Donor Membranes Research Articles

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics.

Despite huge need in the medical domain and significant development efforts, artificial cells to date have limited composition and functionality. Although some artificial cells have proven successful for producing therapeutics or performing in vitro specific reactions, they have not been investigated in vivo to determine whether they preserve their architecture and functionality while avoiding toxicity. Here, these limitations are overcome and customizable cell mimic is achieved—molecular factories (MFs)—by supplementing giant plasma membrane vesicles derived from donor cells with nanometer‐sized artificial organelles (AOs). MFs inherit the donor cell's natural cytoplasm and membrane, while the AOs house reactive components and provide cell‐like architecture and functionality. It is demonstrated that reactions inside AOs take place in a close‐to‐nature environment due to the unprecedented level of complexity in the composition of the MFs. It is further demonstrated that in a zebrafish vertebrate animal model, these cell mimics show no apparent toxicity and retain their integrity and function. The unique advantages of highly varied composition, multicompartmentalized architecture, and preserved functionality in vivo open new biological avenues ranging from the study of biorelevant processes in robust cell‐like environments to the production of specific bioactive compounds.

Roles of Rab proteins in vesicle transport

Rab proteins are a part of a conserved small GTPase family in eukaryotes that are widely expressed in cells, and their activities are strictly regulated within the cell: the transition between the active Rab-GTP and inactive Rab-GDP forms is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activated proteins (GAPs). Functioning as important molecular switches in vesicle transport, Rab proteins interact with several different downstream effector molecules that are involved in cargo selection from the donor membrane, bud development, movement along the cytoskeleton, and anchor of the vesicles to the receptor membrane. In addition, the impaired roles of Rab proteins and disturbed vesicle trafficking caused by abnormal Rab proteins may lead to various diseases, including cancer and neurodegenerative and immune diseases. This article will review the recent research progress on Rab proteins, molecular mechanisms involved in vesicle trafficking, regulation of the Rab protein cycle, and diseases caused by abnormal vesicle transport.

Structure of membrane tethers and their role in fusion.

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so-called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.

ATG2 transports lipids to promote autophagosome biogenesis.

During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.

Research progress in lung transplantation

Lung transplantation is the only effective treatment for end-stage lung disease. The problems, including acute rejection, infection, primary graft dysfunction (PGD) and ischemia/reperfusion injury after lung transplantation, as well as surgical techniques and anesthesia management, donor, cardiopulmonary bypass or extracorporeal membrane oxygenation (ECMO), and the main pathogenesis of the receptor, that restrict the development of lung transplantation seriously, and affect the prognosis of patients. Through the description of above major bottlenecks related to lung transplantation and the factors that seriously affect the survival of lung transplant patients and the current response measures, new ideas for the clinical treatment of lung transplantation are presented.

Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells.

The membrane fusion properties of HCV envelope glycoproteins can be evaluated using several assays. Fusion assays generally require contacts between glycoproteins expressed on a donor membrane, such as those from a cell or a viral particle, and an acceptor membrane that may or may not express cognate viral receptor, such as those from an indicator cell or a liposome. In this chapter, we describe three well-established methods in the field that use either cell surface expression of glycoproteins, HCV pseudoparticles (HCVpp), or cell culture-grown HCV (HCVcc) particles for donor membrane and cells or liposomes as acceptor membrane in which specific components can be included to monitor and quantify fusion. We provide details of cell-cell fusion assay, virus-liposome fusion assay, and finally virus-plasma membrane fusion assay. We also describe inhibitors that can block HCV envelope membrane fusion.

Influenza A Virus NS1 Protein Suppresses JNK1-Dependent Autophagosome Formation Mediated by Rab11a Recycling Endosomes.

Autophagy is an essential process for cellular metabolism and homeostasis, but also functions as one of innate immune responses against pathogen infection. However, in contrast to cellular metabolism and homeostasis pathways, less is known about how virus infection leads to autophagosome formation. Here, we showed that influenza A virus NS1 protein inhibits the formation of autophagosomes. The autophagosome formation was induced by infection with NS1 mutant virus lacking the dsRNA-binding activity for inhibition of innate immune responses (R38AK41A) or the activation of PI3K-Akt signaling pathway (Y89F). R38AK41A mutant infection induced phosphorylation of JNK1 and up-regulated the expression of autophagy-related genes which are downstream of JNK1 signaling pathway. We also found that the amount of phosphorylated TSC2, which activates mTOR, increased in wild type-infected cells but not in Y89F mutant-infected cells. These findings suggest that NS1 inhibits the autophagosome formation through both the inhibition of JNK1 and the activation of PI3K-Akt-mTOR pathway. Further, viral ribonucleoprotein (vRNP) complexes were selectively sequestered into autophagosomes, and knockdown of Rab11a, which is responsible for the apical transport of vRNP complexes, impaired not only engulfment of vRNP complexes by autophagosomes but also the formation of autophagosomes in R38AK41A mutant-infected cells. This indicates that Rab11a-positive recycling endosomes function as a donor membrane for the phagophore elongation and an autophagic receptor for the selective engulfment of viral RNP complexes. Based on these results, we propose that NS1 inhibits JNK1-mediated autophagy induction and the sequestration of vRNP complexes into autophagosomes.

