Light on fluorescent lipids in rafts: a lesson from model membranes

2010 ◽  
Vol 430 (3) ◽  
pp. e7-e9 ◽  
Author(s):  
Nicoletta Kahya
Keyword(s):  
Viral Entry ◽  
Fluorescent Probes ◽  
Membrane Trafficking ◽  
Model Membranes ◽  
Heterogeneous Model ◽  
Phase Partitioning ◽  
Full Characterization ◽  
Systems Model ◽  
Fluorescent Lipids ◽  
Microscopy Data

Tracking fluorescent lipids in cellular membranes has been applied for decades to shed light on membrane trafficking, sorting, endocytosis and exocytosis, viral entry, and to understand the functional relevance of membrane heterogeneity, phase separation and lipid rafts. However, fluorescent probes may display different organizing behaviour from their corresponding endogenous lipids. A full characterization of these probes is therefore required for proper interpretation of fluorescence microscopy data in complex membrane systems. Model membrane studies provide essential clues that guide us to design and interpret our experiments, help us to avoid pitfalls and resolve artefacts in complex cellular environments. In the present issue of the Biochemical Journal, Juhasz, Davis and Sharom demonstrate the importance of testing lipid probes systematically in heterogeneous model membranes of specific composition and well-defined thermodynamic properties. The phase-partitioning behaviour of fluorescent probes, alone and/or in combination, cannot simply be assumed, but has to be fully characterized.

scholarly journals HIV-1 requires Arf6-mediated membrane dynamics to efficiently enter and infect T lymphocytes

2011 ◽  
Vol 22 (8) ◽  
pp. 1148-1166 ◽  
Author(s):  
Laura García-Expósito ◽  
Jonathan Barroso-González ◽  
Isabel Puigdomènech ◽  
José-David Machado ◽  
Julià Blanco ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
Viral Entry ◽  
Membrane Trafficking ◽  
Membrane Dynamics ◽  
Viral Fusion ◽  
Viral Attachment ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis ◽  
Hiv 1

As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4+T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4+T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

scholarly journals Conserved Oligomeric Golgi (COG) Complex Proteins Facilitate Orthopoxvirus Entry, Fusion and Spread

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 707 ◽  
Author(s):  
Susan Realegeno ◽  
Lalita Priyamvada ◽  
Amrita Kumar ◽  
Jessica B. Blackburn ◽  
Claire Hartloge ◽  
...  
Keyword(s):  
Viral Entry ◽  
Membrane Trafficking ◽  
Gene Knockout ◽  
Host Cells ◽  
Viral Fusion ◽  
Genetic Screens ◽  
Viral Spread ◽  
Cog Complex ◽  
Conserved Oligomeric Golgi ◽  
The Individual

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1–COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.

scholarly journals Fluorescent Probes for Lipid Rafts: From Model Membranes to Living Cells

2014 ◽  
Vol 21 (1) ◽  
pp. 97-113 ◽  
Author(s):  
Andrey S. Klymchenko ◽  
Rémy Kreder
Keyword(s):  
Lipid Rafts ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Model Membranes

scholarly journals Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.

1986 ◽  
Vol 103 (4) ◽  
pp. 1221-1234 ◽  
Author(s):  
P S Uster ◽  
R E Pagano
Keyword(s):  
Energy Transfer ◽  
Fluorescent Probes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Model Membranes ◽  
Energy Donor ◽  
Membrane Bound ◽  
Energy Acceptor ◽  
In Cells

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.

Fluorescent probes in model membranes I: anthroyl fatty acid derivatives in monolayers and liposomes of dipalmitoylphosphatidylcholine

Biochemistry ◽  
1977 ◽  
Vol 16 (24) ◽  
pp. 5386-5392 ◽  
Author(s):  
D. Allan Cadenhead ◽  
Benedikt M. J. Kellner ◽  
Kenneth Jacobson ◽  
Demetrios Papahadjopoulos
Keyword(s):  
Fatty Acid ◽  
Fluorescent Probes ◽  
Model Membranes ◽  
Fatty Acid Derivatives

Aggregation and fusion of lipid vesicles induced by diphtheria toxin at low pH: Possible involvement of the P site and the NAD+ binding site

1985 ◽  
Vol 5 (3) ◽  
pp. 243-250 ◽  
Author(s):  
Veronique Cabiaux ◽  
Michel Vandenbranden ◽  
Paul Falmagne ◽  
Jean-Marie Ruysschaert
Keyword(s):  
Energy Transfer ◽  
Binding Site ◽  
Fluorescent Probes ◽  
Diphtheria Toxin ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Lipid Vesicles ◽  
Low Ph ◽  
Model Membranes

Model membranes have been used to study the interaction between diphtheria toxin and lipids. We report here on the ability of this toxin to induc% at low pH, fusion and aggregation of asolectin lipid vesicles. Resonance energy transfer experiments using lipid fluorescent probes make it possible to discriminate between these two processes.

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