Mechanics of biomimetic free-standing lipid membranes: insights into the elasticity of complex lipid compositions.

The creation of free-standing lipid membranes has been so far of remarkable interest to investigate processes occurring in the cell membrane since its unsupported part enables studies in which it is important to maintain cell-like physicochemical properties of the lipid bilayer, that nonetheless depend on its molecular composition. In this study, we prepare pore-spanning membranes that mimic the composition of plasma membranes and perform force spectroscopy indentation measurements to unravel mechanistic insights depending on lipid composition. We show that this approach is highly effective for studying the mechanical properties of such membranes. Furthermore, we identify a direct influence of cholesterol and sphingomyelin on the elasticity of the bilayer and adhesion between the two leaflets. Eventually, we explore the possibilities of imaging in the unsupported membrane regions. For this purpose, we investigate the adsorption and movement of a peripheral protein, the fibroblast growth factor 2, on the complex membrane.

An optogenetic method for the controlled release of single molecules.

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions.

SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.

Neurons in the brain communicate with one another by passing molecules called neurotransmitters across the synapse connecting them together. Mutations in the machinery that controls neurotransmitter release can lead to epilepsy or developmental delays in early childhood, but how exactly is poorly understood. Neurotransmitter release is primarily controlled by three proteins that join together to form the SNARE complex, and another protein called synaptotagmin-1. This assembly of proteins primes vesicles containing neurotransmitter molecules to be released from the neuron. When calcium ions bind to synaptotagmin-1, this triggers vesicles in this readily releasable pool to then fuse with the cell membrane and secrete their contents into the small gap between the communicating neurons. Mutations associated with epilepsy and developmental delays have been found in all components of this release machinery. Here, Kádková, Murach, Østergaard et al. set out to find how three of these mutations, which are found in a protein in the SNARE complex called SNAP25, lead to aberrant neurotransmitter release. Two of these mutations are located in the interface between the SNARE complex and synaptotagmin-1, while the other is found within the bundle of proteins that make up the SNARE complex. In vitro and ex vivo experiments in mice revealed that the two interface mutations led to defects in vesicle priming, while at the same time bypassing the control by synaptotagmin-1, resulting in vesicles spontaneously fusing with the cell membrane in an unregulated manner. These mutations therefore combine loss-of-function and gain-of-function features. In contrast, the bundle mutation did not impact the number of vesicles in the releasable pool but reduced spontaneous and calcium ion evoked vesicle fusion. This was due to the mutation destabilizing the SNARE complex, which reduced the amount of energy available for merging vesicles to the membrane. These findings reveal how SNAP25 mutations can have different effects on synapse activity, and how these defects disrupt the release of neurotransmitters. This experimental framework could be used to study how other synaptic mutations lead to diseases such as epilepsy. Applying this approach to human neurons and live model organisms may lead to the discovery of new therapeutic targets for epilepsy and delayed development.

Phosphoinositide switches in cell physiology - From molecular mechanisms to disease.

Phosphoinositides are amphipathic lipid molecules derived from phosphatidylinositol that represent low abundance components of biological membranes. Rather than serving as mere structural elements of lipid bilayers, they represent molecular switches for a broad range of biological processes, including cell signaling, membrane dynamics and remodeling, and many other functions. Here, we focus on the molecular mechanisms that turn phosphoinositides into molecular switches and how the dysregulation of these processes can lead to disease.

Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

The sterol transporter STARD3 transports sphingosine at ER-lysosome contact sites.

Sphingolipids are important structural components of membranes. Additionally, simple sphingolipids such as sphingosine are highly bioactive and participate in complex subcellular signaling. Sphingolipid deregulation is associated with many severe diseases including diabetes, Parkinson's and cancer. Here, we focus on how sphingosine, generated from sphingolipid catabolism in late endosomes/lysosomes, is reintegrated into the biosynthetic machinery at the endoplasmic reticulum (ER). We characterized the sterol transporter STARD3 as a sphingosine transporter acting at lysosome-ER contact sites. Experiments featuring crosslinkable sphingosine probes, supported by unbiased molecular dynamics simulations, exposed how sphingosine binds to the lipid-binding domain of STARD3. Following the metabolic fate of pre-localized lysosomal sphingosine showed the importance of STARD3 and its actions at contact sites for the integration of sphingosine into ceramide in a cellular context. Our findings provide the first example of interorganellar sphingosine transfer and pave the way for a better understanding of sphingolipid - sterol co-regulation.

Determining the Functional Oligomeric State of Membrane-Associated Protein Oligomers Forming Membrane Pores on Giant Lipid Vesicles.

