrane skeleton. Here, we show that inhibition (>75%) of AE2 expression by antisense oligonucleotides in COS-7 cells results in the fragmentation of the juxtanuclear Golgi apparatus and in structural disorganization of the Golgi stacks, the cisternae becoming generally shorter, distorted, vesiculated and/or swollen. These structural changes occurred without apparent dissociation of the Golgi membrane skeletal protein Ankyrin(195), but were accompanied by the disappearance of the well-focused microtubule-organizing center (MTOC), suggesting the involvement of microtubule reorganization. Similar changes in Golgi structure and assembly of the MTOC were also observed upon transient overexpression of the EGFP-AE2 fusion protein. These data implicate a clear structural role for the AE2 protein in the Golgi and in its cytological positioning around the MTOC.

Animals↗

PubMed1998

NSF is required for the brefeldin A-promoted disassembly of the Golgi apparatus.

N-Ethylmaleimide-sensitive factor (NSF) is required for multiple pathways of vesicle-mediated protein transport. Microinjection of a monoclonal anti-NSF antibody almost completely blocked brefeldin A-promoted Golgi disassembly without affecting the rapid release of beta-COP, a subunit of the Golgi coat proteins (COPI), from the Golgi apparatus. Similar results were obtained using a dominant-negative NSF which is known to compete with endogenous NSF. The present results suggest that an NSF-mediated step is present in the brefeldin A-promoted disassembly of the Golgi apparatus.

Animals↗

PubMed1999

The N-terminal 77 amino acids from tobacco N-acetylglucosaminyltransferase I are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells.

In order to investigate sequences of tobacco N-acetylglucosaminyltransferase I (GnTI), involved in targeting to and retention in the plant Golgi apparatus the cytoplasmic transmembrane stem (CTS) region of the enzyme was cloned in frame with the cDNA of the green fluorescent protein (gfp) and subsequently transiently expressed in Nicotiana benthamiana plants using a tobacco mosaic virus (TMV) based expression vector. Confocal laser scanning microscopy showed small fluorescent vesicular bodies in CTS-gfp expressing cells, while gfp alone expressed in control plants was uniformly distributed in the cytoplasm. The CTS-gfp fusion protein colocalised with immunolabelling observed by an antibody specific for the Golgi located plant Lewis a epitope. Furthermore, treatment with brefeldin A, a Golgi specific drug, resulted in the formation of large fluorescent vesiculated areas. These results strongly suggest a Golgi location for CTS-gfp and as a consequence our findings reveal that the N-terminal 77 amino acids of tobacco GnTI are sufficient to target to and to retain a reporter protein in the plant Golgi apparatus and that TMV based vectors are suitable vehicles for rapid delivery of recombinant proteins to the secretory pathway.

Biological Transport↗

PubMed1999

A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis.

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h-warts, which was mapped at chromosome 6q24-25.1. The h-warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h-warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h-warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

Amino Acid Sequence↗

PubMed2000

Aggregation of ubiquitin and a mutant ALS-linked SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus.

Transgenic mice that express the G93A mutation of human Cu,Zn superoxide dismutase (SOD1(G93A)), found in familial amyotrophic lateral sclerosis (FALS), showed clinical symptoms and histopathological changes of sporadic ALS, including fragmentation of the neuronal Golgi apparatus (GA). The finding of fragmented neuronal GA in asymptomatic mice, months before the onset of paralysis, suggests that the GA is an early target of the pathological processes causing neuronal degeneration. Transgenic mice expressing human SOD1(G93A) have aggregates of mutant protein and ubiquitin in neuronal and glial cytoplasm; they appeared first in the neuropil and later in the perikarya of motor neurons, where they were adjacent to fragmented GA. The aggregates of SOD1(G93A) appeared in neuronal perikarya of asymptomatic mice containing fragmented GA. The numbers of neurons with deposits of SOD1(G93A) and fragmented GA progressively increased with age. Immuno-electron microscopy using colloidal gold showed labeling of ubiquitin and SOD1 over 13 nm thick cytoplasmic filaments. Spinal cord extracts showed a 20-fold increase of SOD1(G93A) in transgenic mice compared to the wild-type protein in controls. The results suggest a causal relationship between the aggregation of mutant SOD1 and ubiquitin, fragmentation of the Golgi apparatus of motor neurons and neurodegeneration.

Amyotrophic Lateral Sclerosis↗

PubMed1996

A new possible parameter for the detection of aneuploidy inducing substances: the analysis of qualitative and quantitative abnormalities of the spindle apparatus.

In the present study, compared to other cytogenetic methods, we measured the number of aneuploid cells directly by analyzing anomalies of the mitotic spindle. Qualitative and quantitative abnormalities of the mitotic spindle apparatus in transformed and non-transformed cell lines in vitro were classified. We treated the different cell lines with well known aneugenic agents as Benomyl and Griseofulvin and investigated the mitotic spindle under different experimental conditions. The spindle apparatus was stained by indirect immunofluorescence and the chromatin was counterstained by fluorescent dyes. The mitotic spindle showed a great sensitivity to the aneuploidy-inducing substances used in our experiments. The spindle-disturbing effect of the tested substances was demonstrated to be dose- dependent. The morphological alterations appeared to be independent of the aneuploidy-inducing test substance used, but showed a relation to the dose and length of treatment. Thus, the analysis of the mitotic spindle may be a useful screening parameter for the detection of aneuploidy-inducing substances and further investigations will provide additional results to specific parameters.

