The ceramide-enriched trans-Golgi compartments reorganize together with other parts of the Golgi apparatus in response to ATP-depletion

Abstract

In this study, the ceramide-enriched trans-Golgi compartments representing sites of synthesis of sphingomyelin and higher organized lipids were visualized in control and ATP-depleted hepatoma and endothelial cells using internalization of BODIPY-ceramide and the diaminobenzidine photooxidation method for combined light-electron microscopical exploration. Metabolic stress induced by lowering the cellular ATP-levels leads to reorganizations of the Golgi apparatus and the appearance of tubulo-glomerular bodies and networks. The results obtained with three different protocols, in which BODIPY-ceramide either was applied prior to, concomitantly with, or after ATP-depletion, revealed that the ceramide-enriched compartments reorganize together with other parts of the Golgi apparatus under these conditions. They were found closely associated with and integrated in the tubulo-glomerular bodies formed in response to ATP-depletion. This is in line with the changes of the staining patterns obtained with the Helix pomatia lectin and the GM130 and TGN46 immuno-reactions occurring in response to ATP-depletion and is confirmed by 3D electron tomography. The 3D reconstructions underlined the glomerular character of the reorganized Golgi apparatus and demonstrated continuities of ceramide positive and negative parts. Most interestingly, BODIPY-ceramide becomes concentrated in compartments of the tubulo-glomerular Golgi bodies, even though the reorganization took place before BODIPY-ceramide administration. This indicates maintained functionalities although the regular Golgi stack organization is abolished; the results provide novel insights into Golgi structure-function relationships, which might be relevant for cells affected by metabolic stress.

Cellular effects of fluorodeoxyglucose: Global changes in the lipidome and alteration in intracellular transport

“…There are two known cellular effects of FDG, the inhibition of glycolysis and protein N-glycosylation, however, none of which seems to be involved in the inhibition of GSL biosynthesis. Under the conditions tested, FDG did not seem to deplete cellular ATP in HEp-2 cells, as (i) there was little effect on cell growth and protein synthesis, (ii) there was no vesiculation of the Golgi observed, which has been shown to occur in response to ATP depletion by 2DG [39], and (iii) the levels of SM were not depleted, although CERT-mediated Cer transport is shown to depend on ATP [38]. The inhibition of GlcCer synthesis by FDG does not seem to be mediated via the inhibition of N-glycosylation either, as 2DG, which is shown to be more efficient in inhibiting N-linked glycosylation than FDG [40], does not reduce cellular GlcCer levels [13].…”

Golgi apparatus dis- and reorganizations studied with the aid of 2-deoxy-d-glucose and visualized by 3D-electron tomography

“…Although 2DG has become a drug of high scientific and medical interest, only few reports have addressed its action with regard to the ultrastructure of cells. Previous studies in our laboratory (Meisslitzer-Ruppitsch et al 2011; Ranftler et al 2015) have shown that treating cultured cells with 2DG leads to extensive changes of the Golgi apparatus. In the present work, we followed the dissociations and re-formations of the Golgi stacks that occur under the influence of 2DG and after administration of the drug had ended.…”

“…For this, we used 2-deoxy- d -glucose (2DG), a nonmetabolizable glucose analogue, which causes a reversible decrease of the cellular levels of adenosine triphosphate (ATP) and which is used in both experimental cell biology and medicine (e.g., Aft et al 2002; Dwarakanath 2009; Kavaliauskiene et al 2015; Zhang et al 2015). Since the Golgi apparatus changes its architecture under the influence of 2DG, this drug was found to be of great value in the context of Golgi apparatus research (del Valle et al 1999; Meisslitzer-Ruppitsch et al 2011; Ranftler et al 2015). …”

“…Based on its chemical structure, 2DG acts by influencing N-glycosylation ( d -mannose analogue) and glycolysis ( d -glucose analogue), thereby changing the cells’ energy level by decreasing the amount of intracellular ATP (del Valle et al 1999; Ranftler et al 2015) and altering the structures of cell organelles and the endomembrane system (del Valle et al 1999; Grieb et al 2004; Wu et al 2009; Meisslitzer-Ruppitsch et al 2011; Ranftler et al 2015). Although 2DG has become a drug of high scientific and medical interest, only few reports have addressed its action with regard to the ultrastructure of cells.…”

Recent progress in histochemistry and cell biology

“…In rat kidney cells these changes occur mainly at the cis-Golgi compartments (del Valle et al 1999). To address the question, if trans-Golgi compartments are also involved in reorganizations, Meisslitzer-Ruppitsch et al (2011) analyzed the ceramide-concentrating trans-Golgi compartments by a combined light and electron microscopic technique applying ATP-depletion. A ceramide, labeled with the fluorochrome boron dipyrromethene (BO-DIPY) was used (Pagano et al 1991).…”