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Sodium dodecyl sulfate–treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum–carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers.

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase β3 (CCTβ3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTβ3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTβ3 and is enhanced by its overexpression. This CCTβ3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTβ3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTβ3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival., The source of phospholipids to generate autophagosomal membranes, particularly after prolonged starvation, is not well characterized. Here, the authors show that CCTβ3, the rate limiting enzyme in phosphatidylcholine synthesis, is activated on lipid droplets and sustains long-term autophagy.

Lipid droplets (LDs) in the cytoplasm are formed in the endoplasmic reticulum (ER) and are connected with various organelles, both structurally and functionally. This is in contrast to LDs in the nucleus, which are separated from organelles in the cytoplasm. How nuclear lipid droplets form and what function they have were not known for many years. Recent results have revealed that nuclear LDs in hepatocytes are derived from lipoprotein precursors in the ER lumen, whereas those in non-hepatocytes and budding yeast newly form in the inner nuclear membrane. Although nuclear LDs are far fewer in number than cytoplasmic LDs, the unique location appears to bestow upon them specific functions, which are potentially linked to nuclear biology. This Review will provide an overview of our current understanding of nuclear LDs, discuss how they are different from cytoplasmic LDs and highlight knowledge gaps that need to be filled in future studies.

The "Guidelines for Cadaver Dissection in Education and Research of Clinical Medicine" drafted by the Japan Surgical Society (JSS) and the Japanese Association of Anatomists in 2012 helped dispel legal concerns over cadaver surgical training (CST) and the usage of donated human bodies for research and development (RD) in the country. Subsequently, in the fiscal year 2018, the Ministry of Health, Labour and Welfare increased the funding for CST, prompting its wider implementation. This study analyzed data obtained in 2012-2021 through the reporting system of the JSS-CST Promotion Committee to map the usage of cadavers for clinical purposes, specifically education and RD, in Japan. We found that the number of medical universities using cadavers for CST and RD programs was just 5 in 2012, and it reached 38 for the decade. Thus, about half of Japan's medical universities implemented such programs over the period. Meanwhile, the total number of programs was 1,173. In the clinical field, the highest number of programs were implemented in orthopedics (27%), followed by surgery (21%), and neurosurgery (12%). Based on the purpose, the most common objective of the programs (approximately 70%) was acquiring advanced surgical techniques. Further, the highest number of programs and participants were recorded in 2019 (295 programs, 6,537 participants). Thus, the guidelines helped expand cadaver usage for clinical purposes in Japan. To further promote the clinical usage of cadavers in medical and dental universities throughout Japan, sharing know-how on operating cadaver laboratories and building understanding among the general public is recommended.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P2 Sperm from VSP-/- mice show more Ca2+ influx upon capacitation than VSP+/- mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P2-sensitive K+ channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca2+ influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P2 in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P2 distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P2 distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P2 distribution plays important roles in ion channel regulation as well as sperm motility.

Orii et al. show that phospholipids distribute symmetrically in the yeast autophagosomal membranes. They also show that de novo–synthesized phosphatidylcholine is incorporated to autophagosomal membranes preferentially and attains the symmetrical distribution within 30 min after synthesis by an Atg9-dependent manner., The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo–synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.

Sołtysik et al. show that the inner nuclear membrane of nonhepatocytes harbors triglyceride synthesis enzymes and generates nuclear lipid droplets (LDs). Seipin knockdown increases nuclear LDs through upregulating lipin-1β transcription and increasing phosphatidic acid levels in the nucleus., Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1β expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the ‘void zone’. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site. This article has an associated First Person interview with the first author of the paper.

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) regulates caveolae formation and caveolae-dependent endocytosis by interacting with caveolae components, which in turn sustains pro-survival signaling toward AKT from multiple RTKs, including EGFR, and MET. We report here a novel function of ROR1 as a scaffold for HRS and STAM1, two essential components of ESCRT-0. The present results show that ROR1 facilitates interactions of HRS and STAM1, thereby preventing the lysosomal degradation of HRS. Furthermore, interaction of ROR1 with STAM1 was found to be required to sustain binding of ROR1 to HRS as well as HRS subcellular localization. Additionally, ROR1 localized in both the limiting membrane and intraluminal vesicles (ILVs) of Rab5-induced multivesicular endosomes (MVEs) containing HRS, CD63, and EEA1 was found to regulate the formation of Rab5-induced MVEs by an association with the GTP-bound form of Rab5 in cancer cells. Notably, ROR1 depletion inhibits CD63-positive MVEs formation and reduces exosomes release. Our findings provide the first evidence that the onco-embryonic antigen receptor ROR1 regulates exosome biogenesis via MVE formation in cancer cells.

Membrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the “void zone”. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the cholesterol-enriched domain in phase-separated artificial membranes. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.Summary statementYeast cells lacking phosphatidylserine generate protein-free plasma membrane domains, and vacuoles contact with this domain. This is the first report of micron-scale plasma membrane domains in living cells.

Fluorescence microscopy is currently one of the more powerful and versatile techniques available for biological studies. With conventional biological immunofluorescence microscopy, caveolin-1 (CAV1) is visualized as numerous small dots, which are often distributed as a linear array or along the edge of the cell. Although its presence, as well as that of other proteins, can be detected by conventional immunofluorescence microscopy, those results do not clarify whether two different proteins exist in the plasma membrane of a specimen or how they are distributed two-dimensionally. Here, we describe an unroofing procedure that clearly reveals CAV1 localization in a single plane of the plasma membrane and also demonstrate a super-resolution structured illumination microscopy technique for observation of CAV1 in the plasma membrane.

Membrane curvature plays a pivotal role in cellular life, including cellular uptake and membrane trafficking. The modulation of membrane curvature provides a novel means of manipulating cellular events. In this report, we show that a nine-residue amphiphilic peptide (R6W3) stimulates endocytic uptake by inducing membrane curvature. Curvature formation on cell membranes was confirmed by observing the cellular distribution of the curvature-sensing protein amphiphysin fused with a yellow fluorescent protein (Amp-YFP). Dot-like signals of Amp-YFP were visible following the addition of R6W3, suggesting curvature formation in cell membranes, leading to endocytic cup and vesicle formation. The promotion of endocytic uptake was confirmed using the endocytosis marker polydextran. Treatment of cells with R6W3 yielded a 4-fold dextran uptake compared with untreated cells. The amphiphilic helical structure of R6W3 was also crucial for R6W3-stimulated endocytic uptake.

Protein prenylation is essential for many cellular processes including signal transduction, cytoskeletal reorganization, and membrane trafficking. Here, we identify a novel type of protein prenyltransferase, which we named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of RabGGTase. Using a biotinylated geranylgeranyl analogue, we identified the Golgi SNARE protein Ykt6 as a substrate of GGTase-III. GGTase-III transfers a geranylgeranyl group to mono-farnesylated Ykt6, generating doubly prenylated Ykt6. The crystal structure of GGTase-III in complex with Ykt6 provides structural basis for Ykt6 double prenylation. In GGTase-III-deficient cells, Ykt6 remained in a singly prenylated form, and the Golgi SNARE complex assembly was severely impaired. Consequently, the Golgi apparatus was structurally disorganized, and intra-Golgi protein trafficking was delayed. Our findings reveal a fourth type of protein prenyltransferase that generates geranylgeranyl-farnesyl Ykt6. Double prenylation of Ykt6 is essential for the structural and functional organization of the Golgi apparatus.

The membrane raft has been a focus of intensive research for the past two decades. Liquid-ordered domains form in artificial liposomes containing sterol and saturated lipids, but their presence in living cell membranes has been controversial. The yeast vacuole is exceptional in that micron-sized raft-like domains form in the stationary phase and under several other conditions. The sterol content of the vacuole in the log phase is much lower than that of liposomes showing liquid-ordered domains, suggesting that sterols may need to be supplied to the vacuole for the raft-like domain formation. We will discuss how lipids and lipid domains are organized in the vacuolar membrane and examine whether evidence is strong enough to conclude that the observed micron-sized domains are rafts., ファイル公開：2019/10/19

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.

Microautophagy is a mode of autophagy in which the lysosomal or vacuolar membrane invaginates and engulfs target components. Oku et al. show that, upon a diauxic shift, yeast microautophagy involves recruitment of ESCRT proteins to the vacuolar membrane, including clathrin-interacting Vps27, and uptake of lipid droplets by the vacuole., Microautophagy refers to a mode of autophagy in which the lysosomal or vacuolar membrane invaginates and directly engulfs target components. The molecular machinery of membrane dynamics driving microautophagy is still elusive. Using immunochemical monitoring of yeast vacuolar transmembrane proteins, Vph1 and Pho8, fused to fluorescent proteins, we obtained evidence showing an induction of microautophagy after a diauxic shift in the yeast Saccharomyces cerevisiae. Components of the endosomal sorting complex required for transport machinery were found to be required for this process, and the gateway protein of the machinery, Vps27, was observed to change its localization onto the vacuolar membrane after a diauxic shift. We revealed the functional importance of Vps27’s interaction with clathrin in this microautophagy that also contributed to uptake of lipid droplets into the vacuole. This study sheds light on the molecular mechanism of microautophagy, which does not require the core Atg proteins.

