Elongated membrane zones boost interactions of diffusing proteins.

Biological membranes are two dimensional, making the discovery of quasi-one-dimensional diffusion of membrane proteins puzzling. Jaqaman et al. (2011) now show that actomyosin and tubulin interact to establish long, thin diffusion corridors, thereby increasing the effective concentration of select membrane proteins to promote their interactions and modulate signaling.

Initiation and evolution of pores formed by influenza fusion peptides probed by lysolipid inclusion.

The fusion peptide (FP) domain is necessary for the fusogenic activity of spike proteins in a variety of enveloped viruses, allowing the virus to infect the host cell, and is the only part of the protein that interacts directly with the target membrane lipid tails during fusion. There are consistent findings of poration by this domain in experimental model membrane systems, and, in certain conditions, the isolated FPs can generate pores. Here, we use molecular dynamics simulations to investigate the specifics of how these FP-induced pores form in membranes with different compositions of lysolipid and POPC. The simulations show that pores form spontaneously at high lysolipid concentrations via hybrid intermediates, where FP aggregates in the cis leaflet tilt to form a funnel-like structure that spans the leaflet and locally reduces the hydrophobic thickness that must be traversed by water to form a pore. By restraining a single FP within an FP aggregate to this tilted conformation, pores can be formed in lower-lysolipid-content membranes, including pure POPC, on the 100-ns timescale, much more rapidly than in unbiased simulations in bilayers with the same composition. The pore formation pathway is similar to the spontaneous formation in high lysolipid concentrations. Depending on the membrane composition, the pores can be metastable (as seen in POPC) or lead to membrane rupture.

Relative quantification of progressive changes in healthy and dysferlin-deficient mouse skeletal muscle proteomes.

INTRODUCTION/AIMS: Individuals with dysferlinopathies, a group of genetic muscle diseases, experience delay in the onset of muscle weakness. The cause of this delay and subsequent muscle wasting are unknown, and there are currently no clinical interventions to limit or prevent muscle weakness. To better understand molecular drivers of dysferlinopathies, age-dependent changes in the proteomic profile of skeletal muscle (SM) in wild-type (WT) and dysferlin-deficient mice were identified. METHODS: Quadriceps were isolated from 6-, 18-, 42-, and 77-wk-old C57BL/6 (WT, Dysf+/+ ) and BLAJ (Dysf-/- ) mice (n = 3, 2 male/1 female or 1 male/2 female, 24 total). Whole-muscle proteomes were characterized using liquid chromatography-mass spectrometry with relative quantification using TMT10plex isobaric labeling. Principle component analysis was utilized to detect age-dependent proteomic differences over the lifespan of, and between, WT and dysferlin-deficient SM. The biological relevance of proteins with significant variation was established using Ingenuity Pathway Analysis. RESULTS: Over 3200 proteins were identified between 6-, 18-, 42-, and 77-wk-old mice. In total, 46 proteins varied in aging WT SM (p < .01), while 365 varied in dysferlin-deficient SM. However, 569 proteins varied between aged-matched WT and dysferlin-deficient SM. Proteins with significant variation in expression across all comparisons followed distinct temporal trends. DISCUSSION: Proteins involved in sarcolemma repair and regeneration underwent significant changes in SM over the lifespan of WT mice, while those associated with immune infiltration and inflammation were overly represented over the lifespan of dysferlin-deficient mice. The proteins identified herein are likely to contribute to our overall understanding of SM aging and dysferlinopathy disease progression.

Perivascular invasion of primary human glioblastoma cells in organotypic human brain slices: human cells migrating in human brain.

INTRODUCTION: Glioblastoma (GBM) is an aggressive primary brain cancer. Lack of effective therapy is related to its highly invasive nature. GBM invasion has been studied with reductionist systems that do not fully recapitulate the cytoarchitecture of the brain. We describe a human-derived brain organotypic model to study the migratory properties of GBM IDH-wild type ex vivo. METHODS: Non-tumor brain samples were obtained from patients undergoing surgery (n = 7). Organotypic brain slices were prepared, and green fluorescent protein (GFP)-labeled primary human GBM IDH-wild type cells (GBM276, GBM612, GBM965) were placed on the organotypic slice. Migration was evaluated via microscopy and immunohistochemistry. RESULTS: After placement, cells migrated towards blood vessels; initially migrating with limited directionality, sending processes in different directions, and increasing their speed upon contact with the vessel. Once merged, migration speed decreased and continued to decrease with time (p < 0.001). After perivascular localization, migration is limited along the blood vessels in both directions. The percentage of cells that contact blood vessels and then continue to migrate along the vessel was 92.5% (- 3.9/ + 2.9)% while the percentage of cells that migrate along the blood vessel and leave was 7.5% (- 2.9/ + 3.9) (95% CI, Clopper-Pearson (exact); n = 256 cells from six organotypic cultures); these percentages are significantly different from the random (50%) null hypothesis (z = 13.6; p < 10-7). Further, cells increase their speed in response to a decrease in oxygen tension from atmospheric normoxia (20% O2) to anoxia (1% O2) (p = 0.033). CONCLUSION: Human organotypic models can accurately study cell migration ex vivo. GBM IDH-wild type cells migrate toward the perivascular space in blood vessels and their migratory parameters change once they contact vascular structures and under hypoxic conditions. This model allows the evaluation of GBM invasion, considering the human brain microenvironment when cells are removed from their native niche after surgery.

