Enhanced transcriptional heterogeneity mediated by NF-κB super-enhancers.

The transcription factor NF-κB, which plays an important role in cell fate determination, is involved in the activation of super-enhancers (SEs). However, the biological functions of the NF-κB SEs in gene control are not fully elucidated. We investigated the characteristics of NF-κB-mediated SE activity using fluorescence imaging of RelA, single-cell transcriptome and chromatin accessibility analyses in anti-IgM-stimulated B cells. The formation of cell stimulation-induced nuclear RelA foci was abolished in the presence of hexanediol, suggesting an underlying process of liquid-liquid phase separation. The gained SEs induced a switch-like expression and enhanced cell-to-cell variability in transcriptional response. These properties were correlated with the number of gained cis-regulatory interactions, while switch-like gene induction was associated with the number of NF-κB binding sites in SE. Our study suggests that NF-κB SEs have an important role in the transcriptional regulation of B cells possibly through liquid condensate formation consisting of macromolecular interactions.

A novel sterol-binding protein reveals heterogeneous cholesterol distribution in neurite outgrowth and in late endosomes/lysosomes.

We identified a mushroom-derived protein, maistero-2 that specifically binds 3-hydroxy sterol including cholesterol (Chol). Maistero-2 bound lipid mixture in Chol-dependent manner with a binding threshold of around 30%. Changing lipid composition did not significantly affect the threshold concentration. EGFP-maistero-2 labeled cell surface and intracellular organelle Chol with higher sensitivity than that of well-established Chol probe, D4 fragment of perfringolysin O. EGFP-maistero-2 revealed increase of cell surface Chol during neurite outgrowth and heterogeneous Chol distribution between CD63-positive and LAMP1-positive late endosomes/lysosomes. The absence of strictly conserved Thr-Leu pair present in Chol-dependent cytolysins suggests a distinct Chol-binding mechanism for maistero-2.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

The roles of the diversity of amphipathic lipids in shaping membranes by membrane-shaping proteins.

Lipid compositions of cells differ according to cell types and intracellular organelles. Phospholipids are major cell membrane lipids and have hydrophilic head groups and hydrophobic fatty acid tails. The cellular lipid membrane without any protein adapts to spherical shapes, and protein binding to the membrane is thought to be required for shaping the membrane for various cellular events. Until recently, modulation of cellular lipid membranes was initially shown to be mediated by proteins recognizing lipid head groups, including the negatively charged ones of phosphatidylserine and phosphoinositides. Recent studies have shown that the abilities of membrane-deforming proteins are also regulated by the composition of fatty acid tails, which cause different degrees of packing defects. The binding of proteins to cellular lipid membranes is affected by the packing defects, presumably through modulation of their interactions with hydrophobic amino acid residues. Therefore, lipid composition can be characterized by both packing defects and charge density. The lipid composition regarding fatty acid tails affects membrane bending via the proteins with amphipathic helices, including those with the ArfGAP1 lipid packing sensor (ALPS) motif and via membrane-deforming proteins with structural folding, including those with the Bin-Amphiphysin-Rvs167 (BAR) domains. This review focuses on how the fatty acid tails, in combination with the head groups of phospholipids, affect protein-mediated membrane deformation.

Transport through recycling endosomes requires EHD1 recruitment by a phosphatidylserine translocase.

P4-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P4-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2-/- mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2+/+ mice. These findings demonstrate the role of P4-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.

Phospholipase Cß1 induces membrane tubulation and is involved in caveolae formation.

Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cß1 (PLCß1) as a new candidate. PLCß1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCß1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCß1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCß1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCß1: plasma membrane remodeling, and in particular, caveolae formation.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Detection of Sphingomyelin Clusters by Raman Spectroscopy.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells that forms specific lipid domains in combination with cholesterol (Chol). Using molecular-dynamics simulation and density functional theory calculation, we identified a characteristic Raman band of SM at ∼1643 cm(-1) as amide I of the SM cluster. Experimental results indicate that this band is sensitive to the hydration of SM and the presence of Chol. We showed that this amide I Raman band can be utilized to examine the membrane distribution of SM. Similarly to SM, ceramide phosphoethanolamine (CerPE) exhibited an amide I Raman band in almost the same region, although CerPE lacks three methyl groups in the phosphocholine moiety of SM. In contrast to SM, the amide I band of CerPE was not affected by Chol, suggesting the importance of the methyl groups of SM in the SM-Chol interaction.

Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase-deficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates.

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family­pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)­strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 µM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

A Bioorthogonal Approach for the Preparation of a Titanium-Binding Insulin-like Growth-Factor-1 Derivative by Using Tyrosinase.

The generation of metal surfaces with biological properties, such as cell-growth-enhancing and differentiation-inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel-inspired bioorthogonal approach to design a 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth-factor-1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.

Sphingomyelin regulates the transbilayer movement of diacylglycerol in the plasma membrane of Madin-Darby canine kidney cells.