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion.

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy.

Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range of cellular processes, including signaling and protein homeostasis. I-CLiPs are multispanning membrane proteins and represent challenging targets in structural and enzyme biology. Here we introduce iCLiPSpy, a simple assay to study I-CLiPs in vivo. To allow easy detection of enzyme activity, we developed a heme-binding reporter based on TNFα that changes color after I-CLiP-mediated proteolysis. Co-expression of the protease and reporter in Escherichia coli (E. coli) results in white or green colonies, depending on the activity of the protease. As a proof of concept, we use this assay to study the bacterial intramembrane-cleaving zinc metalloprotease RseP in vivo. iCLiPSpy expands the methodological repertoire for identifying residues important for substrate binding or activity of I-CLiPs and can in principle be adapted to a screening assay for the identification of inhibitors or activators of I-CLiPs, which is of great interest for proteases being explored as biomedical targets.

Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2.

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2.

Unconventional secretion mediated by direct protein self-translocation across the plasma membranes of mammalian cells.

In recent years, a surprisingly complex picture emerged about endoplasmic reticulum (ER)/Golgi-independent secretory pathways, and several routes have been discovered that differ with regard to their molecular mechanisms and machineries. Fibroblast growth factor 2 (FGF2) is secreted by a pathway of unconventional protein secretion (UPS) that is based on direct self-translocation across the plasma membrane. Building on previous research, a component of this process has been identified to be glypican-1 (GPC1), a GPI-anchored heparan sulfate proteoglycan located on cell surfaces. These findings not only shed light on the molecular mechanism underlying this process but also reveal an intimate relationship between FGF2 and GPC1 that might be of critical relevance for the prominent roles they both have in tumor progression and metastasis.

A Role for Liquid-Ordered Plasma Membrane Nanodomains Coordinating the Unconventional Secretory Pathway of Fibroblast Growth Factor 2?

Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that belongs to a subgroup of extracellular proteins lacking N-terminal signal peptides. Whereas this phenomenon was already recognized in the early 1990s, detailed insights into the molecular mechanisms underlying alternative pathways of protein secretion from eukaryotic cells were obtained only recently. Today, we know about a number of alternative secretory mechanisms, collectively termed unconventional protein secretion (UPS). FGF2 belongs to a subgroup of cargo proteins secreted by direct translocation across the plasma membrane. This feature has been classified as type I UPS and is shared with other unconventionally secreted proteins, such as HIV-Tat and Tau. FGF2 translocation across the membrane is initiated through sequential interactions with the Na,K-ATPase, Tec kinase, and phosphoinositide PI(4,5)P2 at the inner plasma membrane leaflet. Whereas the first two are auxiliary factors of this pathway, the interaction of FGF2 with PI(4,5)P2 triggers the core mechanism of FGF2 membrane translocation. It is based on a lipidic membrane pore that is formed by PI(4,5)P2-induced oligomerization of FGF2. Membrane-inserted FGF2 oligomers are recognized as translocation intermediates that are resolved at the outer plasma membrane leaflet by glypican-1, a heparan sulfate proteoglycan that captures and disassembles FGF2 oligomers on cell surfaces. Here, we discuss recent findings suggesting the molecular machinery mediating FGF2 membrane translocation to be highly organized in liquid-ordered plasma membrane nanodomains, the core process underlying this unusual pathway of protein secretion.

Glypican-1 drives unconventional secretion of fibroblast growth factor 2.

Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse subclasses consisting of syndecans, perlecans, glypicans, and others, Glypican-1 (GPC1) is the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression.

Identification of cis-acting determinants mediating the unconventional secretion of tau.

The deposition of tau aggregates throughout the brain is a pathological characteristic within a group of neurodegenerative diseases collectively termed tauopathies, which includes Alzheimer's disease. While recent findings suggest the involvement of unconventional secretory pathways driving tau into the extracellular space and mediating the propagation of the disease-associated pathology, many of the mechanistic details governing this process remain elusive. In the current study, we provide an in-depth characterization of the unconventional secretory pathway of tau and identify novel molecular determinants that are required for this process. Here, using Drosophila models of tauopathy, we correlate the hyperphosphorylation and aggregation state of tau with the disease-related neurotoxicity. These newly established systems recapitulate all the previously identified hallmarks of tau secretion, including the contribution of tau hyperphosphorylation as well as the requirement for PI(4,5)P2 triggering the direct translocation of tau. Using a series of cellular assays, we demonstrate that both the sulfated proteoglycans on the cell surface and the correct orientation of the protein at the inner plasma membrane leaflet are critical determinants of this process. Finally, we identify two cysteine residues within the microtubule binding repeat domain as novel cis-elements that are important for both unconventional secretion and trans-cellular propagation of tau.

FGF2 and IL-1ß - explorers of unconventional secretory pathways at a glance.

Fibroblast growth factor 2 (FGF2) and interleukin 1ß (IL-1ß) were among the earliest examples of a subclass of proteins with extracellular functions that were found to lack N-terminal secretory signal peptides and were shown to be secreted in an ER- and Golgi-independent manner. Many years later, a number of alternative secretory pathways have been discovered, processes collectively termed unconventional protein secretion (UPS). In the course of these studies, unconventional secretion of FGF2 and IL-1ß were found to be based upon distinct pathways, mechanisms and molecular machineries. Following a concise introduction into various pathways mediating unconventional secretion and transcellular spreading of proteins, this Cell Science at a Glance poster article aims at a focused analysis of recent key discoveries providing unprecedented detail about the molecular mechanisms and machineries driving FGF2 and IL-1ß secretion. These findings are also highly relevant for other unconventionally secreted cargoes that, like FGF2 and IL1ß, exert fundamental biological functions in biomedically relevant processes, such as tumor-induced angiogenesis and inflammation.

Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation.

In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P2-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure-function relationship of membrane-inserted oligomers.

Risk Factors for Failure of Nonoperative Treatment of Posterior Shoulder Labral Tears on Magnetic Resonance Imaging.

INTRODUCTION: There are no reports in the literature describing risk factors for failure of nonoperative treatment of patients with posterior labral tears on magnetic resonance imaging (MRI). The purpose of this study is to identify risk factors for failure of nonoperative treatment in patients with an isolated posterior glenoid labral tear identified on MRI only. Patients with posterior labral tears on MRI who fail to improve with nonoperative treatment likely share a constellation of clinical history, physical exam, and radiographic findings. METHODS: One hundred and fifty-nine active duty military service members under the age of 40 with a posterior labral tear seen on MRI and who were clinically evaluated by a musculoskeletal trained physician were identified. We retrospectively evaluated their records ensuring a minimum of 2 years follow-up after MRI to identify surgical intervention for the posterior labral tear during this time period. Patients were stratified into two groups, those treated with any combination of nonoperative modalities and those treated with posterior labral repair surgery during the 2 years after the MRI. The electronic medical records were reviewed for clinical presentation and physical exam results. We measured multiple radiographic parameters, including glenoid version, size of the tear, and bone loss on MRI. Qualitative and quantitative data were compared between groups using Fisher's exact test and Student's t-test, respectively. This study was conducted under institutional review board approval. RESULTS: Of the 157 patients' shoulders in our study, 52% (n = 82) of patients with posterior labral tears underwent nonoperative treatment while 48% (n = 75) underwent surgery. The significant risk factors associated with surgery were a history of a specific injury, primary presenting complaint of instability, patient reported history of subluxation, inability to trust their shoulder with overhead activity, decreased strength with weight lifting, positive posterior load/shift exam, positive anterior apprehension, increased osseous glenoid retroversion, increased humeral head subluxation ratio, and anterior labral height (P < 0.05). Patients with a chief complaint of pain were much more likely to succeed with nonoperative treatment while those with instability underwent surgery more often. Ten (12.5%) of the surgical procedures included an anterior and posterior labral repair/stabilization procedure. CONCLUSION: Patients with an MRI confirmed posterior labral tear, which present with subjective complaints and physical exam maneuvers consistent with instability, appear less likely to be treated nonoperatively. Increased glenoid retroversion and posterior humeral head subluxation may also predispose patients toward surgical treatment. Additionally, posterior labral tears may extend into the anterior labrum more frequently than is recognized on MRI.

The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast Growth Factor 2.

FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces.

A time-resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling.

Fibroblast growth factor 2 (FGF2) is a cell survival factor with crucial functions in tumor-induced angiogenesis. Here, we describe a novel time-resolved FGF2 signaling assay based upon live cell imaging of neuroblastoma cells. To validate this system, we tested 8960 small molecules for inhibition of FGF2 signaling with kinetic resolution. Hit compounds were validated in dose-response experiments for FGF2 signaling, FGF receptor antagonism, downstream ERK phosphorylation and FGF2-dependent chemoresistance in a cellular leukemia model system. The new screening system for FGF2 signaling inhibitors has unique features, deselecting compounds with pleiotropic effects on cell proliferation and, along with the experimental pipeline reported, great potential for the discovery of new classes of FGF2 signaling inhibitors that block FGF2 dependent tumor cell survival.