Flexible scaffolding made of rigid BARs.

Crescent-shaped BAR domains are generic actors in the creation of membrane curvature. In this issue, Frost et al. (2008) reveal how collective twisting of rigid F-BAR domains on a soft membrane surface may lead to different membrane curvatures.

GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission.

The GTPase dynamin is critically involved in membrane fission during endocytosis. How does dynamin use the energy of GTP hydrolysis for membrane remodeling? By monitoring the ionic permeability through lipid nanotubes (NT), we found that dynamin was capable of squeezing NT to extremely small radii, depending on the NT lipid composition. However, long dynamin scaffolds did not produce fission: instead, fission followed GTPase-dependent cycles of assembly and disassembly of short dynamin scaffolds and involved a stochastic process dependent on the curvature stress imposed by dynamin. Fission happened spontaneously upon NT release from the scaffold, without leakage. Our calculations revealed that local narrowing of NT could induce cooperative lipid tilting, leading to self-merger of the inner monolayer of NT (hemifission), consistent with the absence of leakage. We propose that dynamin transmits GTP's energy to periodic assembling of a limited curvature scaffold that brings lipids to an unstable intermediate.

EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites.

Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites.

Dye Transport through Bilayers Agrees with Lipid Electropore Molecular Dynamics.

Although transport of molecules into cells via electroporation is a common biomedical procedure, its protocols are often based on trial and error. Despite a long history of theoretical effort, the underlying mechanisms of cell membrane electroporation are not sufficiently elucidated, in part, because of the number of independent fitting parameters needed to link theory to experiment. Here, we ask if the electroporation behavior of a reduced cell membrane is consistent with time-resolved, atomistic, molecular dynamics (MD) simulations of phospholipid bilayers responding to electric fields. To avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 10-50-ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes.

Lipid-dependence of target membrane stability during influenza viral fusion.

Although influenza kills about a half million people each year, even after excluding pandemics, there is only one set of antiviral drugs: neuraminidase inhibitors. By using a new approach utilizing giant unilamellar vesicles and infectious X-31 influenza virus, and testing for the newly identified pore intermediate of membrane fusion, we observed ∼30-87% poration, depending upon lipid composition. Testing the hypothesis that spontaneous curvature (SC) of the lipid monolayer controls membrane poration, our Poisson model and Boltzmann energetic considerations suggest a transition from a leaky to a non-leaky fusion pathway depending on the SC of the target membrane. When the target membrane SC is below approximately -0.20 nm-1 fusion between influenza virus and target membrane is predominantly non-leaky while above that fusion is predominantly leaky, suggesting that influenza hemagglutinin (HA)-catalyzed topological conversion of target membranes during fusion is associated with a loss of membrane integrity.

Adaptive optics improves multiphoton super-resolution imaging.

We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 µm from the coverslip surface.

Monolayerwise application of linear elasticity theory well describes strongly deformed lipid membranes and the effect of solvent.

The theory of elasticity of lipid membranes is used widely to describe processes of cell membrane remodeling. Classically, the functional of a membrane's elastic energy is derived under assumption of small deformations; the membrane is considered as an infinitely thin film. This functional is quadratic on membrane surface curvature, with half of the splay modulus as its proportionality coefficient; it is generally applicable for small deformations only. Any validity of this functional for the regime of strong deformations should be verified experimentally. Recently, research using molecular dynamics simulations challenged the validity of this classic, linear model, i.e. the constancy of the splay modulus for strongly bent membranes. Here we demonstrate that the quadratic energy functional still can be applied for calculation of the elastic energy of strongly deformed membranes without introducing higher order terms with additional elastic moduli, but only if applied separately for each lipid monolayer. For cylindrical membranes, both classic and monolayerwise models yield equally accurate results. For cylindrical deformations we experimentally show that the elastic energy of lipid monolayers is additive: a low molecular weight solvent leads to an approximately twofold decrease in the membrane bending stiffness. Accumulation of solvent molecules in the inner monolayer of a membrane cylinder can explain these results, as the solvent partially prevents lipid molecules from splaying there. Thus, the linear theory of elasticity can be expanded through the range from weak to strong deformations-its simplicity and physical transparency describe various membrane phenomena.

Ectodomain Pulling Combines with Fusion Peptide Inserting to Provide Cooperative Fusion for Influenza Virus and HIV.

