The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites.

Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood.

Rim aperture of yeast autophagic membranes balances cargo inclusion with vesicle maturation.

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time.

Phospholipid imbalance impairs autophagosome completion.

Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy.

Designer Liposomic Nanocarriers Are Effective Biofilm Eradicators.

Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation.

Spatial organization and early signaling of the B-cell receptor in CLL.

Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%-10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes.

cSPARCOM: Multi-detector reconstruction by confocal super-resolution correlation microscopy.

Image scanning microscopy (ISM), an upgraded successor of the ubiquitous confocal microscope, facilitates up to two-fold improvement in lateral resolution, and has become an indispensable element in the toolbox of the bio-imaging community. Recently, super-resolution optical fluctuation image scanning microscopy (SOFISM) integrated the analysis of intensity-fluctuations information into the basic ISM architecture, to enhance its resolving power. Both of these techniques typically rely on pixel-reassignment as a fundamental processing step, in which the parallax of different detector elements to the sample is compensated by laterally shifting the point spread function (PSF).

Identification of bacteria-derived HLA-bound peptides in melanoma.

A variety of species of bacteria are known to colonize human tumours, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours.

Assessing changes in the expression levels of cell surface proteins with a turn-on fluorescent molecular probe.

Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were developed and used to image His-tagged-labelled outer membrane protein C (His-OmpC) in live Escherichia coli. One of these probes was designed to light up upon binding, which provided the means to assess changes in the His-OmpC expression levels by taking a simple fluorescence spectrum.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome.

Regulation of axonal morphogenesis by the mitochondrial protein Efhd1.

During development, neurons adjust their energy balance to meet the high demands of robust axonal growth and branching. The mechanisms that regulate this tuning are largely unknown. Here, we show that sensory neurons lacking liver kinase B1 (Lkb1), a master regulator of energy homeostasis, exhibit impaired axonal growth and branching.

ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation.

T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of T cells), are also enriched on microvilli.

Decorating bacteria with self-assembled synthetic receptors.

The responses of cells to their surroundings are mediated by the binding of cell surface proteins (CSPs) to extracellular signals. Such processes are regulated via dynamic changes in the structure, composition, and expression levels of CSPs. In this study, we demonstrate the possibility of decorating bacteria with artificial, self-assembled receptors that imitate the dynamic features of CSPs.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Invited for this month's cover are the group of Prof. Lia Addadi at the Weizmann Institute of Science, Israel and collaborators at the Università Degli Studi di Milano, Italy, and the ALBA Synchrotron Light Source, Spain. The front cover shows how cholesterol crystals form in macrophage cells and in lipid bilayers of different compositions.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Cholesterol crystallization from mixtures of unesterified cholesterol with phospholipids and cholesterol esters is believed to be a key event in atherosclerosis progression. Not much is understood, however, about the influence of the lipid environment on cholesterol crystallization. Here we study cholesterol monohydrate crystal formation from mixed bilayers with palmitoyl-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin.

A Mechanism of Modulating the Direction of Flagellar Rotation in Bacteria by Fumarate and Fumarate Reductase.

Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliG-one of the motor's switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling.

Robo2 regulates synaptic oxytocin content by affecting actin dynamics.

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were enriched with polymerized actin.

Kinetics of Mimivirus Infection Stages Quantified Using Image Flow Cytometry.

Due to the heterogeneity of viruses and their hosts, a comprehensive view of viral infection is best achieved by analyzing large populations of infected cells. However, information regarding variation in infected cell populations is lost in bulk measurements. Motivated by an interest in the temporal progression of events in virally infected cells, we used image flow cytometry (IFC) to monitor changes in Acanthamoeba polyphaga cells infected with Mimivirus.

Autophagy differentially regulates TNF receptor Fn14 by distinct mammalian Atg8 proteins.

Autophagy, a conserved membrane trafficking process, sequesters cytoplasmic components into autophagosomes and targets them for lysosomal degradation. The TNF receptor Fn14 participates in multiple intracellular signaling pathways and is strongly induced upon tissue injury and solid tumorigenesis. While Fn14 is a short-lived protein, the regulation of its levels is largely obscure.

Resolving ESCRT-III Spirals at the Intercellular Bridge of Dividing Cells Using 3D STORM.

The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission.

Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis.

The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.

Colocalization and Disposition of Cellulosomes in as Revealed by Correlative Superresolution Imaging.

Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes.

SNARE priming is essential for maturation of autophagosomes but not for their formation.

Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy.