Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

Current analytical strategies for collecting proteomic data using data-dependent acquisition (DDA) are limited by the low analytical reproducibility of the method. Proteomic discovery efforts that exploit the benefits of DDA, such as providing peptide sequence information, but that enable improved analytical reproducibility, represent an ideal scenario for maximizing measureable peptide identifications in "shotgun"-type proteomic studies. Therefore, we propose an analytical workflow combining DDA with retention time aligned extracted ion chromatogram (XIC) areas obtained from high mass accuracy MS1 data acquired in parallel. We applied this workflow to the analyses of sample matrixes prepared from mouse blood plasma and brain tissues and observed increases in peptide detection of up to 30.5% due to the comparison of peptide MS1 XIC areas following retention time alignment of co-identified peptides. Furthermore, we show that the approach is quantitative using peptide standards diluted into a complex matrix. These data revealed that peptide MS1 XIC areas provide linear response of over three orders of magnitude down to low femtomole (fmol) levels. These findings argue that augmenting "shotgun" proteomic workflows with retention time alignment of peptide identifications and comparative analyses of corresponding peptide MS1 XIC areas improve the analytical performance of global proteomic discovery methods using DDA. Molecular & Cellular Proteomics 13: 10.1074/ mcp.M112.026500, 329-338, 2014.Label-free methods in mass spectrometry-based proteomics, such as those used in common "shotgun" proteomic studies, provide peptide sequence information as well as relative measurements of peptide abundance (1-3). A common data acquisition strategy is based on data-dependent acquisition (DDA) 1 where the most abundant precursor ions are selected for tandem mass spectrometry (MS/MS) analysis (1-2). DDA attempts to minimize redundant peptide precursor selection and maximize the depth of proteome coverage (2). However, the analytical reproducibility of peptide identifications obtained using DDA-based methods result in Ͻ75% overlap between technical replicates (3-4). Comparisons of peptide identifications between replicate analyses have shown that the rate of new peptide identifications increases sharply following two replicate sample injections and gradually tapers off after approximately five replicate injections (4). This phenomenon is due, in part, to the semirandom sampling of peptides in a DDA experiment (5).Alternate label-free methods focused on measuring the abundance of intact peptide ions, such as the accurate mass and time tag (AMT) approach (6 -8, 42), are aimed at differential analyses of extracted ion chromatogram (XIC) areas integrated from high mass accuracy peptide precursor mass spectra (MS1 spectra) exhibiting discrete chromatographic elution times. This method is particularly powerful for the analysis of post-translationally modified (PTM) peptides as pairing the low abundance of PTM candidates with the variabl...

Transport of biomolecules, drugs, and other reagents across the cell's plasma membrane barrier is an inefficient and poorly controlled process, despite its fundamental importance to biotechnology, cell biology, and pharmaceutics. In particular, insufficient membrane permeability frequently limits the accumulation of drugs and reagents in the cytoplasm, undermining their efficacy. While encapsulating drugs in particles increases uptake by cells, inefficient release of drugs from these particles into the cytoplasm ultimately limits drug efficacy. In contrast, gap junctions provide a direct route to the cytoplasm that bypasses the plasma membrane. As transmembrane channels that physically connect the cytoplasm of adjacent cells, gap junctions permit transport of a diverse range of molecules, from ions and metabolites to siRNA, peptides, and chemotherapeutics. To utilize gap junctions for molecular delivery we have developed Connectosomes, cell-derived lipid vesicles that contain functional gap junction channels and encapsulate molecular cargos. Here we show that these vesicles form gap junction channels with cells, opening a direct and efficient route for the delivery of molecular cargo to the cellular cytoplasm. Specifically, we demonstrate that using gap junctions to deliver the chemotherapeutic doxorubicin reduces the therapeutically effective dose of the drug by more than an order of magnitude. Delivering drugs through gap junctions has the potential to boost the effectiveness of existing drugs such as chemotherapeutics, while simultaneously enabling the delivery of membrane-impermeable drugs and reagents.

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.