Endosomal and Phagosomal SNAREs.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein family is of vital importance for organelle communication. The complexing of cognate SNARE members present in both the donor and target organellar membranes drives the membrane fusion required for intracellular transport. In the endocytic route, SNARE proteins mediate trafficking between endosomes and phagosomes with other endosomes, lysosomes, the Golgi apparatus, the plasma membrane, and the endoplasmic reticulum. The goal of this review is to provide an overview of the SNAREs involved in endosomal and phagosomal trafficking. Of the 38 SNAREs present in humans, 30 have been identified at endosomes and/or phagosomes. Many of these SNAREs are targeted by viruses and intracellular pathogens, which thereby reroute intracellular transport for gaining access to nutrients, preventing their degradation, and avoiding their detection by the immune system. A fascinating picture is emerging of a complex transport network with multiple SNAREs being involved in consecutive trafficking routes.

SAC1 degrades its lipid substrate PtdIns4P in the ER to maintain a steep electrochemical gradient on donor membranes

Phosphatidylinositol 4‐phosphate (PtdIns4P) is one of the most functionally diverse molecules utilized by eukaryotic cells. It is both a metabolic hub for other crucial signaling lipids such as PtdIns(4,5)P2 and PtdInsP3, and a key molecule for recruitment of membrane and lipid transport proteins in its own right. Most recently, it has been proposed to form a “phosphoinositide‐motive force”, whereby transport of PtdIns4P molecules down their concentration gradient from the plasma membrane (PM) or Golgi to the ER drives counter transport of other lipids up their own concentration gradients. A critical requirement for this model is that the main PtdIns4P degrading enzyme, the integral ER phosphatase SAC1, hydrolyzes PtdIns4P in the ER in a “cis” configuration. Alternatively, it has been suggested that other functions of PtdIns4P are regulated by SAC1 hydrolyzing PtdIns4P directly in the PM in a “trans” configuration at membrane contact sites (MCS). However, such activity would surely disrupt lipid counter transport. Therefore, we sought to determine whether SAC1 acts in “cis”, “trans”, or both in mammalian cells. Acute chemical ablation of SAC1 activity drives ectopic accumulation of PtdIns4P in the ER, revealing “cis” activity. Furthermore, endogenous or ectopically expressed SAC1 localizes to the ER and Golgi, but does not constitutively or dynamically enrich at ER‐PM MCS. Forced recruitment of SAC1 to experimentally induced MCS does not produce robust “trans” activity on PM PtdIns4P. However, “trans” activity can be induced by adding an approximately 6 nm long helical linker between the ER anchor and the catalytic domain. Together, our results reveal that SAC1 operates in a “cis” configuration. This ensures a “phosphoinositide‐motive force” for lipid transport is effective, and also implies regulation of PM phosphoinositide signaling is tightly linked to non‐vesicular traffic of PtdIns4P at ER‐PM MCS.Support or Funding InformationThis work was supported by National Institutes of Health grant 1R35GM119412‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Layer‐by‐layer Proton Donor and Acceptor Membrane for an Efficient Proton Transfer System in a Polymer Electrolyte Membrane Fuel Cell

AbstractThe high performance proton transfer in polymer electrolyte membranes (PEMs) is known to be achieved through cooperating of proton donors and acceptors. In order to obtain the effective and efficient proton transfer, the present work proposes a membrane with proton donors and proton acceptors in layer‐by‐layer (LbL) structure. By applying sulfonated poly (ether ether ketone) (SPEEK) as the substrate membrane and soaking alternatively with the proton donor polymer, i.e. poly(acrylic acid) (PAA), and the proton acceptor heterocycles, i.e. benzimidazoles (BI), the LbL SPEEK/BI/PAA membranes can be achieved. The systematic variation based on the branching benzimidazole groups suggest that the LbL SPEEK/BI/PAA membrane obtained shows an increase in proton conductivity compared to the neat SPEEK, especially in the case of the membrane with trifunctional benzimidazole (B4) is as high as 1–2.5 orders of magnitude over the temperature range of 30–170 °C. The present work points out the simple surface modification of the proton conductive membrane by layer‐by‐layer approach as a practical and simple way to enhance the performance of polymer electrolyte membrane.

SAC1 degrades its lipid substrate PtdIns4P in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes.

Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a 'cis' configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in 'trans' at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in 'trans', unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate 'cis' activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.

Assembly of COPI and COPII Vesicular Coat Proteins on Membranes.

