Morphological evidence for calcium storage in the chromatoid body of rat spermatids

Morphological evidence for probable Ca2+ storage in the vesicular elements of the rat spermatid chromatoid body is documented using the K-pyroantimonate method, combined with EDTA chelation. Some vesicles are related to the microtubules associated with the chromatoid body. A possible involvement of Ca2+ in the intracellular movement and/or structural integrity of the chromatoid body is discussed.     

Morphological evidence for calcium storage in the chromatoid body of rat spermatids

Summary Morphological evidence for probable Ca²⁺ storage in the vesicular elements of the rat spermatid chromatoid body is documented using the K-pyroantimonate method, combined with EDTA chelation. Some vesicles are related to the microtubules associated with the chromatoid body. A possible involvement of Ca²⁺ in the intracellular movement and/or structural integrity of the chromatoid body is discussed.     

ER-to-Golgi transport and cytoskeletal interactions in animal cells

The endoplasmic reticulum (ER)-Golgi system has been studied using biochemical, genetic, electron and light microscopic techniques. We now understand many aspects of trafficking from the ER to the Golgi apparatus, including some of the signals and mechanisms for selective retention and retrieval of ER resident proteins and export of cargo proteins. Proteins that leave the ER emerge in export complexes or ER exit sites and accumulate in pleiomorphic transport carriers referred to sometimes as VTCs or intermediate compartments. These structures then transit from the ER to the Golgi apparatus along microtubules using the dynein/dynactin motor and fuse with the cis cisterna of the Golgi apparatus. Many proteins (including vSNAREs, ERGIC53/p58 and the KDEL receptor) must cycle back to the ER from pre-Golgi intermediates or the Golgi. We will discuss both the currently favored model that this cycling occurs via 50-nm COPI-coated vesicles and in vivo evidence that suggests retrograde trafficking may occur via tubular structures.     

Protein localization in the plant Golgi apparatus and the <Emphasis Type="Italic">trans</Emphasis>-Golgi network

This review presents plant-specific characteristics of the Golgi apparatus and discusses their impact on retention of membrane proteins in the Golgi or the trans-Golgi network (TGN). The plant Golgi consists of distinct stacks of cisternae that actively move throughout the cytoplasm. The Golgi apparatus is a very dynamic compartment and the site for maturation of N-linked glycans. It is also a factory for complex carbohydrates that are part of the cell wall. The TGN is believed to be the site from where vacuolar proteins are sorted by receptors towards each type of vacuole. To maintain the structure and specific features of the Golgi, resident proteins ought to be maintained in the proper Golgi cisternae or in the TGN. Two families of membrane proteins will be taken as examples for Golgi/TGN retention: (i) the enzymes involved in N-glycosylation processes and (ii) a vacuolar sorting receptor. Although the number of available plant proteins localized in Golgi/TGN is low, the basis of retention appears to be shared over all kingdoms and may result from pure retention and recycling mechanisms. In this review, we will summarize the characteristics of a plant Golgi and will discuss especially their consequences on on the study of this highly dynamic structure. We then choose membrane proteins with a single transmembrane domain to illustrate the signals and mechanisms involved in plants to localize and maintain proteins in the Golgi and the TGN.     

Mechanisms for exporting large-sized cargoes from the endoplasmic reticulum

Cargo proteins exported from the endoplasmic reticulum to the Golgi apparatus are typically transported in coat protein complex II (COPII)-coated vesicles of 60–90 nm diameter. Several cargo molecules including collagens and chylomicrons form structures that are too large to be accommodated by these vesicles, but their secretion still requires COPII proteins. Here, we first review recent progress on large cargo secretions derived especially from animal models and human diseases, which indicate the importance of COPII proteins. We then discuss the recent isolation of specialized factors that modulate the process of COPII-dependent cargo formation to facilitate the exit of large-sized cargoes from the endoplasmic reticulum. Based on these findings, we propose a model that describes the importance of the GTPase cycle for secretion of oversized cargoes. Next, we summarize reports that describe the structures of COPII proteins and how these results provide insight into the mechanism of assembly of the large cargo carriers. Finally, we discuss what issues remain to be solved in the future.     

