ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

TRPV4-dependent Ca2+ influx determines cholesterol dynamics at the plasma membrane.

The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable nonselective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy. To this aim, we labeled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed both inside and outside of caveolae. Agonist-evoked TRPV4 activation remarkably decreased colocalization probability and association rate between TRPV4 and D4H-accessible cholesterol molecules. Interestingly, upon TRPV4 activation, the particle density of D4H-accessible cholesterol molecules was decreased and the D4H-accessible cholesterol molecules in the fast-diffusing state were increased at the plasma membrane. The introduction of skeletal dysplasia-associated R616Q mutation into the CRAC/CARC motif of TRPV4, which reduced the interaction with cholesterol clusters, could not alter the D4H-accessible cholesterol dynamics. Mechanistically, TRPV4-mediated Ca2+ influx and the C-terminal calmodulin-binding site of TRPV4 are essential for modulating the plasmalemmal D4H-accessible cholesterol dynamics. We propose that TRPV4 remodels its surrounding plasmalemmal environment by manipulating cholesterol dynamics through Ca2+ influx.

BCL6B Contributes to Ocular Vascular Diseases via Notch Signal Silencing.

BACKGROUND: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. METHODS: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. RESULTS: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. CONCLUSIONS: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

Macropinoscope: Real-Time Simultaneous Tracking of pH and Cathepsin B Activity in Individual Macropinosomes.

A fluorescent sensor that allows simultaneous analysis of environmental factors in a limited cellular space is useful for understanding precise molecular interactions in live cells and their biological responses. Macropinocytosis is a ubiquitous endocytic pathway for massive uptake of extracellular fluids, resulting in the formation of macropinosomes. Although macropinocytosis may impact intracellular delivery and cancer proliferation, information on the intracellular behaviors of macropinosomes is limited. Here, we aimed to develop a macropinoscope, a sensor that simultaneously detects pH and cathepsin B activity in individual macropinosomes. A macropinosome-specific marker, dextran (70 kDa), was employed as a platform, onto which fluorescein, Oregon Green, and tetramethylrhodamine were loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavable peptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was also mounted; cleavage of the sequence was detected as an increase in sulfo-Cy5 fluorescence. A steep decrease in pH was observed 5-10 min after macropinosome formation, which was accompanied by an immediate increase in cathepsin B activity. Our design concept will lead to the development of other macropinoscopes for the simultaneous detection of other parameters in individual macropinosomes.

SPOP is essential for DNA replication licensing through maintaining translation of CDT1 and CDC6 in HaCaT cells.

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, leads to the ubiquitination of >40 of its target substrates. Since a variety of point mutations in the substrate-binding domain of SPOP have been identified in cancers, including prostate and endometrial cancers, the pathological roles of those cancer-associated SPOP mutants have been extensively elucidated. In this study, we evaluated the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells expressing wild-type SPOP gene. SPOP knockdown using siRNA in HaCaT cells dramatically reduced cell growth and arrested their cell cycles at G1/S phase. The expression of DNA replication licensing factors CDT1 and CDC6 in HaCaT cells drastically decreased on SPOP knockdown as their translation was inhibited. CDT1 and CDC6 downregulation induced p21 expression without p53 activation. Our results suggest that SPOP is essential for DNA replication licensing in non-cancerous keratinocyte HaCaT cells.

Aberrant accumulation of NIK promotes tumor growth by dysregulating translation and post-translational modifications in breast cancer.

BACKGROUND: In vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines. METHODS: We established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein. RESULTS: As a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1. CONCLUSIONS: Our study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.

KCTD10 Biology: An Adaptor for the Ubiquitin E3 Complex Meets Multiple Substrates: Emerging Divergent Roles of the cullin-3/KCTD10 E3 Ubiquitin Ligase Complex in Various Cell Lines.

Protein ubiquitination constitutes a post-translational modification mediated by ubiquitin ligases whereby ubiquitinated substrates are degraded through the proteasomal or lysosomal pathways, or acquire novel molecular functions according to their "ubiquitin codes." Dysfunction of the ubiquitination process in cells causes various diseases such as cancers along with neurodegenerative, auto-immune/inflammatory, and metabolic diseases. KCTD10 functions as a substrate recognition receptor for cullin-3 (CUL3), a scaffold protein in RING-type ubiquitin ligase complexes. Recently, studies by ourselves and others have identified new substrates that are ubiquitinated by the CUL3/KCTD10 ubiquitin ligase complex. Moreover, the type of polyubiquitination (e.g., K27-, K48-, or K63-chain) of various substrates (e.g., RhoB, CEP97, EIF3D, and TRIF) mediated by KCTD10 underlies its divergent roles in endothelial barrier formation, primary cilium formation, plasma membrane dynamics, cell proliferation, and immune response. Here, the physiological functions of KCTD10 are summarized and potential mechanisms are proposed.

PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA positron emission tomography/computed tomography has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear. METHODS: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium (CM) derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the CM. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived CM using a tube formation assay that mimics angiogenesis. RESULTS: Immunohistochemistry of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in 4 of 33 prostate cancer patients (12.1%). We also found that the 10,000g pellet fraction of the LNCaP-derived CM containing PSMA-positive membranes, such as microvesicles transformed HUVECs "PSMA-negative" into "PSMA-positive." Furthermore, treatment of HUVECs with the 10,000g pellet fraction of the LNCaP-derived CM significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner. CONCLUSIONS: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues.

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

ANKFY1 is essential for retinal endothelial cell proliferation and migration via VEGFR2/Akt/eNOS pathway.

Dysregulation of endothelial cell proliferation and migration are hallmarks of angiogenic diseases. Among them, excessive ocular angiogenesis is a major cause of blindness. Vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling plays crucial roles in angiogenesis, endothelial cell proliferation and migration. Here, we showed that ankyrin repeat and FYVE domain containing 1 (ANKFY1), a Rab5-GTP-interacting protein, is required for retinal endothelial cell proliferation and migration. ANKFY1 knockdown significantly suppressed cell growth of human retinal microvascular endothelial cells (HRMECs) in the presence or absence of VEGF. HRMEC migration was also inhibited by depletion of ANKFY1. Western blot analysis showed that ANKFY1 knockdown reduced cell surface VEGFR2 level. In contrast, qRT-PCR analysis indicated that ANKFY1 knockdown had no effect on VEGFR2 mRNA levels. We also found that the attenuation of the protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) pathway in ANKFY1 knockdown HRMECs. In conclusion, our findings revealed novel functions of ANKFY1 in cell growth and migration of retinal endothelial cells.

The Roles of SPOP in DNA Damage Response and DNA Replication.

Speckle-type BTB/POZ protein (SPOP) is a substrate recognition receptor of the cullin-3 (CUL3)/RING type ubiquitin E3 complex. To date, approximately 30 proteins have been identified as ubiquitinated substrates of the CUL3/SPOP complex. Pathologically, missense mutations in the substrate-binding domain of SPOP have been found in prostate and endometrial cancers. Prostate and endometrial cancer-associated SPOP mutations lose and increase substrate-binding ability, respectively. Expression of these SPOP mutants, thus, causes aberrant turnovers of the substrate proteins, leading to tumor formation. Although the molecular properties of SPOP and its cancer-associated mutants have been intensively elucidated, their cellular functions remain unclear. Recently, a number of studies have uncovered the critical role of SPOP and its mutants in DNA damage response and DNA replication. In this review article, we summarize the physiological functions of SPOP as a "gatekeeper" of genome stability.

SNX9 determines the surface levels of integrin ß1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9.

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin ß1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin ß1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.

Cullin-3/KCTD10 E3 complex is essential for Rac1 activation through RhoB degradation in human epidermal growth factor receptor 2-positive breast cancer cells.

Rho GTPase Rac1 is a central regulator of F-actin organization and signal transduction to control plasma membrane dynamics and cell proliferation. Dysregulated Rac1 activity is often observed in various cancers including breast cancer and is suggested to be critical for malignancy. Here, we showed that the ubiquitin E3 ligase complex Cullin-3 (CUL3)/KCTD10 is essential for epidermal growth factor (EGF)-induced/human epidermal growth factor receptor 2 (HER2)-dependent Rac1 activation in HER2-positive breast cancer cells. EGF-induced dorsal membrane ruffle formation and cell proliferation that depends on both Rac1 and HER2 were suppressed in CUL3- or KCTD10-depleted cells. Mechanistically, CUL3/KCTD10 ubiquitinated RhoB for degradation, another Rho GTPase that inhibits Rac1 activation at the plasma membrane by suppressing endosome-to-plasma membrane traffic of Rac1. In HER2-positive breast cancers, high expression of Rac1 mRNA significantly correlated with poor prognosis of the patients. This study shows that this novel molecular axis (CUL3/KCTD10/RhoB) positively regulates the activity of Rac1 in HER2-positive breast cancers, and our findings may lead to new treatment options for HER2- and Rac1-positive breast cancers.

Cullin-3/KCTD10 complex is essential for K27-polyubiquitination of EIF3D in human hepatocellular carcinoma HepG2 cells.

Eukaryotic translation initiation factor 3 subunit D (EIF3D) binds to the 5'-cap of specific mRNAs, initiating their translation into polypeptides. From a pathological standpoint, EIF3D has been observed to be essential for cell growth in various cancer types, and cancer patients with high EIF3D mRNA levels exhibit poor prognosis, indicating involvement of EIF3D in oncogenesis. In this study, we found, by mass spectrometry, that Cullin-3 (CUL3)/KCTD10 ubiquitin (Ub) ligase forms a complex with EIF3D. We also demonstrated that EIF3D is K27-polyubiquitinated at the lysine 153 and 275 residues in a KCTD10-dependent manner in human hepatocellular carcinoma HepG2 cells. Similar to other cancers, high expression of EIF3D significantly correlated with poor prognosis in hepatocellular carcinoma patients, and depletion of EIF3D drastically suppressed HepG2 cell proliferation. These results indicate that EIF3D is a novel substrate of CUL3/KCTD10 Ub ligase and suggest involvement of K27-polyubiquitinated EIF3D in the development of hepatocellular carcinoma.

