scholarly journals Detecting and controlling dye effects in single-virus fusion experiments

2019 ◽  
Author(s):  
R. J. Rawle ◽  
A. M. Villamil Giraldo ◽  
S. G. Boxer ◽  
P. M. Kasson
Keyword(s):  
Membrane Fusion ◽  
Fluorescent Dyes ◽  
Light Exposure ◽  
Viral Proteins ◽  
Live Cells ◽  
Dependent Manner ◽  
Viral Fusion ◽  
Texas Red ◽  
Kinetics Of ◽  
Cellular Fusion

AbstractFluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, while Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.Statement of SignificanceFluorescent dyes are powerful tools for labeling membranes and tracking subcellular objects, and fluorescence dequenching has further been used as a sensitive assay for membrane fusion. Here we show how incorporation of membrane dyes can perturb membrane fusion by influenza virus in a light-dependent manner. We provide a strategy to mitigate this by minimizing dye and light exposure. Finally, we show how in some cases these effects can be due to covalent reaction of some dyes with viral proteins upon illumination. These phenomena may be general and should be carefully controlled for in experiments using such labels.

scholarly journals Extended Synaptotagmin 1 Interacts with Herpes Simplex Virus 1 Glycoprotein M and Negatively Modulates Virus-Induced Membrane Fusion

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Imane El Kasmi ◽  
Bita Khadivjam ◽  
Miki Lackman ◽  
Johanne Duron ◽  
Eric Bonneil ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Viral Proteins ◽  
Life Cycles ◽  
Viral Fusion ◽  
Herpes Simplex Virus 1 ◽  
Synaptotagmin 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTEnveloped viruses typically encode their own fusion machinery to enter cells. Herpesviruses are unusual, as they fuse with a number of cellular compartments throughout their life cycles. As uncontrolled fusion of the host membranes should be avoided in these events, tight regulation of the viral fusion machinery is critical. While studying herpes simplex virus 1 (HSV-1) glycoprotein gM, we identified the cellular protein E-Syt1 (extended synaptotagmin 1) as an interaction partner. The interaction took place in both infected and transfected cells, suggesting other viral proteins were not required for the interaction. Most interestingly, E-Syt1 is a member of the synaptotagmin family of membrane fusion regulators. However, the protein is known to promote the tethering of the endoplasmic reticulum (ER) to the plasma membrane. We now show that E-Syt1, along with the related E-Syt3, negatively modulates viral release into the extracellular milieu, cell-to-cell viral spread, and viral entry, all processes that implicate membrane fusion events. Similarly, these E-Syt proteins impacted the formation of virus-induced syncytia. Altogether, these findings hint at the modulation of the viral fusion machinery by the E-Syt family of proteins.IMPORTANCEViruses typically encode their own fusion apparatus to enable them to enter cells. For many viruses, this means a single fusogenic protein. However, herpesviruses are large entities that express several accessory viral proteins to regulate their fusogenic activity. The present study hints at the additional participation of cellular proteins in this process, suggesting the host can also modulate viral fusion to some extent. Hence E-Syt proteins 1 and 3 seem to negatively modulate the different viral fusion events that take place during the HSV-1 life cycle. This could represent yet another innate immunity response to the virus.

scholarly journals Herpes Simplex Virus 1 gN Partners with gM To Modulate the Viral Fusion Machinery

