Abstract:
:Membrane fission, which divides membrane surfaces into separate compartments, is essential to diverse cellular processes including membrane trafficking and cell division. Quantitative assays are needed to elucidate the physical mechanisms by which proteins drive membrane fission. Toward this goal, several experimental tools have been developed, including visualizing fission products using electron microscopy, measuring membrane shedding from a lipid reservoir, and observing fission of individual membrane tubes pulled from giant vesicles. However, no existing assay of membrane fission provides a quantitative, high-throughput measure of the distribution of vesicle curvatures generated by fission-driving proteins. Toward addressing this challenge, here we describe a novel approach that uses confocal fluorescence imaging to quantify the diameter distribution of membrane vesicles that have been tethered to a coverslip surface following exposure to fission-driving proteins. We employ this assay to measure the progressive appearance of high curvature fission products upon exposure of vesicles to increasing protein concentration. Results from this approach are in quantitative agreement with measurements from electron microscopy, but can be collected with considerably greater throughput, enabling examination of a broad range of experimental conditions. Using the tethered vesicle approach, we have recently found that membrane-bound intrinsically disordered proteins are surprisingly potent drivers of membrane fission. The capacity to drive fission arises from steric pressure generated when disordered domains with large hydrodynamic radii bind to membranes at high local densities. More broadly, the experimental tools described here have the potential to improve our mechanistic understanding of membrane fission in diverse biophysical contexts.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Snead WT,Stachowiak JCdoi
10.1016/bs.mie.2018.08.014subject
Has Abstractpub_date
2018-01-01 00:00:00pages
559-582eissn
0076-6879issn
1557-7988pii
S0076-6879(18)30292-1journal_volume
611pub_type
杂志文章abstract::Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor ac...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)99046-0
更新日期:2005-01-01 00:00:00
abstract::In mice with lupus nephritis qualitative changes in anti-DNA antibodies occur, such as IgG switch and increased cationic charge, to render these antibodies pathogenic. Pathogenic anti-DNA idiotypes can be encoded by genes of a normal mouse strain such as SWR, where they remain dormant. When the normal mice are crossed...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(88)62094-5
更新日期:1988-01-01 00:00:00
abstract::Human embryonic stem cells hold great promise in furthering our treatment of disease and increasing our understanding of early development. This chapter describes protocols for the derivation and maintenance of human embryonic stem cells. In addition, it summarizes briefly several alternative methods for the culture o...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)18005-2
更新日期:2006-01-01 00:00:00
abstract::Sulfate-reducing prokaryotes (SRPs) exploit sulfate as an electron acceptor for anaerobic respiration and exclusively catalyze this essential step of the world's sulfur cycle. Because SRPs are found in many prokaryotic phyla and are often closely related to non-SRPs, 16S rRNA gene-based analyses are inadequate to iden...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)97029-8
更新日期:2005-01-01 00:00:00
abstract::High content, phenotypic screens offer a powerful approach to systems biology at the cellular level. The approach employs cells carrying fluorescently labeled molecules or organelles in 384- or 1536-well microplates, and an automated confocal screening microscope for capturing images from each well. Although some spec...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.09.021
更新日期:2018-01-01 00:00:00
abstract::Among posttranslational modifications of proteins entailed with signal transduction, the redox transition is today brought to the focus as a major biochemical event accounting for the signaling functions of reactive oxygen species. Thermodynamic and kinetic criteria highlight hydroperoxides and protein disulfides as s...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(10)73011-1
更新日期:2010-01-01 00:00:00
abstract::The methods described here concern the use specialized glassware to achieve accurate sub-micromolar to low-millimolar dioxygen concentrations specifically for the study of flavoproteins. Since the early 1960s, these methods have been passed through the lineage of flavin enzymologists almost exclusively as an oral trad...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.03.005
更新日期:2019-01-01 00:00:00
abstract::During the past several years, major progress has been accomplished in the production of "designer cellulosomes," artificial enzymatic complexes that were demonstrated to efficiently degrade crystalline cellulose. This progress is part of a global attempt to promote biomass waste solutions and biofuel production. In d...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-415931-0.00023-9
更新日期:2012-01-01 00:00:00
abstract::Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies shar...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(91)06092-h
更新日期:1991-01-01 00:00:00
abstract::MicroRNA (miRNA) genes are transcribed into long primary transcripts (pri-miRNAs) that get processed into mature miRNAs of about 22 nt in length by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. Various experimental protocols have been developed and modified for genetic and biochemical analyses for ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)27005-3
更新日期:2007-01-01 00:00:00
abstract::Pulsed EPR methods for the study of drug binding to heme-thiolate enzymes such as cytochrome P450 and nitric oxide synthase are discussed. HYSCORE and ENDOR methods to measure (1)H of axial ligands of the heme group are described with illustrations of water serving as the axial ligand in the drug-free enzyme and ligan...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.005
更新日期:2015-01-01 00:00:00
abstract::The Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information archives and freely distributes high-throughput molecular abundance data, predominantly gene expression data generated by DNA microarray technology. The database has a flexible design that can handle diverse styles of bot...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)11019-8