Aneuploidy↗

PubMed1998

Inheritance of the mammalian Golgi apparatus during the cell cycle.

The creation and propagation of the intricate Golgi architecture during the cell cycle poses a fascinating problem for biologists. Similar to the inheritance process for nuclear DNA, the inheritance of the Golgi apparatus consists of biogenesis (replication) and partitioning (mitosis/meiosis) phases, in which Golgi components must double in unit mass, then be appropriately divided between nascent daughter cells during cytokinesis. In this article we focus discussion on the recent advances in the area of Golgi inheritance, first outlining our current understanding of the behaviour of the Golgi apparatus during cell division, then concluding with a more conceptual discussion of the Golgi biogenesis problem. Throughout, we attempt to integrate ultrastructural and biochemical findings with more recent information obtained using live cell microscopy and morphological techniques.

Animals↗

PubMed1999

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.

Acylation↗

PubMed1986

An ultrastructural study of the renal juxtaglomerular apparatus and extraglomerular mesangium in patients with systemic lupus erythematosus.

Experimental studies have indicated that the glomerular mesangium may function by phagocytosis of various circulating substances which are then processed and flow to the juxtaglomerular apparatus (JGA) region and into the intercellular spaces of the macula densa. An electron-micrographic study of the juxtaglomerular apparatus and extramesangial region in systemic lupus erythematosus (SLE) was undertaken. In only five of 39 cases (13%) of SLE were discrete electron-dense "immune-type" deposits noted in the JGA region. In four of those patients, there were also a large number of variously-sized electron-dense deposits in the glomerular mesangial and capillary wall regions, Bowman's capsule, tubular basement membranes, renal interstitium, or in the nearby periglomerular arterioles. The paucity of demonstrable discrete electron-dense deposits in the JGA regions in this study suggests that the deposits noted in SLE in humans may not be commonly processed in this fashion, or alternatively, that the discrete electron-dense deposits are processed and undergo lysis within the glomerular mesangial regions before they are transported to the extraglomerular mesangial regions of the JGA.

Adolescent↗

PubMed1990

What's new in cytoskeleton-organelle interactions? Relationship between microtubules and the Golgi-apparatus.

Several biochemical processes in animal cells are confined to distinct membrane-bounded compartments. Segregation of specialized functions into different compartments necessitates intercompartment transfer of material. This transfer is mediated by carrier vesicles which, by precise sorting and transport mechanisms, are targetted to their correct destinations. Microtubules, major constituents of the cytoskeleton, are involved both in these intracellular transport processes and in the spatial organization of cytoplasmic organelles. Accumulating evidence suggests that various classes of membranous organelles interact with microtubules. The positioning of several organelles, including the Golgi apparatus and lysosomes, depends on an intact interphase microtubule network. Furthermore, it has been shown that many of these organelles, for example Golgi elements, tubules of the endoplasmic reticulum, exocytic or secretory vesicles and lysosomes move along microtubules. In this article we will discuss the role of microtubules in the movement and positioning of elements of the Golgi complex. The first part will summarize structural and functional aspects of microtubules and the Golgi apparatus and review evidence for their interaction. In the second part, the possible physiological relevance of this interaction will be discussed and correlated with other membrane-microtubule interactions. Finally, emerging questions and perspectives in this field are outlined.

Animals↗

PubMed1980

Morphology of the Golgi apparatus in streptozotocin-diabetic rat liver.

The altered morphology of the Golgi apparatus in streptozotocin-diabetic rat liver is described in situ and in preparations of the Golgi-membrane-enriched fraction. The main differences, as compared with controls, are a diminished Golgi apparatus area, the rounding of stacks of cisternae and the absence of secretory vacuoles with VLDL granules. These findings are discussed in view of biochemical alterations found previously in the Golgi-membrane-enriched fraction isolated from streptozotocin-diabetic rat liver.

Animals↗

PubMed2000

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells.

Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum <mitochondria<Golgi apparatus<lysosomes and endosomes< plasma membranes. Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.

Animals↗

PubMed1990

Three-dimensional observation of the Golgi apparatus in rat tracheal gland during the secretory cycle.

The purpose of the present work is three-dimensional clarification of the overall configuration of the Golgi apparatus in rat tracheal serous cells including the sequence of structural modification during the secretory cycle induced by pilocarpine. From the results of both transmission electron microscopy from the serial sections, and scanning electron microscopy using the osmium-DMSO-osmium method based on maceration in dilute osmium tetroxide, the overall configuration of the Golgi apparatus shows that the Golgi stacks are joined together to make an interconnected, single Golgi complex in a cell. Connections, not only between the TGN (trans Golgi network) and rough ER (endoplasmic reticulum), but also between rough ER and cis cisternae are observed. This finding offers alternative routes for movement of substances through the cellular endomembrane system. During the secretory cycle induced by pilocarpine injection, it is evidenced that the disappearance of the TGN is accompanied by rapid discharge of secretory granules and the reconstitution of the TGN is followed by reformation of secretory materials. The beginning of the reconstitution of the TGN is indicated by vesicular buds from the sacculotubular element. These results indicate that the TGN participates directly in membrane recycling and in formation of secretory granules.

Animals↗