In eukaryotes, distinct transport vesicles functionally connect various intracellular compartments. These carriers mediate transport of membranes for the biogenesis and maintenance of organelles, secretion of cargo proteins and peptides, and uptake of cargo into the cell. Transport vesicles have distinct protein coats that assemble on a donor membrane where they can select cargo and curve the membrane to form a bud. A multitude of structural elements of coat proteins have been solved by X-ray crystallography. More recently, the architectures of the COPI and COPII coats were elucidated in context with their membrane by cryo-electron tomography. Here, we describe insights gained from the structures of these two coat lattices and discuss the resulting functional implications.

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic.

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

Sac1 degrades its lipid substrate PI4P in the ER to maintain a steep electrochemical gradient on donor membranes

PI4P metabolism has been proposed to form the basis of an “inositide motive force”, whereby a steep electrochemical gradient of this lipid that is high in a target membrane and low in the endoplasmic reticulum (ER) drives counter transport of other lipids. This mechanism assumes that the ER‐resident Sac1 phosphatase degrades PI4P in the ER (i.e. acting in cis). However, several studies have proposed that the enzyme instead operates in trans at contact sites between the ER and other PI4P rich membranes. We therefore sought to establish whether Sac1 truly acts in cis or trans in living cells. Compared with bona fide plasma membrane (PM)‐ER contact site proteins and broadly distributed ER proteins, Sac1 exhibits a localization consistent with the latter. Strategies to test activity of the enzyme at the PM in living cells revealed far more robust activity when the enzyme met its substrate in cis than at contact sites in trans. Finally, we used chemical and genetic strategies to inactivate Sac1 in yeast and mammalian cells, revealing dramatic accumulation of PI4P in the ER. This result is consistent with a failure to degrade the lipid only after its delivery to the ER. Together, the data show that Sac1 acts in cis, establishing the plausibility of an inositide motive force, and implicating PI4P transfer proteins as essential to phosphoinositide and lipid homeostasis.Support or Funding Information1R35GM119412‐01: NIH(NIGMS) R35

STUDY OF COMPOSITION, PHYSICAL AND CHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF POLYSACCHARIDE COMPLEX OF ARCTIUM LAPPA LEAVES

A method of isolation of polysaccharide complex from the leaves of Burdock (Arctium lappa L.) is described in the article. For the first time the qualitative composition of this complex was determined, belonging to the group of heteroglycans. Some of physical and chemical properties of polysaccharide complex were studied. It is shown that polysaccharide complex increases catalase activity and peroxide resistance of the erythrocyte membranes of healthy donors. The study found that polysaccharide complex of Burdock improves physical performance of test animals.

Cell-free Generation of COPII-coated Procollagen I Carriers

The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the isolation and detection of reconstituted COPII-coated carriers. This cell-free reaction can be used to test conditions that stimulate or suppress the packaging of PC1 into COPII-coated carriers.

Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

ABSTRACTMacroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.

Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma.

Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.

The Composition of the Cell Envelope Affects Conjugation in Bacillus subtilis.

Conjugation in bacteria is the contact-dependent transfer of DNA from one cell to another via donor-encoded conjugation machinery. It is a major type of horizontal gene transfer between bacteria. Conjugation of the integrative and conjugative element ICEBs1 into Bacillus subtilis is affected by the composition of phospholipids in the cell membranes of the donor and recipient. We found that reduction (or elimination) of lysyl-phosphatidylglycerol caused by loss of mpr F caused a decrease in conjugation efficiency. Conversely, alterations that caused an increase in lysyl-phosphatidylglycerol, including loss of ugtP or overproduction of mprF, caused an increase in conjugation efficiency. In addition, we found that mutations that alter production of other phospholipids, e.g., loss of clsA and yfnI, also affected conjugation, apparently without substantively altering levels of lysyl-phosphatidylglycerol, indicating that there are multiple pathways by which changes to the cell envelope affect conjugation. We found that the contribution of mprF to conjugation was affected by the chemical environment. Wild-type cells were generally more responsive to addition of anions that enhanced conjugation, whereas mprF mutant cells were more sensitive to combinations of anions that inhibited conjugation at pH 7. Our results indicate that mprF and lysyl-phosphatidylglycerol allow cells to maintain relatively consistent conjugation efficiencies under a variety of ionic conditions. Horizontal gene transfer is a driving force in microbial evolution, enabling cells that receive DNA to acquire new genes and phenotypes. Conjugation, the contact-dependent transfer of DNA from a donor to a recipient by a donor-encoded secretion machine, is a prevalent type of horizontal gene transfer. Although critically important, it is not well understood how the recipient influences the success of conjugation. We found that the composition of phospholipids in the membranes of donors and recipients influences the success of transfer of the integrative and conjugative element ICEBs1 in Bacillus subtilis Specifically, the presence of lysyl-phosphatidylglycerol enables relatively constant conjugation efficiencies in a range of diverse chemical environments.