更新日期:2006-01-01 00:00:00
abstract::Intracellular calcium (Ca2+) is involved in the regulation of a variety of biological functions in cancer cells, including growth inhibition, tumor invasiveness, and drug resistance. To gain insight into the possible role played by Ca2+ in the development of drug resistance in breast cancer, we performed a comparative...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)14018-5
更新日期:2006-01-01 00:00:00
abstract::Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)28002-4
更新日期:2007-01-01 00:00:00
abstract::The gasdermin (GSDM) family consists of gasdermin A (GSDMA), B (GSDMB), C (GSDMC), D (GSDMD), E or DNFA5 (GSDME), and DFNB59 in human. Expressed in the skin, gastrointestinal tract, and various immune cells, GSDMs mediate homeostasis and inflammation upon activation by caspases and unknown proteases. In particular, GS...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.04.024
更新日期:2019-01-01 00:00:00
abstract::Endosomes are isolated from rat liver using high-speed centrifugation through sucrose density gradients. They are distinguishable from Golgi elements, with which they coisolate, by their capacity to concentrate internalized protein ligands (viz., insulin and epidermal growth factor (EGF)) in receptor-bound intact form...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-397925-4.00017-1
更新日期:2014-01-01 00:00:00
abstract::Gene regulatory grids (GRGs) encompass the space of all the possible transcription factor (TF)-target gene interactions that regulate gene expression, with gene regulatory networks (GRNs) representing a temporal and spatial manifestation of a portion of the GRG, essential for the specification of gene expression. Thus...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.03.003
更新日期:2016-01-01 00:00:00
abstract::Recent work demonstrates that RNA interference (RNAi) can coordinate protein expression. Inhibitory RNAs are expressed naturally in cells as microRNAs (miRNAs) or introduced into cells as small interfering RNAs (siRNAs). Both types of small RNAs can be used at the bench to silence mRNA expression. For many researchers...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(04)92009-5
更新日期:2005-01-01 00:00:00
abstract::Sequence analysis of synthetic peptides using Edman chemistry can be very useful for the elucidation of certain types of synthetic problems, such as residue deletions and the presence of common stable derivatives, and for following the progress of the synthesis itself. However, it can also be a relatively poor techniq...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(97)89056-8
更新日期:1997-01-01 00:00:00
abstract::Paraquat (PQ) is a prototypical redox cycling agent commonly used experimentally to generate reactive oxygen species and oxidative stress. Recently, PQ has also come under investigation as a potential environmental neurotoxin associated with increased risk for neurodegenerative disease developing after chronic exposur...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)04421-2
更新日期:2009-01-01 00:00:00
abstract::T lymphocytes express on their surface a heterodimeric αβ receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibod...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-396962-0.00008-2
更新日期:2012-01-01 00:00:00
abstract::Protein kinase C (PKC), a family of serine/threonine kinases, plays an important role in apoptosis. Several members of the PKC family act as substrates for caspases. In addition, PKCs can also regulate caspase activation and cell death by apoptosis. The cleavage of PKCs separates the regulatory domain from the catalyt...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)01608-X
更新日期:2008-01-01 00:00:00
abstract::In the least-squares fitting of data, there is a unique answer to the question of how the data should be weighted: inversely as their variance. Any other weighting gives less than optimal efficiency and leads to unreliable estimates of the parameter uncertainties. In calibration, knowledge of the data variance permits...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)03810-X
更新日期:2009-01-01 00:00:00
abstract::Integrins are large modular cell-surface receptors that regulate almost every aspect of cellular function through bidirectional signals transmitted across the lipid bilayer. Regulation of integrin activity is accomplished by complex and still incompletely understood biochemical pathways that modify integrin ligand bin...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)26014-8
更新日期:2007-01-01 00:00:00
abstract::This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal pepti...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(86)28100-8
更新日期:1986-01-01 00:00:00
abstract::Perhaps the most important contribution of FLN is that it provides an experimental approach to relate physical changes in the protein to predicted dynamical behavior. It is clear that the sample is inhomogeneously broadened in a continuous manner, consistent with the damped motion of proteins. At the same time configu...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(97)78007-8
更新日期:1997-01-01 00:00:00
abstract::The mapping and characterization of the vasculature using proteomics offers the opportunity to better understand the steps and molecular mechanisms involved in vascular development, and angiogenesis in particular. Proteomics has many key advantages over genomics, especially in directly determining protein expression. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)03008-5
更新日期:2008-01-01 00:00:00
abstract::G-protein-coupled receptors (GPCRs) form a superfamily of membrane proteins that play a crucial role in mediating physiological processes as well as pathogenesis of many critical diseases. They are one of the most successful drug targets, accounting for more than 30% of prescription drugs on the market today. Three-di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05213-6
更新日期:2009-01-01 00:00:00
abstract::There is currently no known genetic disease linked to prolactin (PRL) or its receptor (PRLR) in humans. Recently, we identified three missense variants of the PRLR in patients presenting with breast tumors. Two of them (named PRLR(I146L) and PRLR(I76V)) had been reported earlier, but failed to draw much attention beca...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381298-8.00017-4
更新日期:2010-01-01 00:00:00
abstract::Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now ente...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)56935-4
更新日期:2002-01-01 00:00:00