Abstract: Palmitoylation represents a common motif for anchorage of cytosolic proteins to the plasma membrane. Being reversible, it allows for controlled exchange between cytosolic and plasma membrane-bound subpopulations. In this study, we present a live cell single molecule approach for quantifying the exchange kinetics of plasma membrane and cytosolic populations of fluorescently-labeled Lck - the key Src-family kinase involved in early T cell signaling. Total internal reflection (TIR) fluorescence microscopy was employed for confining the analysis to membrane-proximal molecules. Upon photobleaching Lck-YFP in TIR configuration, fluorescence recovery proceeds first via the cytosol outside of the evanescent field, so that in the early phase fluorescence signal arises predominantly from membrane-proximal cytosolic Lck. The diffusion constant of each molecule allowed us to distinguish whether the molecule has already associated with the plasma membrane or was still freely diffusing in the cytosol. From the number of molecules that inserted during the recovery time we quantified the insertion kinetics: on average, membrane-proximal molecules within the evanescent field needed ~400ms to be inserted. The average lifetime of Lck in the plasma membrane was estimated with 50s; together with the mobility of 0.26mum(2)/s this provides sufficient time to explore the surface of the whole T cell before dissociation into the cytosol. Experiments on palmitoylation deficient Lck mutants yielded similar on-rates, but substantially increased off-rates. We discuss our findings based on a model for the plasma membrane association and dissociation kinetics of Lck, which accounts for reversible palmitoylation on cysteine 3 and 5.
Abstract: In recent years increasing evidence has been reported for the inherent heterogeneity of cell populations. Cell-to-cell variability was particularly found when analyzing protein expression patterns or the responses of cells to different stimuli. Yet, structural features - in particular below the resolution limit of light microscopy - have so far eluded in-depth statistical analysis. We report here for the first time a detailed investigation of the variations in protein mobility between nominally identical cells. Our reasoning was that mobility probes nanometer-sized properties of the moving protein and its local environment, which may be subject to cell-to-cell variability. Single-molecule tracking was employed to characterize the diffusion constant of the glycosylphosphatidylinositol- (GPI-) anchored protein CD59 and the transmembrane protein CD147 in the plasma membrane of T24 cells. Automated and tailored data analysis routines allowed for the analysis of the required large data sets: similar to 200 000 trajectories obtained on similar to 350 cells were analyzed in total. We found up to five-fold higher variability of the diffusion constant between cells compared to the uncertainty for the determination of the diffusion constant on a single cell.
Abstract: The MRS2/MGT gene family in Arabidopsis thaliana belongs to the superfamily of CorA-MRS2-ALR-type membrane proteins. Proteins of this type are characterized by a GMN tripeptide motif (Gly-Met-Asn) at the end of the first of two C-terminal transmembrane domains and have been characterized as magnesium transporters. Using the recently established mag-fura-2 system allowing direct measurement of Mg(2+) uptake into mitochondria of Saccharomyces cerevisiae, we find that all members of the Arabidopsis family complement the corresponding yeast mrs2 mutant. Highly different patterns of tissue-specific expression were observed for the MRS2/MGT family members in planta. Six of them are expressed in root tissues, indicating a possible involvement in plant magnesium supply and distribution after uptake from the soil substrate. Homozygous T-DNA insertion knockout lines were obtained for four members of the MRS2/MGT gene family. A strong, magnesium-dependent phenotype of growth retardation was found for mrs2-7 when Mg(2+) concentrations were lowered to 50 muM in hydroponic cultures. Ectopic overexpression of MRS2-7 from the cauliflower mosaic virus 35S promoter results in complementation and increased biomass accumulation. Green fluorescent protein reporter gene fusions indicate a location of MRS2-7 in the endomembrane system. Hence, contrary to what is frequently found in analyses of plant gene families, a single gene family member knockout results in a strong, environmentally dependent phenotype.
