Teresa Babia

ACTIN MICROFILAMENTS ARE ESSENTIAL FOR THE CYTOLOGICAL POSITIONING AND MORPHOLOGY OF THE GOLGI COMPLEX

The organization and function of the Golgi complex was studied in normal rat kidney cells following disruption of the actin cytoskeleton induced by cytochalasin D. In cells treated with these reagents, the reticular and perinuclear Golgi morphology acquired a cluster shape restricted to the centrosome region. Golgi complex alteration affected all Golgi subcompartments as revealed by double fluorescence staining with antibodies to the cis/middle Mannosidase II and the trans-Golgi network TGN38 proteins or vital staining with the lipid derivate C6-NBD-ceramide. The ultrastructural and stereological analysis showed that the Golgi cisternae remained attached in a stacked conformation, but they were swollen and contained electron-dense intra-cisternal bodies. The Golgi complex cluster remained linked to microtubules since it was fragmented and dispersed after treatment with nocodazole. Moreover, the reassembly of Golgi fragments after the disruption of the microtubuli with nocodazole does not utilize the actin microfilaments. The actin microfilament requirement for the disassembly and reassembly of the Golgi complex and for the ER-Golgi vesicular transport were also studied. The results show that actin microfilaments are not needed for either the retrograde fusion of the Golgi complex with the endoplasmic reticulum promoted by brefeldin A or the anterograde reassembly after the removal of the drug, or the ER-Golgi transport of VSV-G glycoprotein. However, actin microfilaments are directly involved in the subcellular localization and the morphology of the Golgi complex.

Actin Microfilaments Facilitate the Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum in Mammalian Cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum‐to‐Golgi transport monitored with the vesicular stomatitis virus‐G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A‐induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi‐to‐endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI‐coated and uncoated vesicles contain β/γ‐actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi‐to‐endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.

Fluorescent analogues of plasma membrane sphingolipids are sorted to different intracellular compartments in astrocytes; Harmful effects of chronic ethanol exposure on sphingolipid trafficking and metabolism.

Sphingolipids are basic constituents of cellular membranes and are essential for numerous functions such as intracellular signalling. They are transported along the exocytic and endocytic pathways in eukaryotic cells. After endocytosis, fluorescent‐labelled sphingolipids are sorted to distinct intracellular organelles prior to recycling (via early/recycling endosomes) or degradation (late endosomes/lysosomes). Here we examine, in primary cultures of rat astrocytes, the internalisation routes followed by C6‐NBD‐glucosylceramide (NBD‐GlcCer) and C6‐NBD‐sphingomyelin (NBD‐SM) and the effects of ethanol on their endocytic trafficking. Endocytosed plasma membrane NBD‐GlcCer and NBD‐SM are diverted to the Golgi apparatus and lysosomes, respectively. These different internalisation pathways are maintained regardless of the differentiation stage of astrocytes. Chronic ethanol exposure did not alter this endocytic sorting, but delayed the internalisation of both NBD‐sphingolipids. Moreover, ethanol also stimulated the in situ metabolism of NBD‐ceramide to NBD‐GlcCer and NBD‐SM. We conclude that in rat astrocytes internalised plasma membrane NBD‐sphingolipids are sorted to different subcellular compartments. The exposure to chronic ethanol perturbed the lipid endocytic process and stimulated the de novo synthesis of NBD‐sphingolipids, shifting the balance of sphingolipid metabolism in favour of the sphingomyelin pathway.

Endocytosis of NBD-sphingolipids in neurons: Exclusion from degradative compartments and transport to the Golgi complex

Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non‐neuronal cultured cell lines, plasma membrane short‐chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short‐chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD‐labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD‐glucosylceramide or NBD‐sphingomyelin to NBD‐ceramide. Double‐labeling experiments with transferrin or wheat‐germ agglutinin showed that NBD‐sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD‐glucosylceramide and NBD‐sphingomyelin to the Golgi complex.

Dynamical properties of membranes: Application of fluorescent lipid analogs

Recent advances in studies involving the structure and dynamics of membranes have shown that fluorescently-tagged lipid probes have become versatile and, occasionally, indispensable tools in this area of research. These probes are applied in investigations as diverse as those dealing with biophysical aspects of membranes, such as lateral mobility, lipid phase transitions and phase separations (domain formation), nonbilayer formation (hexagonal phases) and lipid translocation, but also in studies of the cell biology of membranes, including membrane flow and lipid trafficking. In this brief overview, some aspects of the properties and the application of fluorescent lipid (-like) probes will be summarized. The primary focus will be on the use of nitro-benzoxadiazole-derivatized lipids (NBD-lipids).

Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential

In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and membrane trafficking steps of the secretory pathway in two human melanoma A375 cell line variants with low (A375‐P) and high metastatic (A375‐MM) potential. Immunofluorescence analysis showed that in A375‐P cells, the Golgi complex showed a collapsed morphology. Conversely, in A375‐MM cells, the Golgi complex presented a reticular and extended morphology. At the ultrastructural level, the Golgi complex of A375‐P cells was fragmented and cisternae were swollen. When the cytoskeleton was analyzed, the microtubular network appeared normal in both cell variants, whereas actin stress fibers were largely absent in A375‐P, but not in A375‐MM cells. In addition, the F‐actin content in A375‐P cells was significantly lower than in A375‐MM cells. These morphological differences in A375‐P cells were accompanied by acceleration and an increase in the endoplasmic reticulum to Golgi and the trans‐Golgi network to cell surface membrane transport, respectively. Our results indicate that in human A375 melanoma cells, metastatic potential correlates with a well‐structured morphofunctional organization of the Golgi complex and actin cytoskeleton.

Immunofluorescence and Immunoelectron Microscopy of Microinjected and Transfected Cultured Cells

Immunofluorescence microscopy is the most common method to analyze expression and localization of a given protein in cells that have been microinjected or transfected previously with the appropriate DNA constructs. It offers the advantage that it is quick, easy to perform, and allows examination of a large number of cells within a short time. However, illumination with UV light is often damaging for the cells, and the fluorescence tends to bleach as a result of the excitation of the fluorochrome by the UV light. Nowadays, these disadvantages have been overcome by sophisticated systems such as video intensification cameras and confocal microscopy. In addition, the information that can be obtained by immunofluorescence microscopy is also restricted by the limited resolution of the optical lens. Moreover, some fixation conditions can induce artifactual changes in the intracellular localization of a significant number of molecules (Melan and Sluder 1992; Griffiths et al. 1993). Therefore, if the precise localization of a protein is being studied, it will always be necessary to look at the fine structure of the cell, using immunoelectron microscopy techniques. Nevertheless, whenever possible, the utilization of immunofluorescence in combination with immunoelectron microscopy is preferable. In this chapter, we describe in detail protocols for immunofluorescence and immunoelectron microscopy analyses which are particularly suited for cells in culture.