Gerhard J. Schuetz

Diffusion Analysis of Lymphocyte Specific Kinase Reveals Immobilisation Hot Spots in Live T-Cell Plasma Membranes

T-Lymphocytes initiate an adaptive immune response after specific binding of the T-cell receptor (TCR) to an antigenic peptide bound to the major histocompatibility complex (peptide-MHC) on an antigen presenting cell. Key events upon TCR-pMHC binding involve the phosphorylation of intracellular tyrosine residues of the TCR and recruitment of adapter molecules, which results in downstream signalling. Initial TCR phosphorylation is primarily carried out by the membrane associated protein lymphocyte specific kinase (Lck) making it a central molecule for T-cell signalling.Recent work on Lck, using superresolution microscopy techniques, has shown the organisation of Lck in nanoclusters in fixed as well as in live cells [1]. However, neither the structural mechanism nor the functional role of this non-random Lck distribution in the plasma membrane are well understood. Possible explanations for Lck clustering may be local lipid heterogeneities as well as interactions with other plasma membrane proteins or the cortical actin cytoskeleton.In order to acquire a better understanding of Lck dynamics in live cells, we carried out superresolution experiments on Lck-mEOS3.2, stably expressed in JCaM 1.6 cells. We used single molecule tracking to elucidate the diffusional behaviour of Lck. Based on the reported existence of nanoclusters, we implemented a simple and practical method to separately analyse Lck diffusion inside and outside these structures. Surprisingly, only Lck-molecules inside clusters were immobilised, whereas molecules outside of clusters exhibited pure Brownian motion. We further found that disruption of the actin cytoskeleton did not influence Lck immobilisations.[1] Rossy, J., et al., Conformational states of the kinase Lck regulate clustering in early T cell signalling. Nature immunology 14, 82-89 (2013).This work was supported by the Austrian Science Fund/FWF project P26337-B21 and P27941-B28.

Imaging of Mobile Stable Nanoplatforms in the Live Cell Plasma Membrane

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of “lipid rafts” or “membrane rafts”. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analogue Bodipy-GM1 which preferentially partitions into liquid ordered phases. For both markers we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, stable platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures the mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

Photoactivation Localization Microscopy (PALM) on Orai1 Channels

Store Operated Calcium Entry (SOCE) is a crucial mechanism for many cellular signalling processes. The two major proteins involved in SOCE are Orai1 (located in the plasma membrane) and STIM1 (located in the membrane of the endoplasmatic reticulum (ER)). Upon depletion of the calcium stores in the ER, the STIM1 co-clusters with the Orai1 in the plasma membrane which results in a calcium influx into the cell.In order to investigate the distribution of the Orai1 in the plasma membrane we used Photoactivation Localization Microscopy (PALM). PALM is a technique that allows overcoming the diffraction limit by photo-activating only a small subset of fluorophores with a laser pulse. At shallow illumination conditions, the active fluorophores are spatially well separated and can be localized with a precision of a few ten nm. Sequential activation, readout and photobleaching allows for recording a complete image of the sample. The Orai1 subunits were fused to photoactivatable GFP (paGFP) and expressed in Chinese hamster ovary cells. PALM revealed submicrometer clusters of Orai1, which show a high degree of colocalization with STIM1-mCherry, as confirmed by two-color microscopy.