Monitoring of myocardial injury by serial measurements of QRS area and T area: The MaastrICCht cohort.

BACKGROUND: Manually derived electrocardiographic (ECG) parameters were not associated with mortality in mechanically ventilated COVID-19 patients in earlier studies, while increased high-sensitivity cardiac troponin-T (hs-cTnT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were. To provide evidence for vectorcardiography (VCG) measures as potential cardiac monitoring tool, we investigated VCG trajectories during critical illness. METHODS: All mechanically ventilated COVID-19 patients were included in the Maastricht Intensive Care Covid Cohort between March 2020 and October 2021. Serum hs-cTnT and NT-proBNP concentrations were measured daily. Conversion of daily 12-lead ECGs to VCGs by a MATLAB-based script provided QRS area, T area, maximal QRS amplitude, and QRS duration. Linear mixed-effect models investigated trajectories in serum and VCG markers over time between non-survivors and survivors, adjusted for confounders. RESULTS: In 322 patients, 5461 hs-cTnT, 5435 NT-proBNP concentrations and 3280 ECGs and VCGs were analyzed. Non-survivors had higher hs-cTnT concentrations at intubation and both hs-cTnT and NT-proBNP significantly increased compared with survivors. In non-survivors, the following VCG parameters decreased more when compared to survivors: QRS area (-0.27 (95% CI) (-0.37 to -0.16, p < .01) µVs per day), T area (-0.39 (-0.62 to -0.16, p < .01) µVs per day), and maximal QRS amplitude (-0.01 (-0.01 to -0.01, p < .01) mV per day). QRS duration did not differ. CONCLUSION: VCG-derived QRS area and T area decreased in non-survivors compared with survivors, suggesting that an increase in myocardial damage and tissue loss play a role in the course of critical illness and may drive mortality. These VCG markers may be used to monitor critically ill patients.

Sphingolipid metabolism enzymes as targets for anticancer therapy.

Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumor cells. Unfortunately, tumor cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics. Ceramide. the central molecule in cellular sphingolipid metabolism, has been recognized as an important mediator of apoptosis. Moreover, an increased cellular capacity for ceramide glycosylation has been identified as a novel multidrug resistance mechanism. Indeed, virtually all multidrug resistant cell types exhibit a deviating sphingolipid composition, most typically an increased level of glucosylceramide. Thus, the enzyme glucosylceramide synthase, which converts ceramide into glucosylceramide, has emerged as a potential target to increase apoptosis and decrease drug resistance of tumor cells. In addition, several other steps in the pathways of sphingolipid metabolism arc altered in multidrug resistant cells, opening a perspective on additional sphingolipid metabolism enzymes as targets for anti-cancer therapy. In this article, we present an overview of the current understanding concerning drug resistance-related changes in sphingolipid metabolism and how interference with this metabolism can be exploited to over come multidrug resistance.

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.

The involvement of sphingolipids in multidrug resistance.

Administration of most chemotherapeutic agents eventually results in the onset of apoptosis, despite the agents' variety in structure and molecular targets. Ceramide, the central molecule in cellular glycosphingolipid metabolism, has recently been identified as an important mediator of this process. Indeed, one of the events elicited by application of many cytotoxic drugs is an accumulation of this lipid. Treatment failure in cancer chemotherapy is largely attributable to multidrug resistance, in which tumor cells are typically cross-resistant to multiple chemotherapeutic agents. Different cellular mechanisms underlying this phenomenon have been described. Of these the drug efflux pump activity of P-glycoprotein and the multidrug resistance-associated proteins are the most extensively studied examples. Recently, an increased cellular capacity for ceramide glycosylation has been recognized as a novel multidrug resistance mechanism. Indeed, virtually all multidrug-resistant cells exhibit a deviating sphingolipid composition, most typically, increased levels of glucosylceramide. On the other hand, several direct molecular interactions between sphingolipids and drug efflux proteins have been described. Therefore, in addition to a role in the multidrug resistance phenotype by which ceramide accumulation and, thus, the onset of apoptosis are prevented, an indirect role for sphingolipids might be envisaged, by which the activity of these efflux proteins is modulated. In this review, we present an overview of the current understanding of the interesting relations that exist between sphingolipid metabolism and multidrug resistance.

Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells.

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT(1) receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPbetaS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.

Regulation of [Ca(2+)](i) homeostasis in MRP1 overexpressing cells.

Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR. MRP1 overexpression was accompanied by a decreased response to thapsigargin. Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells. Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells. Again, these effects were virtually abolished in HT29(col) cells. Our results point to a modulatory role of MRP1 on intracellular calcium concentration ([Ca(2+)](i)) homeostasis which may contribute to the MDR phenotype.

Differential expression of sphingolipids in MRP1 overexpressing HT29 cells.

We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.

1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine.

In this study, we show that an inhibitor of glycosphin-golipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increases the chemosensitivity of neuroblastoma tumor cells for Taxol and vincristine. At noneffective low doses of Taxol or vincristine, the addition of a noneffective dose of PDMP resulted in 70% cytotoxicity, indicating synergy. Such an effect was not observed for etoposide (VP16). PDMP caused an early (6 h) increase in ceramide (Cer) levels, but the excess Cer was metabolically removed in the long-term (96 h). However, upon incubation with PDMP in combination with Taxol, but not with etoposide, Cer levels remained elevated at 96 h. These results suggest that neuroblastoma cells are normally able to metabolically remove excess Cer, but lose this capacity upon exposure to microtubule modulating anticancer agents (Taxol or vincristine). In addition, PDMP treatment resulted in a decreased efflux of [14C]Taxol and [3H]vincristine from neuroblastoma cells, similar to treatment with PSC833 or MK571, suggesting an effect of PDMP on the transporter proteins P-glycoprotein and/or multidrug resistance protein. PDMP did not further reduce [14C]Taxol or [3H]vincristine efflux in PSC833-treated cells, although it did further diminish cell survival under these conditions. We conclude that a combined administration of nontoxic concentrations of PDMP and either Taxol or vincristine results in highly sensitized neuroblastoma cells. This appears to involve a sustained elevation of Cer levels, possibly in concert with increased drug accumulation.

Inhibition of P-glycoprotein activity and chemosensitization of multidrug-resistant ovarian carcinoma 2780AD cells by hexanoylglucosylceramide.

In the present study we show that neutral hexanoyl-(glyco)sphingolipids inhibit P-glycoprotein (Pgp) activity in human ovarian 2780AD cells. By contrast, hexanoylceramide and the gangliosides GM(3) and GM(2) had no effect on Pgp activity, whereas sphingosine had a stimulating effect. In the case of hexanoylglucosylceramide, inhibition of Pgp activity by was reflected by a regained doxorubicin sensitivity of cells, which were grown in medium supplemented with the lipid. Our results lead to the conclusion that a direct transmodulation of Pgp activity by glycolipids occurs, depending on lipid headgroup structure, which can result in reduced resistance to the chemotherapeutic agent doxorubicin.

PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes.

We reported that an inhibitor of sphingolipid biosynthesis, D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25-38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component beta-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.

Leukemia-induced bone marrow depression: effects of gangliosides on erythroid cell production.

Bone marrow depression is a common feature in hematological malignancies or other bone marrow-involving cancers. The mechanism of this hemopoietic suppression resulting in pancytopenia and especially anemia has not been elucidated. Gangliosides can be shed by cancer cells. Therefore, we investigated the effects of exogenously added gangliosides on erythropoiesis in a human and murine in vitro system. A dose-dependent inhibition of murine colony-forming-unit-erythroid (CFU-E) and burst-forming-unit-erythroid (BFU-E) colony growth was observed. Furthermore the maturation of BFU-Es into CFU-Es was inhibited. The inhibition by gangliosides was not abolished by increasing the dose of erythropoietin (10 U/ml). FACS-analysis studies with human CD34+ cells cultured with gangliosides (GM3), erythropoietin (EPO) and stem cell factor (SCF) demonstrated a strong inhibition on cell growth. This resulted in a significantly higher percentage of immature cells (CD34+/GpA-, 24% vs. 3%), and a lower percentage of mature erythroid cells (CD34-/GpA+, 36% vs. 89%). Under these circumstances the effects on erythroid cell growth were much higher than on other cell lineages. The inhibitory effect of gangliosides isolated from acute lymphoblastic leukemic patients on in vitro erythropoiesis suggests that in vivo hemopoietic suppression might have its origin in the gangliosides present and probably shed by the malignant cells in the microenvironment and plasma. Our results show that gangliosides inhibit erythropoiesis in vitro at several stages of development, by a mechanism involving modulation of the maturation of erythroid cells.

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.

N-Ras induces alterations in Golgi complex architecture and in constitutive protein transport.

