Organization and composition of the striated roots supporting the Golgi apparatus,the so-called parabasal apparatus,in parabasalid flagellates

Summary— In parabasalid flagellates, trichomonads and hypermastigids, the stack of cisternae of the Golgi apparatus are supported by striated roots attached to the basal bodies of flagella forming the so-called parabasal apparatus. Monoclonal antibodies raised for several trichomonad species, Monocercomonas, Trichomonas and Tetratrichomonas, label the parabasal fibre in immunofluorescence or immunogold staining and protein bands in immunoblotting. Several antibodies cross-react between trichomonad species, and one of them labels the homologous parabasal fibre in the hypermastigids: Trichonympha, Joenia, Pseudotrichonympha and Holomastigotoides. Considering the molecular mass range of the labelled proteins (100–135 kDa) and the lack of antibody cross-reactivity with the striated root proteins (centrin, assemblin, kinetodesmal protein, ciliary root proteins of epithelial ciliated cells) of other organisms, these proteins recognized by these antibodies seem to represent a new class of protein forming striated roots. The occurrence and significance of parabasal organization in eukaryogenesis is discussed.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Structure of Golgi apparatus

Summary Golgi apparatus (GA) of eukaryotic cells consist of one or more stacks of flattened saccules (cisternae) and an array of fenestrae and tubules continuous with the peripheral edges of the saccules. Golgi apparatus also are characterized by zones of exclusion that surround each stack and by an assortment of vesicles (or vesicle buds) associated with both the stacks and the peripheral tubules of the stack cisternae. Each stack (sometimes referred to as Golgi apparatus, Golgi complex, or dictyosome) is structurally and functionally polarized, reflecting its role as an intermediate between the endoplasmic reticulum, the cell surface, and the lysosomal system of the cell. There is probably only one GA per cell, and all stacks of the GA appear to function synchronously. All Golgi apparatus are involved in the generation and movement of product and membrane within the cell or to the cell exterior, and these functions are often reflected as structural changes across the stacks. For example, in plants, both product and membrane appear to maturate from the cis to the trans poles of the stacks in a sequential, or serial, manner. However, there is also strong ultrastructural evidence in plants for a parallel input to the stack saccules, probably through the peripheral tubules. The same modes of functioning probably also occur in animal GA; although here, the parallel mode of functioning almost surely predominates. In some cells at least, GA stacks give rise to tubular-vesicular structures that resemble the trans Golgi network. Rudimentary GA, consisting of tubular-vesicular networks, have been identified in fungi and may represent an early stage of GA evolution.     

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).     

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three‐dimensional localization using confocal microscopy of immunofluorescence‐labelled GA‐related markers [GM130 (cis‐Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin], uroepithelial differentiation‐related markers (UPs), MTs (microtubules; α‐tubulin) and intermediate filaments [CK7 (cytokeratin 7) and CK20], we found that in non‐differentiated, UP‐negative UCs the GA is mostly organized as a single ribbon‐like structure close to the nucleus, whereas in differentiated, UP‐positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans‐Golgi/TGN enzyme β1,4‐galactosyltransferase) fused to fluorescent protein showed that Golgi‐resident enzyme cycles freely within ribbon‐like GA but not within fragmented GA. By CLEM (correlative light—electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and the accumulation of intermediate filaments in the apical cytoplasm that could affect the kinetics of MT star leading to the peripheral fragmentation of the GA in the differentiated UCs. Conclusions. The fragmentation of the GA and the subsequent spreading of GA to the cell periphery represent one of the key events that promote the uniform delivery of UPs over the entire APM of differentiating UCs and thus are of major importance in the final proper formation and maintenance of the blood—urine barrier.     

Golgi apparatus and slime secretion in plants: the early implications and recent models of membrane traffic

Summary The function of the Golgi apparatus in the secretion of plant slimes is reviewed. It is shown how the research on slime secretion has increased the knowledge on the structure and dynamics of dictyosomes. Current models on intradictyosomal membrane traffic-anterograde progression of complete cisternae or anterograde movement of lateral vesicles with stationary cisternae-are discussed in the light of old and new results on slime secreting plant cells.Dedicated to Hilton H. Mollenhauer on the occasion of his retirement     

Cell biology of the plant Golgi apparatus

The higher plant Golgi apparatus, comprising many individual stacks of membrane bounded cisternae, is one of the most enigmatic of the cytoplasmic organelles. Not only can the stacks receive material from the endoplasmic reticulum, process it and target it to the correct cellular destination, but they can also synthesise and export complex carbohydrates and lipids and most likely act as one end point of the endocytic pathway. In many cells such processing and sorting can take place while the stacks are moving within the cytoplasm and, remarkably, the organelle manages to retain its structural integrity. This review considers some of the latest data and views on transport both to and from the Golgi and the mechanisms by which such activity is regulated.     