Fluorescence microscopy is currently one of the more powerful and versatile techniques available for biological studies. With conventional biological immunofluorescence microscopy, caveolin-1 (CAV1) is visualized as numerous small dots, which are often distributed as a linear array or along the edge of the cell. Although its presence, as well as that of other proteins, can be detected by conventional immunofluorescence microscopy, those results do not clarify whether two different proteins exist in the plasma membrane of a specimen or how they are distributed two-dimensionally. Here, we describe an unroofing procedure that clearly reveals CAV1 localization in a single plane of the plasma membrane and also demonstrate a super-resolution structured illumination microscopy technique for observation of CAV1 in the plasma membrane.

ER‐phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER‐phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro‐ER‐phagy has been discovered, the molecules and mechanisms mediating micro‐ER‐phagy remain unknown. Here, we first show that micro‐ER‐phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1‐Spo7 phosphatase complex and the ESCRT machinery as key components for micro‐ER‐phagy. Third, we demonstrate that macro‐ and micro‐ER‐phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro‐ER‐phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis., Micro‐ER‐phagy requires the Nem1‐Spo7 phosphatase complex and ESCRT proteins for membrane scission during uptake of multilamellar ER whorls into the lysosome.

The voltage-sensing phosphatase (VSP) is a unique protein that shows voltage-dependent phosphoinositide phosphatase activity. Here we report that VSP is activated in mice sperm flagellum and generates a unique subcellular distribution pattern of PtdIns(4,5)P(2). Sperm from VSP(−/−) mice show more Ca(2+) influx upon capacitation than VSP(+/−) mice and abnormal circular motion. VSP-deficient sperm showed enhanced activity of Slo3, a PtdIns(4,5)P(2)-sensitive K(+) channel, which selectively localizes to the principal piece of the flagellum and indirectly enhances Ca(2+) influx. Most interestingly, freeze-fracture electron microscopy analysis indicates that normal sperm have much less PtdIns(4,5)P(2) in the principal piece than in the midpiece of the flagellum, and this polarized PtdIns(4,5)P(2) distribution disappeared in VSP-deficient sperm. Thus, VSP appears to optimize PtdIns(4,5)P(2) distribution of the principal piece. These results imply that flagellar PtdIns(4,5)P(2) distribution plays important roles in ion channel regulation as well as sperm motility.

Lipid droplets (LDs) are not an inert storage of excessive lipids, but play various roles in cellular lipid metabolism. Autophagy involves several mechanisms for the degradation of cellular components, and is related to many aspects of lipid metabolism. LD and autophagic membranes often distribute in proximity, but their relationship is complex. LDs can be degraded by autophagy, but LDs are also generated as a result of autophagy or support the execution of autophagy. Moreover, several proteins crucial for autophagy were shown to affect different aspects of LD formation. This article aims to categorize this multifaceted and seemingly entangled LD-autophagy relationship and to discuss unresolved issues.

TMEM16K, a membrane protein carrying 10 transmembrane regions, has phospholipid scramblase activity. TMEM16K is localized to intracellular membranes, but whether it actually scrambles phospholipids inside cells has not been demonstrated, due to technical difficulties in studying intracellular lipid distributions. Here, we developed a freeze-fracture electron microscopy method that enabled us to determine the phosphatidylserine (PtdSer) distribution in the individual leaflets of cellular membranes. Using this method, we found that the endoplasmic reticulum (ER) of mammalian cells harbored abundant PtdSer in its cytoplasmic leaflet and much less in the luminal leaflet, whereas the outer and inner nuclear membranes (NMs) had equivalent amounts of PtdSer in both leaflets. The ER and NMs of budding yeast also harbored PtdSer in their cytoplasmic leaflet, but asymmetrical distribution in the ER was not observed. Treating mouse embryonic fibroblasts with the Ca(2+) ionophore A23187 compromised the cytoplasmic leaflet-dominant PtdSer asymmetry in the ER and increased PtdSer in the NMs, especially in the nucleoplasmic leaflet of the inner NM. This Ca(2+)-induced PtdSer redistribution was not observed in TMEM16K-null fibroblasts, but was recovered in these cells by reexpressing TMEM16K. These results indicate that, similar to the plasma membrane, PtdSer in the ER of mammalian cells is predominantly localized to the cytoplasmic leaflet, and that TMEM16K directly or indirectly mediates Ca(2+)-dependent phospholipid scrambling in the ER.

