佐藤 久美

Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P(2) at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-delta1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P(2) distribution quantitatively. By using this method, we found that PI(4,5)P(2) is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P(2) was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P(2) level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P(2) level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P(2) level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P(2) pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.

Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5mM methyl-beta-cyclodextrin or 10-20microg/ml nystatin for 1h, or metabolically by 20microM mevastatin and 200microM mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50microm in length. The increase of LC3-II by methyl-beta-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.

Lipid droplets (LDs) are organelles that store neutral lipids, but their regulatory mechanism is not well understood. In the present study, we identified Rab18 as an LD component of HepG2 cells by proteomic analysis, and confirmed its localization by immunohistochemistry and western blotting. Wild-type and dominant-active Rab18 localized to LDs but the dominant-negative form did not. Endogenous Rab18 coexisted with adipocyte differentiation-related protein (ADRP) in LDs, but the labeling intensity of the two proteins showed clear reciprocity. Consistent with this observation, overexpression of Rab18 induced a decrease in the amounts of ADRP in LDs in HepG2 and BALB/c 3T3 cells. Furthermore, Rab18 overexpression caused close apposition of LDs to membrane cisternae connected to the rough ER. Two other procedures that decrease ADRP, i.e. RNA interference and brefeldin A treatment, induced the same morphological change, indicating that decrease in ADRP was the cause of the LD-ER apposition. In accordance with similar structures found between ER and other organelles, we propose that the ER membrane apposed to LDs should be named the LD-associated membrane, or LAM. The present results suggested that Rab18 regulates LAM formation, which is likely to be involved in mobilizing lipid esters stored in LDs.

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.