Inheriting a structural scaffold for Golgi biosynthesis

In animal cells, the Golgi complex undergoes reversible disassembly during mitosis. The disassembly/reassembly process has been intensively studied in order to understand the mechanisms that govern organelle assembly and inheritance during cell division. A long-standing controversy in the field has been whether formation of Golgi structure is template-mediated or self-organizes from components of the endoplasmic reticulum. A recent study1 however, has demonstrated that a subset of proteins that form a putative Golgi matrix can be inherited during cell division in the absence of membrane input from the endoplasmic reticulum. The outcome of this study suggests that a templating mechanism for the formation of Golgi structure may exist. This study has important implications for understanding mechanisms that govern Golgi biogenesis.     

Golgi inheritance under a block of anterograde and retrograde traffic

In mitosis, the Golgi complex is inherited following its dispersion, equal partitioning and reformation in each daughter cell. The state of Golgi membranes during mitosis is controversial, and the role of Golgi-intersecting traffic in Golgi inheritance is unclear. We have used brefeldin A (BFA) to perturb Golgi-intersecting membrane traffic at different stages of the cell cycle and followed by live cell imaging the fate of Golgi membranes in those conditions. We observed that addition of the drug on cells in prometaphase prevents mitotic Golgi dispersion. Under continuous treatment, Golgi fragments persist throughout mitosis and accumulate in a Golgi-like structure at the end of mitosis. This structure localizes at microtubule minus ends and contains all classes of Golgi markers, but is not accessible to cargo from the endoplasmic reticulum or the plasma membrane because of the continuous BFA traffic block. However, it contains preaccumulated cargo, and intermixes with the reforming Golgi upon BFA washout. This structure also forms when BFA is added during metaphase, when the Golgi is not discernible by light microscopy. Together the data indicate that independent Golgi fragments that contain all classes of Golgi markers (and that can be isolated from other organelles by blocking anterograde and retrograde Golgi-intersecting traffic) persist throughout mitosis.     

Inheritance of the endoplasmic reticulum and Golgi apparatus

Yeast and mammalian cells use a variety of different mechanisms to ensure that the endoplasmic reticulum and Golgi apparatus are inherited by both daughter cells on cell division. In yeast, endoplasmic reticulum inheritance involves both active microtubule and passive actin-based mechanisms, while the Golgi is transported into the forming daughter cell by an active actin-based mechanism. Animal cells actively partition the endoplasmic reticulum and Golgi apparatus, but association with the mitotic spindle-rather than the actin cytoskeleton-appears to be the mechanism     

An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.     

Golgi apparatus partitioning during cell division

This review discusses the mitotic segregation of the Golgi apparatus. The results from classical biochemical and morphological studies have suggested that in mammalian cells this organelle remains distinct during mitosis, although highly fragmented through the formation of mitotic Golgi clusters of small tubules and vesicles. Shedding of free Golgi-derived vesicles would consume Golgi clusters and disperse this organelle throughout the cytoplasm. Vesicles could be partitioned in a stochastic and passive way between the two daughter cells and act as a template for the reassembly of this key organelle. This model has recently been modified by results obtained using GFP- or HRP-tagged Golgi resident enzymes, live cell imaging and electron microscopy. Results obtained with these techniques show that the mitotic Golgi clusters are stable entities throughout mitosis that partition in a microtubule spindle-dependent fashion. Furthermore, a newer model proposes that at the onset of mitosis, the Golgi apparatus completely loses its identity and is reabsorbed into the endoplasmic reticulum. This suggests that the partitioning of the Golgi apparatus is entirely dependent on the partitioning of the endoplasmic reticulum. We critically discuss both models and summarize what is known about the molecular mechanisms underlying the Golgi disassembly and reassembly during and after mitosis. We will also review how the study of the Golgi apparatus during mitosis in other organisms can answer current questions and perhaps reveal novel mechanisms.     

Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities

Golgi inheritance during mammalian cell division occurs through the disassembly, partitioning, and reassembly of Golgi membranes. The mechanisms responsible for these processes are poorly understood. To address these mechanisms, we have examined the identity and dynamics of Golgi proteins within mitotic membranes using live cell imaging and electron microscopy techniques. Mitotic Golgi fragments, seen in prometaphase and telophase, were found to localize adjacent to endoplasmic reticulum (ER) export domains, and resident Golgi transmembrane proteins cycled rapidly into and out of these fragments. Golgi proteins within mitotic Golgi haze-seen during metaphase-were found to redistribute with ER markers into fragments when the ER was fragmented by ionomycin treatment. The temperature-sensitive misfolding mutant ts045VSVG protein, when localized to the Golgi at the start of mitosis, became trapped in the ER at the end of mitosis in cells shifted to 40 degrees C. Finally, reporters for Arf1 and Sar1 activity revealed that Arf1 and Sar1 undergo sequential inactivation during mitotic Golgi breakdown and sequential reactivation upon Golgi reassembly at the end of mitosis. Together, these findings support a model of mitotic Golgi inheritance that involves inhibition and subsequent reactivation of cellular activities controlling the cycling of Golgi components into and out of the ER.     

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.     

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.     

Capacity of the Golgi apparatus for cargo transport prior to complete assembly

In yeast, particular emphasis has been given to endoplasmic reticulum (ER)-derived, cisternal maturation models of Golgi assembly while in mammalian cells more emphasis has been given to golgins as a potentially stable assembly framework. In the case of de novo Golgi formation from the ER after brefeldin A/H89 washout in HeLa cells, we found that scattered, golgin-enriched, structures formed early and contained golgins including giantin, ranging across the entire cis to trans spectrum of the Golgi apparatus. These structures were incompetent in VSV-G cargo transport. Second, we compared Golgi competence in cargo transport to the kinetics of addition of various glycosyltransferases and glycosidases into nascent, golgin-enriched structures after drug washout. Enzyme accumulation was sequential with trans and then medial glycosyltransferases/glycosidases found in the scattered, nascent Golgi. Involvement in cargo transport preceded full accumulation of enzymes or GPP130 into nascent Golgi. Third, during mitosis, we found that the formation of a golgin-positive acceptor compartment in early telophase preceded the accumulation of a Golgi glycosyltransferase in nascent Golgi structures. We conclude that during mammalian Golgi assembly components fit into a dynamic, first-formed, multigolgin-enriched framework that is initially cargo transport incompetent. Resumption of cargo transport precedes full Golgi assembly.     

Unraveling the Golgi ribbon

The Golgi apparatus lies at the heart of the secretory pathway where it receives, modifies and sorts protein cargo to the proper intracellular or extracellular location. Although this secretory function is highly conserved throughout the eukaryotic kingdom, the structure of the Golgi complex is arranged very differently among species. In particular, Golgi membranes in vertebrate cells are integrated into a single compact entity termed the Golgi ribbon that is normally localized in the perinuclear area and in close vicinity to the centrosomes. This organization poses a challenge for cell division when the single Golgi ribbon needs to be partitioned into the two daughter cells. To ensure faithful inheritance in the progeny, the Golgi ribbon is divided in three consecutive steps in mitosis, namely disassembly, partitioning and reassembly. However, the structure of the Golgi ribbon is only present in higher animals and Golgi disassembly during mitosis is not ubiquitous in all organisms. Therefore, there must be unique reasons to build up the Golgi in this particular conformation and to preserve it over generations. In this review, we first highlight the diversity of the Golgi architecture in different organisms and revisit the concept of the Golgi ribbon. Following on, we discuss why the ribbon is needed and how it forms in vertebrate cells. Lastly, we conclude with likely purposes of mitotic ribbon disassembly and further propose mechanisms by which it regulates mitosis.     

花生胚乳细胞化的超微结构观察

花生（ＡｒａｃｈｉｓｈｙｐｏｇｅａｅＬ．）心形胚期的胚乳游离核多瓣裂,或具长尾状结构。胚乳细胞质内有大量线粒体、质体、高尔基体、小泡及少量内质网。中央细胞壁有壁内突。球胚及心形胚期常见胚乳瘤。心形胚晚期,胚乳开始细胞化,胚乳细胞壁形成有３种方式,分别存在于不同的胚珠中：（１）从胚囊壁产生自由生长壁形成初始垂周壁,具有明显的电子密度深的中层,其生长主要靠末端的高尔基体小泡及内质网囊泡的融合。两相邻的自由生长壁末端或其分枝末端相连形成胚乳细胞。（２）核有丝分裂后产生细胞板,细胞板向外扩展并可分枝。间期的非姊妹核间也观察到形成了细胞板。小泡与微管参与细胞板的扩展,高尔基体和内质网是小泡的主要来源。细胞板的扩展末端相互连接,形成胚乳细胞的前身。小泡继续加入细胞板的组成,以后形成胚乳细胞壁。（３）胚乳细胞质中,出现一些比较大的不规则形的片段性泡状结构,它们可能来源于高尔基体小泡,这些片段性泡状结构随机相连形成细胞壁,未见微管参与。胚乳细胞外切向壁及经向壁上有壁内突。     