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and ESCRT proteins as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that ESCRT proteins directly function in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

Phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] is a phosphoinositide that plays important roles in signal transduction, endocytosis, and cell migration among others. The intracellular distribution of PtdIns(3,4)P2 has mainly been studied by observing the distribution of GFP-tagged PtdIns(3,4)P2-binding protein domains in live cells and by labeling with anti-PtdIns(3,4)P2 antibody in fixed cell samples, but these methods only offer low spatial resolution results and may have pitfalls. In the present study, we developed an electron microscopic method to observe the PtdIns(3,4)P2 distribution using the SDS-treated freeze-fracture replica labeling method. The recombinant GST-tagged pleckstrin homology (PH) domain of TAPP1 was used as the binding probe, and its binding to PtdIns(3,4)P2 in the freeze-fracture replica was confirmed by using liposomes containing different phosphoinositides and by the lack of labeling by a mutant probe, in which one amino acid in the PH domain was substituted. The method was applied to NIH3T3 cell samples and showed that the increase of PtdIns(3,4)P2 in cells treated with hydrogen peroxide occurs in the cytoplasmic leaflet of the plasma membrane, except in the caveolar membrane. The present method can define the distribution of PtdIns(3,4)P2 at a high spatial resolution and will facilitate our understanding of the physiological function of this less studied phosphoinositide.

New functionalities of phosphoinositides (PIs) are being revealed continuously, and the scale of the membrane area studied is becoming smaller, from the micrometer range like the entire surface of organelles to the nanometer range as in subdomains of organelles. Concurrently, function of less abundant PIs, such as PI(3,4)P2 and PI(3,5)P2, attracts increasing attention. In accordance with the progress, finer and more accurate information on PI distribution is required. The fluorescence biosensor method utilizing PI-binding domains and/or immunolabeling with anti-PI antibodies are used for this purpose in most studies but both methods are known to have caveats. In this article, we examined how PI distribution was defined in recent studies and discussed whether methodological uncertainty has any bearing on the results.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2–1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.

The origin and physiological significance of lipid droplets (LDs) in the nucleus is not clear. Here we show that nuclear LDs in hepatocytes are derived from apolipoprotein B (ApoB)-free lumenal LDs, a precursor to very low-density lipoproprotein (VLDL) generated in the ER lumen by microsomal triglyceride transfer protein. ApoB-free lumenal LDs accumulate under ER stress, grow within the lumen of the type I nucleoplasmic reticulum, and turn into nucleoplasmic LDs by disintegration of the surrounding inner nuclear membrane. Oleic acid with or without tunicamycin significantly increases the formation of nucleoplasmic LDs, to which CTP:phosphocholine cytidylyltransferase α (CCTα) is recruited, resulting in activation of phosphatidylcholine (PC) synthesis. Perilipin-3 competes with CCTα in binding to nucleoplasmic LDs, and thus, knockdown and overexpression of perilipin-3 increases and decreases PC synthesis, respectively. The results indicate that nucleoplasmic LDs in hepatocytes constitute a feedback mechanism to regulate PC synthesis in accordance with ER stress.

Because chemical fixatives like aldehydes do not work on most lipid molecules in the membrane, small-scale lipid distribution cannot be identified by immunoelectron microscopy in cells fixed by conventional methods. Here we describe a method for physically stabilizing membranes through quick-freezing and freeze-fracture replica formation and for specifically labeling gangliosides for electron microscopy. This method enables the ultrahigh-resolution mapping of membrane lipids including gangliosides within the two-dimensional plane of membranes.

Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP:phosphocholine cytidylyltransferase α and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) sustains prosurvival signalling directly downstream of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinoma. Here we report an unanticipated function of this receptor tyrosine kinase (RTK) as a scaffold of cavin-1 and caveolin-1 (CAV1), two essential structural components of caveolae. This kinase-independent function of ROR1 facilitates the interactions of cavin-1 and CAV1 at the plasma membrane, thereby preventing the lysosomal degradation of CAV1. Caveolae structures and prosurvival signalling towards AKT through multiple RTKs are consequently sustained. These findings provide mechanistic insight into how ROR1 inhibition can overcome EGFR–tyrosine kinase inhibitor (TKI) resistance due to bypass signalling via diverse RTKs such as MET and IGF-IR, which is currently a major clinical obstacle. Considering its onco-embryonic expression, inhibition of the scaffold function of ROR1 in patients with lung adenocarcinoma is an attractive approach for improved treatment of this devastating cancer., Resistance to receptor tyrosine kinase inhibitors is a major obstacle in treatment of lung adenocarcinoma. Yamaguchi et al. identify the orphan receptor ROR1 as a potential target to overcome this resistance, by virtue of its role in promoting cell survival through stabilisation of caveolae.