Faculty Bibliography

PspA, -B and -C regulate the bacterial phage shock protein stress response by controlling the PspF transcription factor. Here, we have developed complementary approaches to study the behaviour of these proteins at their endogenous levels in Yersinia enterocolitica. First, we observed GFP-tagged versions with an approach that resolves individual protein complexes in live cells. This revealed that PspA, -B and -C share common behaviours, including a striking contrast before and after induction. In uninduced cells, PspA, -B and -C were highly mobile and widely distributed. However, induction reduced mobility and the proteins became more organized. Combining mCherry- and GFP-tagged proteins also revealed that PspA colocalizes with PspB and PspC into large stationary foci, often located close to the pole of induced cells. In addition, co-immunoprecipitation assays provided the first direct evidence supporting the model that PspA switches binding partners from PspF to PspBC upon induction. Together, these data suggest that PspA, -B and -C do not stably interact and are highly mobile before induction, perhaps sampling the status of the membrane and each other. However, an inducing signal promotes PspABC complex formation and their relocation to discrete parts of the membrane, which might then be important for mitigating envelope stress.

Ventricular ATP-sensitive potassium (K(ATP)) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K(ATP) channel-associated proteins. We investigated whether the association of K(ATP) channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K(ATP) channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K(ATP) channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K(ATP) channels are clustered within nanometer distances from junctional proteins. The local K(ATP) channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K(ATP) channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K(ATP) channel current density (activated by metabolic inhibition) was approximately 40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K(ATP) channels was absent in the PKP2 deficient mice, but the K(ATP) channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K(ATP) channel distribution within a cardiac myocyte. The higher K(ATP) channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.

The initial step of viral infection is the binding of a virus onto the host cell surface. This first viral-host interaction would determine subsequent infection steps and the fate of the entire infection process. A basic understating of the underlining mechanism of initial virus-host binding is a prerequisite for establishing the nature of viral infection. Bacteriophage λ and its host Escherichia coli serve as an excellent paradigm for this purpose. λ phages bind to specific receptors, LamB, on the host cell surface during the infection process. The interaction of bacteriophage λ with the LamB receptor has been the topic of many studies, resulting in wealth of information on the structure, biochemical properties and molecular biology of this system. Recently, imaging studies using fluorescently labeled phages and its receptor unveil the role of spatiotemporal dynamics and divulge the importance of stochasticity from hidden variables in the infection outcomes. The scope of this article is to review the present state of research on the interaction of bacteriophage λ and its E. coli receptor, LamB.

BACKGROUND: Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization"). OBJECTIVE: To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling. METHODS: Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp. RESULT: Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells. CONCLUSIONS: Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Several super-resolution techniques exist, yet most require multiple lasers, use either large or weakly emitting fluorophores, or involve chemical manipulation. Here we show a simple technique that exceeds the standard diffraction limit by 5-15x on fixed samples, yet allows the user to localize individual fluorophores from among groups of crowded fluorophores. It relies only on bright, organic fluorophores and a sensitive camera, both of which are commercially available. Super-resolution is achieved by subtracting sequential images to find the fluorophores that photobleach (temporarily or permanently), photoactivate, or bind to the structure of interest in transitioning from one frame to the next. These fluorophores can then be localized via Gaussian fitting with selective frame averaging to achieve accuracies much better than the diffraction limit. The signal-to-noise ratio decreases with the square root of the number of nearby fluorophores, producing average single-molecule localization errors that are typically <30 nm. Surprisingly, one can often extract signal when there are approximately 20 fluorophores surrounding the fluorophore of interest. Examples shown include microtubules (in vitro and in fixed cells) and chromosomal DNA

We report the first two-photon (2P) microscopy of individual quantum dots (QDs) in an aqueous environment with both widefield and point-scan excitations at nanometer accuracy. Thiol-containing reductants suppress QD blinking and enable measurement of the 36 nm step size of individual Myosin V motors in vitro. We localize QDs with an accuracy of 2-3 nm in all three dimensions by using a 9 x 9 matrix excitation hologram and an array detector, which also increases the 3D scan imaging rate by 80-fold. With this 3D microscopy we validate the LamB receptor distribution on E. coli and the endocytosis of EGF-receptors in breast cancer cells

Viral infection begins with the binding of a virus to a specific target on the surface of the host cell, followed by viral genome delivery into the host and a continuation of the infection process. Before binding occurs, the virus must first find its receptor by a process whose details are largely unknown. We applied high-resolution fluorescence microscopy and single-particle tracking to elucidate the target-finding process in bacteriophage lambda as it infects an Escherichia coli cell. By monitoring the motion of individual viruses through the early stages of infection, we identified a unique spatial focusing process that allows a virus to arrive from its initial random landing site to its destination at the cell pole. The search process is governed by the interaction between the virus and the LamB receptors, and by the spatial organization of the receptor network on the cell surface. Our findings allowed us to develop a theoretical model for the target-finding process that reproduces the key features observed in experiment. We discuss the possible implications of our findings for the process of viral receptor-finding in higher systems

Rad52 promotes the annealing of complementary strands of DNA bound by replication protein A (RPA) during discrete repair pathways. Here, we used a fluorescence resonance energy transfer (FRET) between two fluorescent dyes incorporated into DNA substrates to probe the mechanism by which human Rad52 (hRad52) interacts with and mediates annealing of ssDNA-hRPA complexes. Human Rad52 bound ssDNA or ssDNA-hRPA complex in two, concentration-dependent modes. At low hRad52 concentrations, ssDNA was wrapped around the circumference of the protein ring, while at higher protein concentrations, ssDNA was stretched between multiple hRad52 rings. Annealing by hRad52 occurred most efficiently when each complementary DNA strand or each ssDNA-hRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52-ssDNA complexes. In contrast to the wild type protein, hRad52(RQK/AAA) and hRad52(1-212) mutants with impaired ability to bind hRPA protein competed with hRPA for binding to ssDNA and failed to counteract hRPA-mediated duplex destabilization highlighting the importance of hRad52-hRPA interactions in promoting efficient DNA annealing

In recent years, advancements in single-biomolecule probing techniques have provided critical information on and greater insight into the nature of biomolecules. Of significance is the application of single-molecule fluorescence resonance energy transfer (smFRET) to probe isolated events and changes at the nanometer scales. In particular, the study of helicases using smFRET has supplied much information regarding the nature and dynamics of these enzymes and provided a toolbox for further investigations. In this chapter we provide a general guide for the construction and execution of single-molecule FRET assays for the study of helicase properties and functionalities

The Rad52 protein has critical functions in distinct pathways of the homology-directed DNA repair, one of which is to promote the annealing of complementary strands of DNA. Both yeast and human Rad52 proteins organize into ring-shaped oligomers with the predominant form being a heptamer. Despite the wealth of information obtained in previous investigations, how Rad52 mediates homology search and annealing remains unclear. Here, we developed single-molecule fluorescence resonance energy transfer approaches to probe hRad52-mediated DNA annealing events in real time. We found that annealing proceeds in successive steps involving rearrangements of the ssDNA-hRad52 complex. Moreover, after initial pairing, further search for extended homology occurs without dissociation. This search process is driven by an interaction between 2 overlapping nucleoprotein complexes. In light of these observations we propose a model for hRad52-mediated DNA annealing where ssDNA release and dsDNA zippering are coordinated through successive rearrangement of overlapping nucleoprotein complexes