Cytoskeleton mediating transport between the ER system and the Golgi apparatus in the green alga Scenedesmus acutus

In the green alga Scenedesmus acutus, Golgi bodies are located near the nucleus and supplied with transition vesicles that bud from the outer nuclear envelope membrane. Using this alga, we have shown previously that thiamine pyrophosphatase (TPPase), a marker enzyme of Golgi bodies, migrates in vesicles from the Golgi bodies to the ER via the nuclear envelope in the presence of BFA (Noguchi et al., Protoplasma 201, 202-212, 1998). In this study we demonstrate that both cytochalasin B and oryzalin (microtubule-disrupting agent) inhibit the BFA-induced migration of TPPase from Golgi bodies to the nuclear envelope. However, only actin filaments--not microtubules--can be detected between the nuclear envelope and the Golgi bodies in both BFA-treated and untreated cells. These observations suggest that actin filaments mediate the BFA-induced retrograde transport of vesicles. This mechanism differs from that found in mammalian cells, in which microtubules mediate BFA-induced retrograde transport by the elongation of membrane tubules from the Golgi cisternae. We also discuss the non-participation of the cytoskeleton in anterograde transport from the nuclear envelope to the Golgi bodies.     

Phospholipase D in the Golgi apparatus

Phospholipase D has long been implicated in vesicle formation and vesicular transport through the secretory pathway. The Golgi apparatus has been shown to exhibit a plethora of mechanisms of vesicle formation at different stages to accommodate a wide variety of cargo. Phospholipase D has been found on the Golgi apparatus and is regulated by ADP-ribosylation factors which are themselves regulators of vesicle trafficking. Moreover, the product of phospholipase D activity, phosphatidic acid, as well as its degradation product diacylglycerol, have been implicated in vesicle fission and fusion events. Here we summarize recent advances in the understanding of the role of phospholipase D at the Golgi apparatus.     

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.     

Targeting of proteins to the Golgi apparatus

The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recyclingtrans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.     

The arrangement of the Golgi complex beads is not controlled by the cytoskeleton

Exposure of insect fat body to treatments which disrupt microtubules (colchicine, vinblastine sulfate and cold treatment) blocks intracellular transport between the Golgi complex and the plasma membrane but does not affect Golgi complex bead rings or transport from rough endoplasmic reticulum to the Golgi complex. Drugs which disrupt microfilaments (cytochalasins B and D) do not affect the bead rings or intracellular transport of secretory proteins at any level. Thus, intracellular transport between the rough endoplasmic reticulum and the Golgi complex and the arrangement of the beads in rings are both independent of the cytoskeleton. The ring arrangement is presumably maintained by interconnection(s) with rough endoplasmic reticulum membrane.     

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.     

The mitotic phosphatase cdc25C at the Golgi apparatus

cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G(2)/M transition. The activity of cdc25C is regulated by phosphorylation which by itself is implicated in regulating the subcellular localization. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G(2)/M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures which by immunofluorescence analysis as well as by biochemical subfractionation turned out to be the Golgi apparatus. It will be further shown that cdc25C at the Golgi fraction is an active phosphatase suggesting an additional and new role of cdc25C at the Golgi apparatus.     

Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks

We have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jellyfish green fluorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves. All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks. Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum. This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin filaments. Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton. These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems. However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) significantly slowed down the recovery of Golgi fluorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.     

Sorting out glycosylation enzymes in the Golgi apparatus

The study of glycosylation and glycosylation enzymes has been instrumental for the advancement of Cell Biology. After Neutra and Leblond showed that the Golgi apparatus is the main site of glycosylation, elucidation of oligosaccharide structures by Baenziger and Kornfeld and subsequent mapping of glycosylation enzymes followed. This enabled development of an in vitro transport assay by Rothman and co-workers using glycosylation to monitor intra Golgi transport which, complemented by yeast genetics by Schekman and co-workers, provided much of the fundamental insights and key components of the secretory pathway that we today take for granted. Glycobiology continues to play a key role in Cell Biology and here, we look at the use of glycosylation enzymes to elucidate intra Golgi transport.     

How apoptotic cells aid in the removal of their own cold dead bodies

Apoptotic cell clearance facilitates the removal of aged, damaged, infected or dangerous cells although minimizing perturbation of surrounding tissues, and is a vital process in the development and homeostasis of multicellular organisms. Importantly, failure to correctly execute programmed cell death and subsequent corpse clearance is broadly associated with chronic inflammatory and/or autoimmune diseases such as systemic lupus erythematosus. Apoptotic cells develop dramatic morphological changes including contraction, membrane blebbing and apoptotic body formation, which were among the first and most readily identifiable features of cellular suicide. However, understanding the purpose of apoptotic cell morphological changes has proven to be elusive, and recent studies have made somewhat surprising, and occasionally opposing, conclusions about the contribution of blebbing to phagocytic clearance and prevention of inflammatory/autoimmune disease. We review the evidence indicating how apoptotic blebs actively promote corpse recognition, uptake, and generation of auto-reactive antibodies.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.     

JVG9, a benzimidazole derivative,alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Trypanosoma cruzi has a particular cytoskeleton that consists of asubpellicular network of microtubules and actin microfilaments. Therefore, it is anexcellent target for the development of new anti-parasitic drugs. Benzimidazole2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown toinhibit the in vitro growth of many protozoa. Therefore, to find efficientanti-trypanosomal (trypanocidal) drugs, our group has designed and synthesisedseveral benzimidazole derivatives. One, named JVG9(5-chloro-1H-benzimidazole-2-thiol), has been found to be effectiveagainst T. cruzi bloodstream trypomastigotes under both in vitroand in vivo conditions. Here, we present the in vitro effects observed by laserscanning confocal and scanning electron microscopy on T. cruzitrypomastigotes. Changes in the surface and the distribution of thecytoskeletal proteins are consistent with the hypothesis that the trypanocidalactivity of JVG9 involves the cytoskeleton as a target.