Lipid droplets (LDs) are independent organelles that are composed of a lipid ester core and a surface phospholipid monolayer. Recent studies have revealed many new proteins, functions, and phenomena associated with LDs. In addition, a number of diseases related to LDs are beginning to be understood at the molecular level. It is now clear that LDs are not an inert store of excess lipids but are dynamically engaged in various cellular functions, some of which are not directly related to lipid metabolism. Compared to conventional membrane organelles, there are still many uncertainties concerning the molecular architecture of LDs and how each function is placed in a structural context. Recent findings and remaining questions are discussed.

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

A novel actin filament (F-actin)–binding protein with a molecular mass of ∼205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

The Ca2+ pump in the plasma membrane plays a key role in the fine control of the cytoplasmic free Ca2+ concentration. In the present study, its subcellular localization was examined with immunocytochemical techniques using a specific antibody generated against the erythrocyte membrane Ca2+ pump ATPase. By immunofluorescence microscopy of cultured cells, the labeling with the antibody was seen as numerous small dots, often distributed in linear arrays or along cell edges. Immunogold EM of cryosections revealed that the dots correspond to caveolae, or smooth invaginations of the plasma membrane. The same technique applied to mouse tissues in vivo showed that the Ca2+ pump is similarly localized in caveolae of endothelial cells, smooth muscle cells, cardiac muscle cells, epidermal keratinocytes and mesothelial cells. By quantitative analysis of the immunogold labeling, the Ca2+ pump in capillary endothelial cells and visceral smooth muscle cells was found to be concentrated 18-25-fold in the caveolar membrane compared with the noncaveolar portion of the plasma membrane. In renal tubular and small intestinal epithelial cells, which have been known to contain the Ca2+ pump but do not have many caveolae, most of the labeling was randomly distributed in the basolateral plasma membrane, although caveolae were also positively labeled. The results demonstrate that the caveolae in various cells has the plasmalemmal Ca2+ pump as a common constituent. In conjunction with our recent finding that an inositol 1,4,5-trisphosphate receptor-like protein exists in the caveolae (Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. 1992. J. Cell Biol. 119:1507-1513), it is inferred that the smooth plasmalemmal invagination is an apparatus specialized for Ca2+ intake and extrusion from the cytoplasm.