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Three-dimensional single-molecule microscopy of bacterial regulatory proteins within a pole-localized microdomain

Since the first optical detection of a single molecule 29 years ago, the development of single-molecule microscopy and spectroscopy has revolutionized the study of complex chemical systems. As reviewed in Chapter 1, by imaging and computationally localizing individual fluorescent dyes or proteins within a sample, their positions can be localized with typical precisions (10-40 nm) an order of magnitude or better than the optical diffraction limit of visible light (~250 nm laterally and ~500 nm axially). This technique is critical to super-resolution fluorescence microscopy and single-molecule tracking, which are now regularly used to measure the nanoscale structures, biomolecular motions, and stochastic chemical processes underlying the biology of cells. This dissertation comprises two intertwined single-molecule imaging projects: 1) optical and analytical methods development for three-dimensional (3D) single-molecule tracking and super-resolution microscopy, and 2) the application of these methods to understand the nanoscale organization and dynamics of proteins at the poles of the bacterium Caulobacter crescentus. Without modification, a single-molecule microscope only improves imaging resolution in the lateral (xy) dimension, but biological cells are intrinsically 3D. To improve the imaging resolution in z, the detection path of a standard widefield microscope can be modified using Fourier processing to encode z position in the pattern of light formed by each fluorescent emitter and detected on the camera. Chapter 2 reviews the development of a two-color 3D single-molecule microscope that uses the double-helix point spread function pattern to encode 3D position, while Chapter 3 describes how to correctly align and to calibrate the fine aberrations of such a microscope to achieve nanoscale imaging accuracy in multiple color channels simultaneously. The bacterium Caulobacter crescentus is a model organism for the study of cell polarization and asymmetric cell division. Chapters 4 and 5 describe work performed in collaboration with Prof. Lucy Shapiro and her laboratory in the Department of Developmental Biology in the Stanford University School of Medicine to study how the tips, or poles, of Caulobacter cells use proteins to act as nanoscale spatial landmarks that polarize cells and induce spatially organized development. The polar organizing protein PopZ is one such critical landmark, and Chapter 4 describes results obtained from 3D super-resolution imaging of PopZ. Such imaging showed that PopZ forms 150-200 nm space-filling polar microdomains of roughly uniform density, and that proteins of the chromosome partitioning machinery (ParA and ParB) exhibit different spatial behaviors (recruitment vs. tethering) relative to the PopZ microdomain depending on their biochemistry and role in the chromosome replication process. Chapter 5 discusses the combination of single-molecule tracking and super-resolution imaging to study the polar localization of the signaling molecules of that activate the master regulator protein CtrA. Precise 3D imaging and tracking showed that PopZ acts as a selectively permeable localization hub that slows the motion of signaling proteins. In combination with reaction-diffusion modeling and transcriptional assays, these microscopic measurements indicated that the PopZ microdomain acts to sequester the CtrA signaling pathway within the pole and spatially pattern transcriptional output within the predivisional Caulobacter cell.