Abstract. Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rabll. Rabll was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rabll mutants that are preferentially in the GTP-or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rabll-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rabll-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rabll proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rabll-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rabll in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rabll in controlling traffic through the recycling endosome.

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane traf®cking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind speci®cally, and with high af®nity, to PI(3)P both in vitro and in vivo. When expressed in ®broblasts, a tagged probe localized to endosomes, as detected by¯uorescence microscopy. Electron microscopy of untransfected ®broblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells de®cient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27D yeast cells, which have impaired endosome to vacuole traf®cking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in de®ning the early endosome and the subsequent steps leading to multivesicular endosome formation.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profiles and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae.Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein Thyl, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.Caveolae are specialized domains of the plasma membrane present in most mammalian cell types. While the function of these structures remains controversial, their morphology has been studied in great detail (1). Caveolae generally appear as 50-to 90-nm vesicular invaginations of the plasma membrane with a characteristic bulb shape. Clusters of connected caveolae resembling bunches of grapes are often seen in adipocytes, endothelial cells, and fibroblasts. In contrast to clathrin-coated pits, caveolae do not show a well-defined coat by conventional transmission electron microscopy. However, filamentous material arranged in spiral or striated arrays on the cytoplasmic side of caveolae has been revealed by other electron microscopy techniques (2, 3) and is generally referred to as the caveolar coat. VIP21-caveolin, an integral membrane protein originally identified as a substrate for v-src (4) and a component of Golgi-derived transport vesicles (5), was localized in caveolae by immunogold staining and proposed to play a structural role (6, 7). We have previously reported that the absence of detectable VIP21-caveolin correlates with the lack of caveolae in a lymphocyte cell line (8). Similar results were obtained in N2a neuroblastoma cells (9). Here we express VIP21-caveolin in our model lymphocyte cell line and analyze the plasma membrane morphology by electron microscopy. Consistent with its proposed structural role, expression of VIP21-caveolin induces formation of caveolae.

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.

Abstract. In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.

VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N-and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures.