Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin

ZW10 participates in the termination of the spindle checkpoint during mitosis by interacting with dynamitin, a subunit of the dynein accessory complex dynactin. We previously showed that ZW10 is attached to the endoplasmic reticulum through RINT-1 in interphase HeLa cells and involved in membrane transport between the endoplasmic reticulum and Golgi. Although a recent study demonstrated that ZW10 is localized in the Golgi in COS7 cells, the mechanism that regulates ZW10 localization remains unknown. In this study we showed a correlation between the Golgi localization of ZW10 and the centrosomal accumulation of dynactin. The amounts of ZW10 associated with dynactin were larger in cells where ZW10 was present in the Golgi than those where ZW10 was not in the Golgi. The targeting of ZW10 to the perinuclear Golgi region was found to depend on the perinuclear accumulation of dynactin, suggesting that dynactin regulates ZW10 localization.     

The interaction of two tethering factors, p115 and COG complex, is required for Golgi integrity

The vesicle-tethering protein p115 functions in endoplasmic reticulum-Golgi trafficking. We explored the function of homologous region 2 (HR2) of the p115 head domain that is highly homologous with the yeast counterpart, Uso1p. By expression of p115 mutants in p115 knockdown (KD) cells, we found that deletion of HR2 caused an irregular assembly of the Golgi, which consisted of a cluster of mini-stacked Golgi fragments, and gathered around microtubule-organizing center in a microtubule-dependent manner. Protein interaction analyses revealed that p115 HR2 interacted with Cog2, a subunit of the conserved oligomeric Golgi (COG) complex that is known another putative cis-Golgi vesicle-tethering factor. The interaction between p115 and Cog2 was found to be essential for Golgi ribbon reformation after the disruption of the ribbon by p115 KD or brefeldin A treatment and recovery by re-expression of p115 or drug wash out, respectively. The interaction occurred only in interphase cells and not in mitotic cells. These results strongly suggested that p115 plays an important role in the biogenesis and maintenance of the Golgi by interacting with the COG complex on the cis-Golgi in vesicular trafficking.     

Interaction of Golgin‐84 with the COG Complex Mediates the Intra‐Golgi Retrograde Transport

The coiled‐coil Golgi membrane protein golgin‐84 functions as a tethering factor for coat protein I (COPI) vesicles. Protein interaction analyses have revealed that golgin‐84 interacts with another tether, the conserved oligomeric Golgi (COG) complex, through its subunit Cog7. Therefore, we explored the function of golgin‐84 as the tether for COPI vesicles of intra‐Golgi retrograde traffic. First, glycosylic maturation of both plasma membrane (CD44) and lysosomal (lamp1) glycoproteins was distorted in golgin‐84 knockdown (KD) cells. The depletion of golgin‐84 caused fragmentation of the Golgi with the mislocalization of Golgi resident proteins, resulting in the accumulation of vesicles carrying intra‐Golgi soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and cis‐Golgi membrane protein GPP130. Similar observations were obtained by diminution of the COG complex, suggesting a strong correlation between the two tethers. Indeed, COG complex‐dependent (CCD) vesicles that accumulate in Cog3 or Cog7 KD cells carried golgin‐84. Surprisingly, the interaction between golgin‐84 and another candidate tethering partner CASP (CDP/cut alternatively spliced product) decreased in Cog3 KD cells. These results indicate that golgin‐84 on COPI vesicles interact with the COG complex before SNARE assembly, suggesting that the interaction of golgin‐84 with COG plays an important role in the tethering process of intra‐Golgi retrograde vesicle traffic.     

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).     

Rab29 activation of the Parkinson's disease‐associated LRRK2 kinase

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector‐binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans‐Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild‐type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29‐mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2‐mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.     

蛋白的高尔基体定位

高尔基体既是蛋白质修饰、分选、水解加工的场所,又是分泌物质的转运站,每时每刻都有大量的蛋白进出高尔基体。在这种情况下,高尔基体仍能保持完整且高度有序的结构,表明高尔基体驻留蛋白有精确的定位信号,以保证它们定位于正确的区隔,而不会沿着分泌途径被运输出去。高尔基体内有几种不同类别的膜蛋白,包括糖基转移酶、周缘膜蛋白、病毒蛋白和受体等。研究显示,有多种定位信号和定位机制参与了蛋白的高尔基体定位。     

Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes

In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.     

Ultrastructural changes in the Golgi apparatus and secretory granules of HL-60 cells treated with the imino sugar N-butyldeoxynojirimycin

The imino sugar N-butyldeoxynojirimycin inhibits the N-linked oligosaccharide processing enzymes α-glucosidases I and II, and the ceramide specific glucosyltransferase which catalyses the first step in glucosphingolipid biosynthesis. We have studied the effects of this compound on the ultrastructure of HL-60 cells to identify novel activities of this compound. Treatment of HL-60 cells with this imino sugar results in several morphological changes within the cell, none of which result in cytotoxicity. The plasma membrane stains heavily with potassium ferrocyanide within 30 min following addition of the compound to the medium, and there is then a time dependent involvement of all other intracellular membranes. Secretory granules become enlarged and lose their dense core morphology and appear either empty and vacuolated or have low density contents. However, the most striking effect of NB-DNJ treatment is on the Golgi apparatus. The Golgi exhibits a time-dependent change from typical Golgi morphology to a structure almost completely devoid of cisternae and consisting predominantly of vesicles. All the observed changes are fully reversible on withdrawal of the compound.     

1998: The centenary of the discovery of the Golgi apparatus

1998 is the year of the centenary of the discovery of the Golgi apparatus. This event is considered in its historical context: the first cell theory of 1838–1839, the first polemics in cytology and the research on the cell organelles at the turn of the century. The first approaches to clarify the physiological significance of the apparatus is traced from Golgi (1909) to Bowen (1929).     

Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.     

Differentiation of binuclear spermatozoa in mice

In an electron microscopy study of abnormal spermatogenesis in mice, we have found that two discrete haploid nuclei may be located in a single spermatid cytoplasm after the second meiotic division. The spermatid continues to differentiate and forms a binucleate spermatozoon with both nuclei separately packaged within the sperm head. The Golgi apparatus of the double spermatid forms a single proacrosome that attaches to both nuclei. Apparently, one acrosomal structure differentiates to cover and compartmentalize the two haploid nuclei within the sperm head. Chromatin condensation appears normal. The head morphology and number of flagella vary in mature spermatozoa produced by this process. This work demonstrates one pathway by which polyploid spermatids continue to differentiate to spermatozoa after failure of cytoplasmic division or possibly cellular fusion.