Subcellular Architecture of Regulatory Protein Complexes at the Bacterial Pole Recent advances in microscopic imaging with single fluorescent molecules have led to super-resolution information providing the ability to observe objects with resolution beyond the standard optical diffraction limit of ~250 nm in the visible. At the same time, the complexity of bacterial organization has become more and more apparent, and given that the human body contains more prokaryotic cells than eukaryotic cells, it is essential to understand our microbial partners, for scientific benefit and for prevention of pathology. Much of the organization in ?-proteobacteria occurs in the cell pole, the anchor not only for the flagellum, but also for the chromosomal origin, the chemotactic apparatus and for critical regulatory and signaling subsystems that coordinate cell cycle progression. While approximate information is available about the cell pole, many mysteries remain, and high resolution information on the identity and precise relative locations of polar proteins is required to understand and ultimately influence bacterial biology. This application proposes a new line of research to understand the subcellular organization of regulatory proteins at the Caulobacter cell pole at unprecedented resolution. Such an effort requires the close integration of biochemical genetics with advanced three-dimensional (3D) super-resolution fluorescence imaging beyond the optical diffraction limit, in order to fully quantify the locations and spatial interactions of key proteins at the bacterial cell pole down to a precision of ~20-30 nm in x, y, and z. Caulobacter crescentus is a powerful model of cellular differentiation by virtue of its asymmetric cell division cycle, of which one of the PIs is expert. The new imaging methodology in which the other PI is expert relies on two components: (a) a two- color method for 3D imaging in cells with the double-helix point spread function (DH-PSF) microscope, which allows precise 3D imaging over a large depth of field, and (b) single-molecule active control microscopy, which provides super-resolution detail by sequentially imaging and localizing sparse subsets of individual emitters. Three thrusts define this program:
Aim 1 : Development of advanced two-color, 3D imaging with the DH- PSF microscope: Methods for localizing relative locations of pairs of polar proteins with precision extending down to ~20nm in x, y, and z will be developed and validated.
Aim 2 : Super-resolution 3D imaging of benchmark protein assemblies to define the coordinate system of the pole. The polar reference coordinate system will be defined by performing precise 3D imaging of TipN, McpA, CreS, and PopZ, key polar markers.
Aim 3 : Define 3D structural organization and dynamics of key regulatory protein assemblies at the bacterial cell pole. By combining an array of mutant strains with two-color 3D super-resolution imaging, we will establish the spatial organization of multiple pairs of regulatory proteins at the bacterial cell pole. Dynamical information in live cells will be extracted from imaging at differen times of the cell cycle, thus providing an unprecedented view of the structure as well as the dynamics controlling bacterial cell organization and function.

Public Health Relevance

By combining new methods for three-dimensional high resolution optical imaging in living cells with expertise in bacterial cell biology, this research ill define the structural organization of the bacterial cell pole, a site of critical regulatory functin, in unprecedented detail. Such precise information about how bacteria actually work will bear directly upon biotechnological and biomedical problems where microbes are either essential symbionts or pathogens. The ability to specifically and noninvasively measure positions of key proteins and their superstructures at high resolution in live cells without requiring ionizing radiation or low temperatures will have strong implications for biomedical imaging and analysis of eukaryotic cells whose cellular structures and behavior are altered in the progress of disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-BCMB-P (02))
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Deatherage, James F
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Stanford University
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Lee, Marissa K; Rai, Prabin; Williams, Jarrod et al. (2014) Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy. J Am Chem Soc 136:14003-6
Ptacin, Jerod L; Gahlmann, Andreas; Bowman, Grant R et al. (2014) Bacterial scaffold directs pole-specific centromere segregation. Proc Natl Acad Sci U S A 111:E2046-55
Gahlmann, Andreas; Moerner, W E (2014) Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging. Nat Rev Microbiol 12:9-22
Lee, Yin Loon; Santé, Joshua; Comerci, Colin J et al. (2014) Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function. Mol Biol Cell 25:2919-33
Calderon, Christopher P; Weiss, Lucien E; Moerner, W E (2014) Robust hypothesis tests for detecting statistical evidence of two-dimensional and three-dimensional interactions in single-molecule measurements. Phys Rev E Stat Nonlin Soft Matter Phys 89:052705
Sahl, Steffen J; Moerner, W E (2013) Super-resolution fluorescence imaging with single molecules. Curr Opin Struct Biol 23:778-87
Gahlmann, Andreas; Ptacin, Jerod L; Grover, Ginni et al. (2013) Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. Nano Lett 13:987-93
Lee, Marissa K; Williams, Jarrod; Twieg, Robert J et al. (2013) Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopyýýý Chem Sci 42:220-225
Biteen, Julie S; Goley, Erin D; Shapiro, Lucy et al. (2012) Three-dimensional super-resolution imaging of the midplane protein FtsZ in live Caulobacter crescentus cells using astigmatism. Chemphyschem 13:1007-12
Lew, Matthew D; Lee, Steven F; Ptacin, Jerod L et al. (2011) Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Proc Natl Acad Sci U S A 108:E1102-10

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