Demonstration by light and ultrastructural immunoperoxidase study of alpha-fetoprotein-positive non-hepatoma cells and hepatoma cells during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis

Immunoreaction of alpha-fetoprotein (AFP) has been described in cholangiolar "oval" cells in the early stage of 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in the oval cells was in the perinuclear space, rough endoplasmic reticulum and Golgi apparatus. In livers with hyperplastic nodules there were two different types of foci containing AFP-positive cells. One type had a normal nucleocytoplasmic ratio and was seen in well-preserved hepatic trabecular structures, and the other had less cytoplasm and occurred in trabecular structures in disarray. AFP-immunoreactivity in the former type was visible in the perinuclear space and rough endoplasmic reticulum but scarce in the Golgi apparatus, and in the latter type it was present in the proliferative smooth endoplasmic reticulum and in several parts of Golgi apparatus in the submembranous or pericanalicular areas. In livers with hepatocarcinoma, AFP immunoreaction was detected in well-differentiated hepatocellular carcinomas, and the subcellular location of AFP was in the perinuclear space, rough endoplasmic reticulum and many developed Golgi complexes. Therefore, AFP-positive cells in livers with hyperplastic nodules are a new cell population in hepatocarcinogenesis, and each type is morphologically different from the oval cell.     

Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.     

Secretion of invertase in mitotic yeast cells.

In mammalian cells intracellular transport is inhibited during mitosis. Here we show that in the yeast Saccharomyces cerevisiae secretion continues uninterrupted during mitosis. S. cerevisiae cells were arrested in mitosis by treating wild-type cells with the microtubule-inhibitor nocodazole, or by incubating a temperature-sensitive cell division cycle mutant (cdc16) at the restrictive temperature. Secretion of invertase into the periplasmic space was equally efficient in mitotic and in unsynchronized cells. Electron microscopy of nocodazole-treated mitotic wild-type cells revealed stretches of rough endoplasmic reticulum, strongly fenestrated Golgi cisternae and clusters of vesicles with the diameter of 30-90 nm. Secretion of invertase was inhibited in mitotic sec7 cells at the restrictive temperature, but continued at the permissive temperature. Sec7 is a mutant strain where intracellular traffic is blocked in unsynchronized cells in the Golgi complex at the restrictive temperature. Thus, the elements of the mitotic Golgi complex appear to be able to support intracellular traffic.     

Changes in cell polarity during mitosis in rat parotid acinar cells.

We studied the ultrastructure and cytochemistry of mitotic parotid acinar cells in vivo after induction of mitosis by isoproterenol injection. With entrance of the cells into the division cycle, the Golgi apparatus lost its characteristic stacked structure and internal polarity among the cisternae, appearing as fragments distributed throughout the cytoplasm. These fragments consisted of electron-lucent vesiculotubular structures and electron-dense 70-nm vesicles; neither component showed thiamine pyrophosphatase activity, a marker for trans cisternae of the Golgi apparatus, but the 70-nm vesicles showed a positive reaction for osmium impregnation, indicating retention of the cis nature. The rough endoplasmic reticulum was dilated and fragmented. Recovery of the structure of Golgi apparatus and rearrangement of rough endoplasmic reticulum occurred in daughter cells during telophase. These changes were the same as those observed after drug-induced inhibition of protein transport. The secretory granules were not dispersed but were divided into two groups with which centrioles were closely associated. Both groups migrated with the centrioles as far as the next interphase. The distribution of 5'-nucleotidase on the luminal plasma membrane showed no change during the process of division, thus demonstrating that surface polarity was maintained during mitosis. These changes in organelle structure and distribution may be due to the conversion of cell function from a secretory to a mitotic action.     