Glycolipid transfer protein and intracellular traffic of glucosylceramide

Glycolipid transfer protein (GL-TP), a nonglycosylated protein with a molecular weight of 22,000 K, has been purified from pig brain. The protein transfers, by a carrier mechanism, glycolipids with a beta-glucosyl or beta-galactosyl residue directly linked to either ceramide or diacylglycerol. GL-TP appears to be present in most animal cells, and evidence has been obtained which indicates that it is a cytoplasmic protein. Little is known about the function of GL-TP. Current evidence indicates that glycosphingolipid glycosylation occurs at the luminal side of the Golgi apparatus, except for the glucosylation of ceramide, which has been shown to occur at the cytoplasmic side of the Golgi or endoplasmic membrane. It appears most likely that GL-TP participates in the intracellular traffic of glucosylceramide.     

Dynamics of proteins in Golgi membranes: comparisons between mammalian and plant cells highlighted by photobleaching techniques

In less than a decade the green fluorescent protein (GFP) has become one of the most popular tools for cell biologists for the study of dynamic processes in vivo. GFP has revolutionised the scientific approach for the study of vital organelles, such as the Golgi apparatus. As Golgi proteins can be tagged with GFP, in most cases without altering their targeting and function, it is a great substitute to conventional dyes used in the past to highlight this compartment. In this review, we cover the application of GFP and its spectral derivatives in the study of Golgi dynamics in mammalian and plant cells. In particular, we focus on the technique of selective photobleaching known as fluorescence recovery after photobleaching, which has successfully shed light on essential differences in the biology of the Golgi apparatus in mammalian and plant cells.     

Interaction of volkensin with HeLa cells: binding,uptake, intracellular localization,degradation and exocytosis

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10^-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.Received 21 April 2004; received after revision 26 May 2004; accepted 9 June 2004     

Internalization of polypeptide hormones and receptor recycling

Conclusion The insulin receptor is an integral protein of the plasma membrane of the cell. It is composed of two subunits: an subunit, which binds the hormone, and a subunit which is a tyrosine specific protein kinase capable of undergoing autophosphorylation. These independent subunits are synthesized by way of a higher molecular weight single chain precursor and thus are the product of a single gene^{29, 49, 85} localized to chromosome 19^{29, 91}. Assuming that the insulin receptor is synthesized in the same fashion as other integral membrane glycoproteins, then the nucleus, the rough endoplasmic reticulum, and the Golgi apparatus are involved in its biosynthesis. Further, there must be some form of transport of the mature receptor subunits to the plasma membrane where they are inserted.By contrast, the endocytotic route involves coated pits, coated vesicles, large clear vesicles or endosomes, multivesicular bodies and other lysosomal forms. In addition, it is possible that some other as yet unidentified organelle is involved in recycling (fig. 8). At the present time, with respect to the insulin receptor, the biosynthetic pathway and the endocytotic pathway appear to be separate. Further, it does not appear that either pathway, i. e. synthesis or endocytosis, exerts a regulatory function over the other.     

Molecular aspects of membrane fission in the secretory pathway

Membrane fission is essential in various intracellular dissociative transport steps. The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated. Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface. Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex. Received 10 May 2002; received after revision 20 June 2002; accepted 26 June 2002 RID="*" ID="*"Corresponding author.     

La vitellogenesi inArbacia lixula eSphaerechinus granularis (Echin. Echin.)

Summary The researches carried out both under the light and electron microscopes permitted us to ascertain that, in EchinodermataArbacia lixula andSphaerechinus granularis, yolk globules form by aggregation of ribonucleoproteic particles. It appears that the globules themselves in the first stage of their formation show such physico-chemical conditions as to condition the precipitation of silver in the method used to reveal Golgi apparatus.     