Abstract: Communication between cells is crucial for proper functioning of multicellular organisms. The recently discovered membranous tubes, named tunneling nanotubes, that directly bridge neighboring cells may offer a very specific and effective way of intercellular communication. Our experiments on RT4 and T24 urothelial cell lines show that nanotubes that bridge neighboring cells can be divided into two types. The nanotubes of type I are shorter and more dynamic than those of type II, and they contain actin. laments. They are formed when cells explore their surroundings to make contact with another cell. The nanotubes of type II are longer and more stable than type I, and they have cytokeratin. laments. They are formed when two already connected cells start to move apart. On the nanotubes of both types, small vesicles were found as an integral part of the nanotubes (that is, dilatations of the nanotubes). The dilatations of type II nanotubes do not move along the nanotubes, whereas the nanotubes of type I frequently have dilatations (gondolas) that move along the nanotubes in both directions. A possible model of formation and mechanical stability of nanotubes that bridge two neighboring cells is discussed.
Abstract: We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.
Abstract: Members of the CorA- Mrs2-Alr1 superfamily of Mg2+ transporters are ubiquitous among pro- and eukaryotes. The crystal structure of a bacterial CorA protein has recently been solved, but the mode of ion transport of this protein family remained obscure. Using single channel patch clamping we unequivocally show here that the mitochondrial Mrs2 protein forms a Mg2+-selective channel of high conductance ( 155 pS). It has an open probability of similar to 60% in the absence of Mg2+ at the matrix site, which decreases to similar to 20% in its presence. With a lower conductance (similar to 45 pS) the Mrs2 channel is also permeable for Ni2+, whereas no permeability has been observed for either Ca2+, Mn2+, or Co2+. Mutational changes in key domains of Mrs2p are shown either to abolish its Mg2+ transport or to change its characteristics toward more open and partly deregulated states. We conclude that Mrs2p forms a high conductance Mg2+ selective channel that controls Mg2+ influx into mitochondria by an intrinsic negative feedback mechanism.
Abstract: The nuclear gene MRS2 in Saccharomyces cerevisiae encodes an integral protein (Mrs2p) of the inner mitochondrial membrane. It forms an ion channel mediating influx of Mg2+ into mitochondria. Orthologues of Mrs2p have been shown to exist in other lower eukaryotes, in vertebrates and in plants. Characteristic features of the Mrs2 protein family and the distantly related CorA proteins of bacteria are the presence of two adjacent transmembrane domains near the C terminus of Mrs2p one of which ends with a F/Y-G-M-N motif. Two coiled-coil domains and several conserved primary sequence blocks in the central part of Mrs2p are identified here as additional characteristics of the Mrs2p family. Gain-of-function mutations obtained upon random mutagenesis map to these conserved sequence blocks. They lead to moderate increases in mitochondrial Mg2+ concentrations and concomitant positive effects on splicing of mutant group II intron RNA. Site-directed mutations in several conserved sequences reduce Mrs2p-mediated Mg2+ uptake. Mutants with strong effects on mitochondrial Mg2+ concentrations also have decreased group II intron splicing. Deletion of a nonconserved basic region, previously invoked for interaction with mitochondrial introns, lowers intramitochondrial Mg2+ levels as well as group II intron splicing. Data presented support the notion that effects of mutations in Mrs2p on group II intron splicing are a consequence of changes in steady-state mitochondrial Mg2+ concentrations.
Abstract: Steady-state concentrations of mitochondrial Mg2+ previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg2+ transport protein CorA suggested a role for Mrs2p in Mg2+ influx into the organelle, other functions in cation homeostasis could not be excluded. Making use of the fluorescent dye mag-fura 2 to measure free Mg2+ concentrations continuously, we describe here a high capacity, rapid Mg2+ influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Deltapsi and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases influx rates 5-fold, while the deletion of the MRS2 gene abolishes this high capacity Mg2+ influx. Mg2+ efflux from isolated mitochondria, observed with low Deltapsi only, also requires the presence of Mrs2p. Cross-linking experiments revealed the presence of Mrs2p-containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo- oligomers. Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.