2014 ◽  
Vol 89 (4) ◽  
pp. 2313-2323 ◽  
Author(s):  
Imane El Kasmi ◽  
Roger Lippé
Keyword(s):  
Herpes Simplex ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Viral Proteins ◽  
Viral Fusion ◽  
Herpes Simplex Virus 1 ◽  
Induced Membrane ◽  
Nuclear Membranes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) capsids are assembled in the nucleus, where they incorporate the viral genome. They then transit through the two nuclear membranes and are wrapped by a host-derived envelope. In the process, several HSV-1 proteins are targeted to the nuclear membranes, but their roles in viral nuclear egress are unclear. Among them, glycoprotein M (gM), a known modulator of virus-induced membrane fusion, is distributed on both the inner and outer nuclear membranes at the early stages of the infection, when no other viral glycoproteins are yet present there. Later on, it is found on perinuclear virions and ultimately redirected to the trans-Golgi network (TGN), where it cycles with the cell surface. In contrast, transfected gM is found only at the TGN and cell surface, hinting at an interaction with other viral proteins. Interestingly, many herpesvirus gM analogs interact with their gN counterparts, which typically alters their intracellular localization. To better understand how HSV-1 gM localization is regulated, we evaluated its ability to bind gN and discovered it does so in both transfected and infected cells, an interaction strongly weakened by the deletion of the gM amino terminus. Functionally, while gN had no impact on gM localization, gM redirected gN from the endoplasmic reticulum (ER) to the TGN. Most interestingly, gN overexpression stimulated the formation of syncytia in the context of an infection by a nonsyncytial strain, indicating that gM and gN not only physically but also functionally interact and that gN modulates gM's activity on membrane fusion.IMPORTANCEHSV-1 gM is an important modulator of virally induced cell-cell fusion and viral entry, a process that is likely finely modulated in time and space. Until now, little was known of the proteins that regulate gM's activity. In parallel, gM is found in various intracellular locations at different moments, ranging from nuclear membranes, perinuclear virions, the TGN, cell surface, and mature extracellular virions. In transfected cells, however, it is found only on the TGN and cell surface, hinting that its localization is modulated by other viral proteins. The present study identifies HSV-1 gN as a binding partner for gM, in agreement with their analogs in other herpesviruses, but most excitingly shows that gN modulates gM's impact on HSV-1-induced membrane fusion. These findings open up new research avenues on the viral fusion machinery.

Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg
Keyword(s):  
Lipid Bilayers ◽  
Binding Kinetics ◽  
Supported Lipid Bilayers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Supported Lipid Bilayer ◽  
Head Groups ◽  
Lipid Diffusion ◽  
Kinetics Of ◽  
Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

scholarly journals Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes

2007 ◽  
Vol 27 (9) ◽  
pp. 3456-3469 ◽  
Author(s):  
Shaohui Huang ◽  
Larry M. Lifshitz ◽  
Christine Jones ◽  
Karl D. Bellve ◽  
Clive Standley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Insulin Signaling ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Tirf Microscopy ◽  
Adipocyte Plasma Membranes ◽  
Kinetics Of

ABSTRACT Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by ∼4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

1932 ◽  
Vol 16 (2) ◽  
pp. 233-242 ◽  
Author(s):  
B. G. Wilkes ◽  
Elizabeth T. Palmer
Keyword(s):  
Physiological Activity ◽  
Outer Region ◽  
Living Cells ◽  
Yeast Cells ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Relationship Of ◽  
Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

scholarly journals Light, Alertness, and Alerting Effects of White Light: A Literature Overview

2018 ◽  
Vol 33 (6) ◽  
pp. 589-601 ◽  
Author(s):  
Renske Lok ◽  
Karin C. H. J. Smolders ◽  
Domien G. M. Beersma ◽  
Yvonne A. W. de Kort
Keyword(s):  
White Light ◽  
Light Exposure ◽  
Neural Mechanisms ◽  
Dependent Manner ◽  
Experimental Paradigm ◽  
Research Paradigms ◽  
Lighting Conditions ◽  
Literature Overview ◽  
Dose Dependent ◽  
As Effects

Light is known to elicit non–image-forming responses, such as effects on alertness. This has been reported especially during light exposure at night. Nighttime results might not be translatable to the day. This article aims to provide an overview of (1) neural mechanisms regulating alertness, (2) ways of measuring and quantifying alertness, and (3) the current literature specifically regarding effects of different intensities of white light on various measures and correlates of alertness during the daytime. In general, the present literature provides inconclusive results on alerting effects of the intensity of white light during daytime, particularly for objective measures and correlates of alertness. However, the various research paradigms employed in earlier studies differed substantially, and most studies tested only a limited set of lighting conditions. Therefore, the alerting potential of exposure to more intense white light should be investigated in a systematic, dose-dependent manner with multiple correlates of alertness and within one experimental paradigm over the course of day.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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