In the yeast Hansenula polymorpha, peroxisome formation from the ER is independent of Pex19p, but involves the function of p24 proteins

The peroxin Pex19p is important for the formation of functional peroxisomal membranes. Here we show that Hansenula polymorpha Pex19p is also required for peroxisome inheritance. Peroxisome inheritance is partly defective when Pex19p farnesylation is blocked, whereas deletion of PEX19 resulted in a severe defect in partitioning of peroxisomal structures. Time lapse imaging revealed that in newly formed buds, which had not inherited a peroxisome from the mother cell, new peroxisomes are formed that derive from the nuclear envelope/endoplasmic reticulum. This process was impaired upon deletion of EMP24 and ERP3, genes that encode p24 proteins. p24 Proteins are components of coated vesicles that mediate trafficking between the endoplasmic reticulum and Golgi apparatus. In an H. polymorpha wild-type background, deletion of EMP24 and ERP3 resulted in a strong reduction of organelle number in conjunction with an increase in the size of individual peroxisomes. This observation suggests that p24 proteins also play a role in peroxisome development in wild-type H. polymorpha cells.     

The trans Golgi face in rat small intestinal absorptive cells

In the small intestine cell differentiation from immature crypt cells to mature absorptive cells localized along the villi is accompanied by alterations in the organization of the trans Golgi side. In immature crypt cells the transmost Golgi cisterna is usually located closely adjacent to the other cisternae thus being a component of the stack. Concomitantly with cell differentiation the transmost cisterna of an increasing number of Golgi stacks sets off from the other cisternae being then located at various distances to the stacks. This transmost cisterna has, as in several other cell types, been interpreted as "GERL" (Golgi associated endoplasmic reticulum lysosomes [20, 28]) and thus, has been postulated to represent a specialized region of the endoplasmic reticulum. Our results, however, have shown that the cytochemical staining pattern which has been used as a basis for the differentiation of GERL from Golgi components is not present in crypt cells nor in mature absorptive cells of the proximal small intestine: identical cisternae react for thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. Thiamine pyrophosphatase and inosine diphosphatase--enzymes characteristic for Golgi cisternae--are apparent over transmost cisternae defined as GERL, too, and in addition, acid phosphatase--postulated as GERL-marker--is demonstrable over stacked Golgi cisternae. This overlapping cytochemical reaction, as well as the alterations during cell differentiation, indicate that those structures which have been described as GERL are to be interpreted as Golgi components rather than as endoplasmic reticulum. On the other hand, endoplasmic reticulum is a constant component of the trans Golgi face in undifferentiated crypt-base cells and in maturing cells of the crypt-top region. From its localization closely adjacent to trans Golgi cisternae it may be termed "Golgi-associated endoplasmic reticulum"; however, these cisternae of endoplasmic reticulum are constantly devoid of acid phosphatase. No indications exist for continuities with the thiamine pyrophosphatase-, inosine diphosphatase-, and acid phosphatase-positive transmost Golgi cisternae, and for an engagement in production of lysosomes.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Progress in studies of ZW10, a proper chromosome segregation protein

The conserved protein ZW10 is found in various organisms. It is localized on the kinetochores or spindle microtubules during cell division. ZW10 regulates not only the segregation of homologous chromosomes, each consisting of attached sister chromatids (during the first meiotic division), but also the separation of individual chromatids (during mitosis and the second meiotic division). ZW10 is required for proper chromosome segregation during both mitosis and meiosis. The effects of zwl0 mutations are similar for both equational and reductional divisions, giving rise to anaphases with lagging chromosomes and/or unequal numbers of chromosomes at the two poles. The localization of ZW10 is similar during mitosis, meiosis I, and meiosis II. In interphase the distribution of ZW10 changes; it is localized in the endoplasmic reticulum, Golgi apparatus, and in the cytosol and is involved in membrane trafficking between the endoplasmic reticulum and Golgi apparatus. ZW10 forms a subcomplex with RINT-1 and p31 which are involved in a larger complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE that is implicated in membrane trafficking. The text was submitted by the authors in English.     

Ultrastructure of the replication of the grasshopper crystalline-array virus in Schistocerca americana compared with other picornaviruses

The replication of the grasshopper crystalline-array virus (CAV), a picornavirus, was studied in thoracic muscles and pericardial cells. Two types of intracellular vesicles were evident that apparently functioned in viral synthesis. Those with smooth membranes were predominant in perinuclear regions and appeared to originate as protrusions from the outer nuclear membrane; those with particle-associated membranes were evident throughout the cytoplasm and appeared to be derived from existing membranous structures such as Golgi bodies and endoplasmic reticulum. In general, the replication of the grasshopper-CAV appears similar to the replication of picornaviruses in vertebrate systems.