Enveloped viruses include the most dangerous human and animal pathogens, in particular coronavirus, influenza virus, and human immunodeficiency virus (HIV). For these viruses, receptor binding and entry are accomplished by a single viral envelope protein (termed the fusion protein), the structural changes of which trigger the remodeling and merger of the viral and target cellular membranes. The number of fusion proteins required for fusion activity is still under debate, and several studies report this value to range from 1 to 9 for type I fusion proteins. Here, we consider the earliest stage of viral fusion based on the continuum theory of membrane elasticity. We demonstrate that membrane deformations induced by the oblique insertion of amphipathic fusion peptides mediate the lateral interaction of these peptides and drive them to form into a symmetric fusion rosette. The pulling force produced by the structural rearrangements of the fusion protein ectodomains gives additional torque, which deforms the membrane and additionally stabilizes the symmetric fusion rosette, thus allowing a reduction in the number of fusion peptides needed for fusion. These findings can resolve the large range of published cooperativity indices for HIV, influenza, and other type I fusion proteins.

Dynamic Relationship of the SNARE Complex with a Membrane.

Fusion of secretory granules and synaptic vesicles with the plasma membrane is driven by SNARE protein interactions. Intensive investigations in vitro, which include x-ray crystallography, cryoelectron microscopy, and NMR analyses by numerous groups, have elucidated structures relevant to the function of these proteins. Although function depends on the proteins being membrane bound, for experimental reasons, most of the studies have used cytosolic domains, as exemplified by the groundbreaking studies that elucidated the structure of a tetrapeptide helical bundle formed by interaction of the cytosolic domains of syntaxin1A, SNAP25 (two peptides) and synaptobrevin 2. Because the cytosolic fragments were unfettered by membrane attachments, it is likely that the tetrapeptide helical bundle reflects the lowest energy state, such as that found in the "cis" interactions of the SNARE motifs after fusion when they co-localize in the plasma membrane. Much more difficult to study and still poorly understood are critical "trans" interactions between the synaptic vesicle SNARE protein synaptobrevin 2 and the plasma membrane syntaxin1A/SNAP25 complex that initiate the fusion event. In a series of articles from the laboratory of Lukas Tamm, the spontaneous orientation of the SNARE motif of membrane-bound, full-length syntaxin1A with respect to the membrane hosting syntaxin's transmembrane domain was investigated with nanometer precision under a variety of conditions, including those that model aspects of the "trans" configuration. The studies rely on fluorescence interference-contrast microscopy, a technique that utilizes the pattern of constructive and destructive interference arising from incoming and reflected excitation and emission light at the surface of a silicon chip that has been layered with oxidized silicon of varying depths. This Perspective discusses their findings, including the unexpected influence of the degree of lipid unsaturation on the orientation of the SNARE complex.

Response to Blast-like Shear Stresses Associated with Mild Blast-Induced Brain Injury.

Toward the goal of understanding the pathophysiology of mild blast-induced traumatic brain injury and identifying the physical forces associated with the primary injury phase, we developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a timescale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we have characterized the shear transients experienced by the cells and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume. This system is compatible with high-resolution, time-lapse optical microscopy. Using this system, we demonstrate that blast-like shear transients produced with minimal pressure transients and submillisecond rise times activate calcium responses in dissociated human CNS cultures. Cells respond with increased cytosolic free calcium to a threshold shear stress between 8 and 21 Pa; the propagation of this calcium response is a result of purinergic signaling. We propose that this system models, in vitro, the fundamental injury wave produced by shear forces consequent to blast shock waves passing through density inhomogeneity in human CNS cells.

Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering.

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.

Subcutaneous adipose tissue imaging of human obesity reveals two types of adipocyte membranes: Insulin-responsive and -nonresponsive.

In adipose tissue, resistance to insulin's ability to increase glucose uptake can be induced by multiple factors, including obesity. Impaired insulin action may take place at different spatial loci at the cellular or subcellular level. To begin to understand the spatial response to insulin in human subcutaneous adipose tissue (hSAT), we developed a quantitative imaging method for activation of a major signaling node in the glucoregulatory insulin signaling pathway. After treatment with insulin or control media, biopsied tissues were immunostained for Akt phosphorylation at Thr-308/9 (pAkt) and then imaged by confocal fluorescence microscopy automated to collect a large grid of high resolution fields. In hSAT from 40 men and women with obesity, substantial heterogeneity of pAkt densities in adipocyte membranes were quantified in each image mosaic using a spatial unit of at least twice the size of the point spread function. Statistical analysis of the distribution of pAkt spatial units was best fit as the weighted sum of two separate distributions, corresponding to either a low or high pAkt density. A "high pAkt fraction" metric was calculated from the fraction of high pAkt distributed units over the total units. Importantly, upon insulin stimulation, tissues from the same biopsy showed either a minimal or a substantial change in the high pAkt fraction. Further supporting a two-state response to insulin stimulation, subjects with similar insulin sensitivity indices are also segregated into either of two clusters identified by the amount of membrane-localized pAkt.

Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes.

Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting membranes. Parasite Ca2+ , kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. Here, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live-cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite [Ca2+ ]i , the PV began rounding. Then after ~1.5 min, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next ~6 min, there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress programme when newly formed parasites mobilised and erythrocyte membranes permeabilised and then ruptured-a dramatic finale to the parasite cycle of replication.

Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.