Glycolipid transfer protein and intracellular traffic of glucosylceramide

Summary Glycolipid transfer protein (GL-TP), a nonglycosylated protein with a molecular weight of 22,000 K, has been purified from pig brain. The protein transfers, by a carrier mechanism, glycolipids with a -glucosyl or -galactosyl residue directly linked to either ceramide or diacylglycerol. GL-TP appears to be present in most animal cells, and evidence has been obtained which indicates that it is a cytoplasmic protein. Little is known about the function of GL-TP. Current evidence indicates that glycosphingolipid glycosylation occurs at the luminal side of the Golgi apparatus, except for the glucosylation of ceramide, which has been shown to occur at the cytoplasmic side of the Golgi or endoplasmic membrane. It appears most likely that GL-TP participates in the intracellular traffic of glucosylceramide.     

Membrane traffic in the secretory pathway

It is generally thought that microtubule-associated motors insure long-range movements of the secretory vesicles from the center of the cell to its periphery, while myosins insure short-range movements at the cell periphery. However, several of the myosins that have been reported during the last decade to be involved in the exocytic pathway are not processive, meaning that they do not have the ability to move cargos along actin polymers. We will review here the possible mechanisms by which these myosins could contribute to the traffic of secretory proteins from the Golgi complex to the plasma membrane.     

A preliminary report on the fine structure of Tripneustes esculentus eggs.

The fine structure of Tripneustes esculentus eggs was studied with the aid of an electron microscope. Cells obtained from this West Indies sea urchin showed cortical granules, mitochondria forming a rosette around lipid granules, endoplasmic reticula, Golgi apparatus and annulated lamellae. These structures appear identical to those seen in eggs of the Atlantic sea urchin: Arbacia punctulata.     

Coated vesicles in the rat adrenal glomerular zone after a low-sodium diet.

In rats subjected to a low-sodium diet, a great activity was observed of the coated vesicles at Golgi complex and cell surfaces of glomerular adrenal zone. These findings are related to the function of these organoids in the uptake and transport of necessary substances under stimulating conditions of the zone.     

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ER–Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER–Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER–Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER–Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ER–Golgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS.     

Coated vesicles in the rat adrenal glomerular zone after a low-sodium diet

Summary In rats subjected to a low-sodium diet, a great activity was observed of the coated vesicles at Golgi complex and cell surfaces of glomerular adrenal zone. These findings are related to the function of these organoids in the uptake and transport of necessary substances under stimulating conditions of the zone.     

A trapper keeper for TRAPP, its structures and functions

During biosynthesis many membrane and secreted proteins are transported from the endoplasmic reticulum, through the Golgi and on to the plasma membrane in small transport vesicles. These transport vesicles have to undergo budding, movement, tethering, docking, and fusion at each organelle of the biosynthetic pathway. The transport protein particle (TRAPP) complex was initially identified as the tethering factor for endoplasmic reticulum (ER)—derived COPII vesicles, but the functions of TRAPP may extend to other areas of biology. Three forms of TRAPP complexes have been discovered to date, and recent advances in research have provided new insights on the structures and functions of TRAPP. Here we provide a comprehensive review of the recent findings in TRAPP biology.     

Localization of cholesterol in the Golgi apparatus of cardiac muscle cells

Summary Filipin (a polyene) interacts with cholesterol in membranes, producing distinctive deformations that can be detected by freeze-fracture. The distribution of filipin-induced deformations in the Golgi apparatus of cardiac myocytes suggests a role for this organelle in the transformation of cholesterol-poor membrane to cholesterol-rich membrane.Acknowledgments. This work was supported by a grant (No. 779) from the British Heart Foundation. I thank Dr E. Massey and Mr A. Slade for their help. Filipin was generously donated by Upjohn Ltd, Crawley, Sussex, U.K.     

Ultrastructural demonstration of transferrin synthesis in the adult rat liver]

An ultrastructural immunoenzymatic study allowed us to demonstrate in Rat adult hepatocytes the presence of transferrin on the rough endoplasmic reticulum and in the Golgi apparatus. Such localizations suggest that the different steps of transferrin synthesis could possibly be related topographically to them. These results further an approach to the problems of maturation and secretion of transferrin.