Cullin 3 regulates ADAMs-mediated ectodomain shedding of amphiregulin.

A disintegrin and metalloproteinase (ADAM) family are crucial enzymes for ectodomain shedding of multiple substrates and are involved in diverse biologic and pathologic processes. However, the molecular mechanism underlying substrate selectivity of ADAMs is poorly understood. In this study, we observed that disruption of actin polymerization by pharmacological inhibitors, latrunculin A (LatA) and cytochalasin D (CyD), induced ectodomain shedding of epidermal growth factor (EGF) family ligands. Induced shedding activity by LatA or CyD was suppressed by a metalloprotease inhibitor KB-R7785, indicating that ADAMs-mediated shedding is tightly controlled by actin cytoskeleton. We also investigated roles of cullin family, a component of cullin-RING based E3 ubiquitin ligases, in ectodomain shedding, since cullin family is implicated in the regulation of cytoskeletal dynamics. Knockdown of cullin 3 (Cul3) by a specific siRNA inhibited ectodomain shedding of amphiregulin (AREG), a member of EGF family, and responses were associated with activation of RhoA GTPase and induction of stress fiber formation. On the other hand, the RhoA inhibitor C3 transferase rescued AREG shedding reduced by Cul3 knockdown. These results describe a novel molecular mechanism of Cul3 to regulate AREG shedding by modulating cytoskeletal dynamics in a RhoA dependent manner.

Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.

Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.

Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.

Cholesterol is an essential component of metazoan cellular membranes and it helps to maintain the structural integrity and fluidity of the plasma membrane. Here, we developed a cholesterol biosensor, termed D4H, based on the fourth domain of Clostridium perfringens theta-toxin, which recognizes cholesterol in the cytosolic leaflet of the plasma membrane and organelles. The D4H probe disassociates from the plasma membrane upon cholesterol extraction and after perturbations in cellular cholesterol trafficking. When used in combination with a recombinant version of the biosensor, we show that plasmalemmal phosphatidylserine is essential for retaining cholesterol in the cytosolic leaflet of the plasma membrane. In vitro experiments reveal that 1-stearoy-2-oleoyl phosphatidylserine can induce phase separation in cholesterol-containing lipid bilayers and shield cholesterol from cholesterol oxidase. Finally, the altered transbilayer distribution of cholesterol causes flotillin-1 to relocalize to endocytic organelles. This probe should be useful in the future to study pools of cholesterol in the cytosolic leaflet of the plasma membrane and organelles.

Sequential breakdown of 3-phosphorylated phosphoinositides is essential for the completion of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and solutes are internalized into cells. Macropinocytosis starts with the formation of membrane ruffles at the plasma membrane and ends with their closure. The transient and sequential emergence of phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 in the membrane ruffles is essential for macropinocytosis. By making use of information in the Caenorhabditis elegans mutants defective in fluid-phase endocytosis, we found that mammalian phosphoinositide phosphatase MTMR6 that dephosphorylates PI(3)P to PI, and its binding partner MTMR9, are required for macropinocytosis. INPP4B, which dephosphorylates PI(3,4)P2 to PI(3)P, was also found to be essential for macropinocytosis. These phosphatases operate after the formation of membrane ruffles to complete macropinocytosis. Finally, we showed that KCa3.1, a Ca(2+)-activated K(+) channel that is activated by PI(3)P, is required for macropinocytosis. We propose that the sequential breakdown of PI(3,4,5)P3 → PI(3,4)P2 → PI(3)P → PI controls macropinocytosis through specific effectors of the intermediate phosphoinositides.

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes-The Update.

The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

Molecular probes to visualize the location, organization and dynamics of lipids.

Cellular lipids play crucial roles in the cell, including in energy storage, the formation of cellular membranes, and in signaling and vesicular trafficking. To understand the functions and characteristics of lipids within cells, various methods to image lipids have been established. In this Commentary, we discuss the four main types of molecular probes that have significantly contributed to our understanding of the cell biology of lipids. In particular, genetically encoded biosensors and antibodies will be discussed, and how they have been used extensively with traditional light and electron microscopy to determine the subcellular localization of lipids and their spatial and temporal regulation. We highlight some of the recent studies that have investigated the distribution of lipids and their ability to cluster using super-resolution and electron microscopy. We also examine methods for analyzing the movement and dynamics of lipids, including single-particle tracking (SPT), fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). Although the combination of these lipid probes and the various microscopic techniques is very powerful, we also point out several potential caveats and limitations. Finally, we discuss the need for new probes for a variety of phospholipids and cholesterol.