Roland Schwarzer

An Amphiphilic Perylene Imido Diester for Selective Cellular Imaging

A new amphiphilic membrane marker based on a water-soluble dendritic polyglycerol perylene imido dialkylester has been designed, synthesized, and its optical properties characterized. In water it forms fluorescently quenched micellar self-aggregates, but when incorporated into a lipophilic environment, it monomerizes, and the highly fluorescent properties of the perylene core are recovered. These properties make it an ideal candidate for the imaging of artificial and cellular membranes as demonstrated by biophysical studies.

Organization of fluorescent cholesterol analogs in lipid bilayers - lessons from cyclodextrin extraction.

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.

The cholesterol‐binding motif of the HIV‐1 glycoprotein gp41 regulates lateral sorting and oligomerization

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM‐FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO‐K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO‐K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell‐to‐cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.

DBD dyes as fluorescent probes for sensing lipophilic environments

Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM).

Recruitment of SH-Containing peptides to lipid and biological membranes through the use of a palmitic acid functionalized with a Maleimide Group

This study presents a novel and easily applicable approach to recruit sulfhydryl-containing biomolecules to membranes by using a palmitic acid which is functionalized with a maleimide group. Notably, this strategy can also be employed with preformed (biological) membranes. The applicability of the assay is demonstrated by characterizing the binding of a Rhodamine-labeled peptide to lipid and cellular membranes using methods of fluorescence spectroscopy, lifetime measurement, and microscopy. Our approach offers new possibilities for preparing biologically active liposomes and manipulating living cells.

Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level.

Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin

Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor‐binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker.

Gp41 dynamically interacts with the TCR in the immune synapse and promotes early T cell activation

The HIV-1 glycoprotein gp41 critically mediates CD4+ T-cell infection by HIV-1 during viral entry, assembly, and release. Although multiple immune-regulatory activities of gp41 have been reported, the underlying mechanisms of these activities remain poorly understood. Here we employed multi-colour single molecule localization microscopy (SMLM) to resolve interactions of gp41 proteins with cellular proteins at the plasma membrane (PM) of fixed and live CD4+ T-cells with resolution of ~20–30 nm. We observed that gp41 clusters dynamically associated with the T cell antigen receptor (TCR) at the immune synapse upon TCR stimulation. This interaction, confirmed by FRET, depended on the virus clone, was reduced by the gp41 ectodomain in tight contacts, and was completely abrogated by mutation of the gp41 transmembrane domain. Strikingly, gp41 preferentially colocalized with phosphorylated TCRs at the PM of activated T-cells and promoted TCR phosphorylation. Gp41 expression also resulted in enhanced CD69 upregulation, and in massive cell death after 24–48 hrs. Our results shed new light on HIV-1 assembly mechanisms at the PM of host T-cells and its impact on TCR stimulation.

Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains.

We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac‐R7‐GG‐KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine‐containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol‐rich domains in a GPMV model, thus documenting the potential of our approach for membrane‐partitioning studies.

Chasing Raft Localisation Signals: FLIM-FRET Reveals CRAC Mediated Microdomain Association of the Human Immunodeficiency Virus Glycoprotein gp41

The glycoprotein gp41 is the only transmembrane protein of the human immunodeficiency virus (HIV). In concert with gp120 it mediates host cell infection by binding cellular receptors and later on triggering membrane fusion. Hitherto, little is known about the role of gp41 during assembly and budding of the newly synthesized virus particles. It has been reported that membranes of HIV-infected cells as well as virions are enriched in cholesterol, hence it was suggested that HIV-1 may take advantage of cholesterol and sphingolipids during budding. Both findings point to an important function of lipid microdomains, the so called rafts, in the late virus lifecycle.This work focuses on the detection of Forster Resonance Energy Transfer (FRET) between a raft marker, a GPI-anchored cyan fluorescent protein (CFP), and gp41 fusion proteins labeled with yellow fluorescent proteins (YFP)to elucidate raft clustering. Since energy transfer is highly dependent on the distance between the participating molecules, efficient FRET can be considered as a strong indication for close proximity of raft marker and fusion proteins and, therefore for colocalization in lipid microdomains. Fluorescence lifetime imaging microscopy (FLIM) was employed to accurately investigate FRET in living cells. In combination with acceptor fluorescence analysis the ascertained energy transfer efficiencies provide reliable information about clustering independent of expression level and fluorophore concentration. Several gp41 chimera were produced to address the role of different protein domains for raft association but also intracellular distribution and trafficking. The impact of truncations of the cytoplasmic tail as well as mutations of, the cholesterol recognition amino acid consensus (CRAC) domain, intrinsic trafficking signals and a palmitoylation site were studied in this context.