Quantitative Superresolution Research Articles

Quantitative Storm using Organic Dyes

Quantitative super-resolution imaging is important for understanding and determining the exact stoichiometry of molecular complexes or oligomerization states of proteins within a cell. This has been done previously using fluorescence proteins, however these proteins require extensive computational correction due to their blinking. Organic dyes are commonly used for super-resolution imaging because of their high photon numbers, unfortunately blinking corrections for these dyes are very difficult. Here, we develop a technique for quantitative super resolution microscopy that allows us to count the absolute number of molecules using organic “caged” dyes, which do not blink. We calibrate our system and find that caged-fluorescein and caged-carborhodamine, when conjugated with SNAP-tag labeling, allow for quantitative counting of targets. We apply this technique to the characterization of the oligomerization states of chemokine receptors in mammalian cells.

Cell Cycle Phase Determines Critical Temperature in Plasma Membrane Vesicles

Giant plasma membrane vesicles (GPMVs) isolated from RBL-2H3 cells appear uniform at physiological temperatures, contain coexisting liquid-ordered and liquid-disordered phases at low temperatures, and experience micron-sized critical fluctuations close to their critical temperature. We observe a broad distribution of critical temperatures in GPMVs isolated from a dish of cells even though individual vesicles have a well-defined critical temperature. Interestingly, we find that densely plated cells yield GPMVs with lower average critical temperatures than GPMVs from less densely plated cells. Since it is known that cellular doubling times are reduced in cells plated at high density due to contact inhibition, we hypothesized that critical temperatures are linked to the cell cycle. To test this hypothesis, we isolated GPMVs from cells at various stages in their cell cycle. We plated cells in low serum media to produce a population of cells with arrested growth, and found low critical temperatures in GPMVs isolated from these cells (∼10°C). Critical temperatures recovered to typical values (∼20°C) after incubating the growth-arrested cells in serum rich media for 24h. Populations of cells are synchronized at S, G2, M, and G1 stages using a double Thymidine block that arrests cells at the border between G1 and S phases. Critical temperatures are elevated in GPMVs from populations of cells in cell cycle phases that immediately precede cell division (G2 and M) compared to the other stages (G1 and S). These results suggest that the magnitude of plasma membrane heterogeneity may be dependent on the cell cycle. We are currently investigating how position in the cell cycle influences the organization of plasma membrane proteins using quantitative super-resolution microscopy with multiple colors. We are also investigating cell cycle position's impact on outcomes of functional processes known to depend on lipid heterogeneity.

Roles played by capsid-dependent induction of membrane curvature and Gag-ESCRT interactions in tetherin recruitment to HIV-1 assembly sites.

Tetherin/BST-2 (here called tetherin) is an antiviral protein that restricts release of diverse enveloped viruses from infected cells through physically tethering virus envelope and host plasma membrane. For HIV-1, specific recruitment of tetherin to assembly sites has been observed as its colocalization with the viral structural protein Gag or its accumulation in virus particles. Because of its broad range of targets, we hypothesized that tetherin is recruited through conserved features shared among various enveloped viruses, such as lipid raft association, membrane curvature, or ESCRT dependence. We observed that reduction of cellular cholesterol does not block tetherin anti-HIV-1 function, excluding an essential role for lipid rafts. In contrast, mutations in the capsid domain of Gag, which inhibit induction of membrane curvature, prevented tetherin-Gag colocalization detectable by confocal microscopy. Disruption of Gag-ESCRT interactions also inhibited tetherin-Gag colocalization when disruption was accomplished via amino acid substitutions in late domain motifs, expression of a dominant-negative Tsg101 derivative, or small interfering RNA (siRNA)-mediated depletion of Tsg101 or Alix. However, further analyses of these conditions by quantitative superresolution localization microscopy revealed that Gag-tetherin coclustering is significantly reduced but persists at intermediate levels. Notably, this residual tetherin recruitment was still sufficient for the full restriction of HIV-1 release. Unlike the late domain mutants, the capsid mutants defective in inducing membrane curvature showed little or no coclustering with tetherin in superresolution analyses. These results support a model in which both Gag-induced membrane curvature and Gag-ESCRT interactions promote tetherin recruitment, but the recruitment level achieved by the former is sufficient for full restriction.

Temporal and Spatial Remodeling of Host Cell Plasma Membrane during HIV Assembly Revealed by Quantitative Superresolution Microscopy

The budding of enveloped retrovirus, such as HIV, involves oligomerization of the structural protein Gag at the plasma membrane and the incorporation of a patch of the host cell plasma membrane in the viral particle. Previous studies indicate that certain host proteins and viral envelope proteins are enriched in the membrane of virus particles. Since the viral membrane is derived from the host membrane, such specialized composition of viral membrane suggests viral assembly and budding involve differentiation of the host cell plasma-membrane at the assembly site. However, the mechanism of the membrane remodeling process during viral assembly is not understood. using a combination of quantitative superresolution microscopy (PALM and STORM) and diffraction-limited TIRF microscopy, we have investigated the spatial and temporal differentiation of the host cell plasma-membrane during budding of HIV. We find that in absence of Gag, the viral envelope protein is randomly distributed in the plasma membrane. However, oligomerization of Gag at the membrane leads to dramatic redistribution of the envelope proteins at the site of Gag assembly. We also see enrichment of GPI-anchored protein such as CD59 at the assembly site, while a model transmembrane protein EGFP-GT46 is actively excluded from the membrane patch. Gag is anchored to the plasma membrane via myristoylated lipid anchor and electrostatic interactions. Interestingly, we find that myristoylated inner leaflet anchored proteins are enriched while proteins with farnesyl and geranyl lipid anchors are depleted at the assembly site. Taken together, our results indicate that the oligomerization of HIV Gag at the plasma-membrane creates a specialized microenvironment, which leads to the differentiation of the local plasma membrane. This provides new insights about the temporal and spatial remodeling of plasma membrane during assembly and budding of retroviruses.