Diacylglycerol (DAG) is a key component in lipid metabolism and signaling. Previous model membrane studies using DAG analogs suggest their rapid membrane transbilayer movement. However, little is known about the DAG distribution and dynamics in cell membranes. Using live-cell fluorescence microscopy, we monitored the transbilayer movement of DAG with the yellow fluorescent protein-tagged C1AB domain from protein kinase C-γ (EYFP-C1AB), which selectively binds DAG. When HeLa cells were treated with Bacillus cereus phospholipase C (Bc-PLC) to produce DAG on the outer leaflet of the plasma membrane, intracellularly expressed EYFP-C1AB probe accumulated at the plasma membrane, indicating the transbilayer movement of the outer leaflet DAG to the inner leaflet. This Bc-PLC-induced translocation of EYFP-C1AB probe to the plasma membrane was not observed in the sphingolipid-enriched plasma membrane of Madin-Darby canine kidney cells, but was recovered after cell treatment with sphingomyelinase or preincubation with an inhibitor of sphingolipid biosynthesis. The inhibitory effect of sphingomyelin (SM) on the transbilayer movement of DAG was reproduced in model membranes using a fluorescent short-chain DAG analog. These results demonstrate that the SM content on the outer leaflet regulates the transbilayer movement of DAG in the plasma membrane, thus providing new insights into the dynamics of DAG in cell pathophysiology.

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains.

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-ß-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.

Quantitative Imaging Analysis of NF-κB for Mathematical Modeling Applications.

Mathematical models can integrate different types of experimental datasets, reconstitute biological systems in silico, and identify previously unknown molecular mechanisms. Over the past decade, mathematical models have been developed based on quantitative observations, such as live-cell imaging and biochemical assays. However, it is difficult to directly integrate next-generation sequencing (NGS) data. Although highly dimensional, NGS data mostly only provides a "snapshot" of cellular states. Nevertheless, the development of various methods for NGS analysis has led to much more accurate predictions of transcription factor activity and has revealed various concepts regarding transcriptional regulation. Therefore, fluorescence live-cell imaging of transcription factors can help alleviate the limitations in NGS data by supplementing temporal information, linking NGS to mathematical modeling. This chapter introduces an analytical method for quantifying dynamics of nuclear factor kappaB (NF-κB) which forms aggregates in the nucleus. The method may also be applicable to other transcription factors regulated in a similar fashion.

The SH3 binding site in front of the WH1 domain contributes to the membrane binding of the BAR domain protein endophilin A2.

The Bin-Amphiphysin-Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott-Aldrich syndrome protein (N-WASP). Here, we re-examined the binding sites of the SH3 domain of endophilin in N-WASP by machine learning-based prediction and identified the previously unrecognized binding site. In addition to the well-recognized PRM at the central proline-rich region, we found a PRM in front of the N-terminal WASP homology 1 (WH1) domain of N-WASP (NtPRM) as a binding site of the endophilin SH3 domain. Furthermore, the diameter of the membrane tubules in the presence of NtPRM mutant was narrower and wider than that in the presence of N-WASP and in its absence, respectively. Importantly, the NtPRM of N-WASP was involved in the membrane localization of endophilin A2 in cells. Therefore, the NtPRM contributes to the binding of endophilin to N-WASP in membrane remodeling.

Small GTPase Cdc42, WASP, and scaffold proteins for higher-order assembly of the F-BAR domain protein.

The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor. The assembly kinetics by fluorescence resonance energy transfer monitoring indicated that the GAS7 assembly on liposomes started within seconds and was further increased by the presence of these proteins. The regulated GAS7 assembly was abolished by Wiskott-Aldrich syndrome mutations both in vitro and in cellular phagocytosis. Therefore, Cdc42 and the scaffold proteins that commonly bind to the BAR domain superfamily proteins promoted GAS7 assembly.

Fusion of lipid vesicles with planar lipid bilayers induced by a combination of peptides.

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRF) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRF microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.

Impact of Intrinsic and Extrinsic Factors on Cellular Sphingomyelin Imaging with Specific Reporter Proteins.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. Although SM is enriched in the outer leaflet of the cell plasma membrane, lipids are also observed in the inner leaflet of the plasma membrane and intracellular organelles such as endolysosomes, the Golgi apparatus and nuclei. SM is postulated to form clusters with glycosphingolipids (GSLs), cholesterol (Chol), and other SM molecules through hydrophobic interactions and hydrogen bonding. Thus, different clusters composed of SM, SM/Chol, SM/GSL and SM/GSL/Chol with different stoichiometries may exist in biomembranes. In addition, SM monomers may be located in the glycerophospholipid-rich areas of membranes. Recently developed SM-binding proteins (SBPs) distinguish these different SM assemblies. Here, we summarize the effects of intrinsic factors regulating the lipid-binding specificity of SBPs and extrinsic factors, such as the lipid phase and lipid density, on SM recognition by SBPs. The combination of different SBPs revealed the heterogeneity of SM domains in biomembranes.

A nuclear envelop-associated baculovirus protein promotes intranuclear lipid accumulation during infection.

Although it has been well-accepted that baculoviruses produce a virus envelop within the nucleus, the redistribution of membrane lipids in infected cells has not been demonstrated. Here, we characterize a baculovirus protein (Bm5/Ac13: renamed BION; baculovirus protein associated with both the inner- and outer nuclear membranes) that localizes to both the inner- and outer nuclear membranes and show that the nuclear membrane (NE) protein promotes formation of a virus-induced intranuclear structure, the peristromal region (PR). Consistent with its role in virus envelopment, the PR was found to contain viral membrane proteins and lipids, suggesting PR formation proceeds through intranuclear lipid accumulation. About 50% of the cells infected with a bion-deficient virus exhibited no polyhedra production due to lack of the PR. Association of BION with the NE rather than the PR may contribute to the formation of the PR and polyhedra via NE-to-PR lipid transport.

Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes.

Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5-10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.