Aberrant glycosylation of proteins and lipids is a common feature of many tumor cell types, and is often accompanied by alterations in membrane traffic and an anomalous localization of Golgi-resident proteins and glycans. These observations suggest that the Golgi complex is a key organelle for at least some of the functional changes associated with malignant transformation. To gain insight into this possibility, we have analyzed changes in the structure and function of the Golgi complex induced by the conditional expression of the transforming N-Ras(K61) mutant in the NRK cell line. A remarkable and specific effect associated with this N-Ras-induced transformation was a conspicuous rearrangement of the Golgi complex into a collapsed morphology. Ultrastructural and stereological analyses demonstrated that the Golgi complex was extensively fragmented. The collapse of the Golgi complex was also accompanied by a disruption of the actin cytoskeleton. Functionally, N-Ras-transformed KT8 cells showed an increase in the constitutive protein transport from the trans-Golgi network to the cell surface, and did not induce the appearance of aberrant cell surface glycans. The Golgi complex collapse, the actin disassembly, and the increased constitutive secretion were all partially inhibited by the phospholipase A2 inhibitor 4-bromophenylacyl bromide. The results thus suggest the involvement of the actin cytoskeleton in the shape of the Golgi complex, and intracellular phospholipase A2 in its architecture and secretory function.

Modulation of carcinoembryonic antigen release by glucosylceramide--implications for HT29 cell differentiation.

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short-chain analog of the lipid, hexanoyl(C6)-GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C6-GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C6-GlcCer. Control experiments reveal that a potential interference of the well-known modulator of cell physiology, ceramide (Cer), can be excluded. Long-term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.

Inhibition of hemopoiesis in vitro by neuroblastoma-derived gangliosides.

Hemopoiesis is disturbed in bone marrow-involving cancers like leukemia and neuroblastoma. Shedding of gangliosides by tumor cells may contribute to this tumor-induced bone marrow suppression. We studied in vitro the inhibitory effects of murine neuroblastoma cells (Neuro-2a and C1300) and their gangliosides on hemopoiesis using normal murine hemopoietic progenitor colony-forming assays. Transwell cultured neuroblastoma cells showed a dose-dependent inhibition on hemopoiesis, indicating that a soluble factor was responsible for this effect. Furthermore, the supernatant of Neuro-2a cultured cells inhibited hemopoietic proliferation and differentiation. To determine whether the inhibitory effect was indeed due to shed gangliosides and not, for instance, caused by cytokines, the effect of DL-threo-1 -phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) on Neuro-2a cells was studied. DL-PDMP is a potent inhibitor of glucosylceramide synthase, resulting in inhibition of the synthesis and shedding of gangliosides. The initially observed inhibitory effect of supernatant of Neuro-2a cells was abrogated by culturing these cells for 3 days in the presence of 10 microM DL-PDMP. Moreover, gangliosides isolated from Neuro-2a cell membranes inhibited hemopoietic growth. To determine whether the described phenomena in vitro are a reflection of bone marrow suppression occurring in vivo, gangliosides isolated from plasma of neuroblastoma patients were tested for their effects on human hemopoietic progenitor colony-forming assays. These human neuroblastoma-derived gangliosides inhibited normal erythropoiesis (colony-forming unit-erythroid/burst-forming unit-erythroid) and myelopoiesis (colony-forming unit-granulocyte/macrophage) to a higher extent compared with gangliosides isolated from control plasma. Altogether these results suggest that gangliosides shed by neuroblastoma cells inhibit hemopoiesis and may contribute to the observed bone marrow depression in neuroblastoma patients.

PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism.

In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not "rescue" the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1, 3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Interferon-gamma-induced differentiation and apoptosis of HT29 cells: dissociation of (glucosyl)ceramide signaling.

Recently, (glyco)sphingolipids (SL) like ceramide (Cer) and glucosylceramide (GlcCer) have been shown to be involved in signaling pathways leading to differentiation and apoptosis in several cell types, including the colon adenocarcinoma cell line HT29. Intracellular levels of Cer can be modulated by ligands such as interferon-gamma (IFN gamma). In the present study we show that IFN gamma, depending on its concentration, has both differentiation- and apoptosis-inducing effects on HT29 cells. Since both phenomena have been related to SL-mediated signaling in other cell types, we next examined whether IFN gamma was able to induce changes in the SL levels of HT29 cells. Remarkably, no significant changes in these levels could be revealed, implying that SL are not involved in IFN gamma-induced differentiation and/or apoptosis of HT29 cells. This observation provides evidence for the hypothesis that SL-mediated signaling pathways might be more cell type specific than is generally assumed.

Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated.

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degreesC, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.

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.