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Structured RNAs play a range of essential roles in the cell, including central functions in protein synthesis and gene regulation. These important RNAs include ribozymes, which catalyze chemical reactions, and riboswitches, mRNA elements which modulate gene expression in response to metabolite ligands. Structured RNAs ultimately enact their function by folding from linear sequences into elaborate three-dimensional conformations with specific biochemical properties. A mechanistic understanding of the relationships between RNA sequence, folding, and function should allow for increasingly sophisticated engineering of RNAs with desired functions, as well as the design of drugs targeting naturally occurring RNAs. In my PhD thesis research, I delved into these relationships in the context of the glmS ribozyme riboswitch. This widespread bacterial riboswitch regulates the production of an essential cell wall precursor, glucosamine 6-phosphate (GlcN6P), making it a compelling target for antibiotic development efforts. The key step for gene regulation is a self-cleavage reaction performed by the ribozyme riboswitch only when GlcN6P is bound in its active site. I investigated this structured RNA using two powerful and complementary approaches: (1) single-molecule force spectroscopy using a high-resolution optical tweezers apparatus to study folding, ligand binding, and catalysis, and their interrelationship; and (2) massively-parallel biochemistry on an RNA array to systematically dissect the sequence-dependence of self-cleavage activity and mutational interactions between residues.

The bacterium Caulobacter crescentus divides asymmetrically. This lends to its suitability as a model organism for studying the bacterial cell cycle as well as the asymmetry inherent in all life. Central to Caulobacter's cell cycle is its single circular chromosome, which encodes genetic elements whose expression patterns are coordinated in a temporally oscillating transcriptional network. But there is more to the bacterial chromosome than genetic elements like genes and their promoters; in reality, the chromosome is a DNA polymer with physical constraints, capabilities, and complexities outside of its role as a template for transcription. For instance, DNA in bacteria is supercoiled and compacted by three orders of magnitude. At the ~1Mb level, the E. coli nucleoid is characterized by insulated macrodomains and flexible unstructured regions. At the ~10 kb level the bacterial nucleoid is arranged into so-called microdomains by nucleoid-associated proteins (NAPs). This structuring of DNA functions in segregation, replication, decatenation, and double- and single-strand break repair. In the work presented in this thesis I leverage four Next Generation Sequencing (NGS)-based technologies to examine the regulation and function of four physical and non-genic features of the Caulobacter chromosome: its covalent modifications, essential intergenic regions, and global accessibility, as well as a novel, essential nucleoid-associated protein. First, in Chapter 2, I explore the covalent modification systems in Caulobacter using SMRT sequencing technologies. By measuring the methylation state of every base pair in the chromosome at five times in the cell cycle, I demonstrate that DNA methylation by the cell cycle-regulated methyltransferase CcrM is in fact dynamic, changing from fully methylated to hemimethylated at the time of replication fork passage. Importantly, the master transcriptional regulators ctrA and dnaA have promoters that become activated or repressed respectively once hemimethylated. From this perspective, the methylation data supports a model in which the replicating chromosome acts like a clock, with the cell cycle timing of ctrA activation and dnaA repression precisely synchronized with replication fork passage. I then use the SMRT sequencing data to predict which additional promoters besides PctrA and PdnaA are possibly controlled by methylation state. In addition, I also report that some of the CcrM target sites were found to be constitutively unmethylated. Work by our colleagues has shown that at one such unmethylated region of the chromosome called Gap 7, the DNA-binding proteins MucR1/2 block CcrM from its cognate site. In Chapter 3, I investigate Caulobacter's essential intergenic regions, called Gap regions. These non-genic elements of unknown function appeared as essential regions of the chromosome during Transposon-sequencing (Tn-seq) experiments. By performing molecular genetics experiments I demonstrated that at least some Gaps are nonessential. Furthermore, I deduce that at least two of those nonessential Gaps appeared in the Tn-seq screen because they are specifically protected from transposase, the enzyme that carries out transposition, in vivo. I end this chapter by exploring the effects of placing the intergenic regions encompassing Gaps onto high copy plasmids. These experiments demonstrate that such perturbations actually cause elongation and division defects in Caulobacter in a growth rate-dependent manner. I predict that some of these transposase-protected regions may be blocked by the binding of specific proteins that function in the Caulobacter cell cycle. In Chapter 4, I build further support for these conclusions by developing and performing ATAC-seq in Caulobacter. By measuring transposase accessibility of the entire chromosome in vivo, ATAC-seq more definitively and globally divides Gaps into non-disruptable regions, which are relatively protected from transposase enzyme, and true candidate essential regions. Thus, uncharacterized non-genic elements of the chromosome may perform essential functions in Caulobacter. I also performed ATAC-seq on a strain lacking the novel nucleoid-associated protein GapR, which binds over many Gaps in vivo, to test whether this protein is responsible for forming Gap regions where it binds the chromosome. While GapR does not affect local DNA accessibility, global analysis of ATAC-seq data provides evidence that GapR plays key roles in shaping megabase-scale properties of the Caulobacter nucleoid. In WT (wild type) cells, this global accessibility pattern is reminiscent of the macrodomain-level structure of the E. coli chromosome, which features insulated origin and terminus-proximal regions as well as two highly flexible origin-flanking regions. These domains compose the largest scale of chromosome organization known in prokaryotes and are thought to help control the fidelity of chromosome segregation. Such macrodomains have not been observed explicitly before in Caulobacter. Overall, ATAC-seq shows that the chromosome is varied in accessibility along its length, reflecting how transcription, local DNA structure, NAPs like GapR, and possibly other protein-DNA interactions, alongside other unknown factors, may together yield globally varying enzyme access to the nucleoid. In Chapter 5, I focus on GapR, a novel, conserved and essential DNA binding protein introduced above. ChIP-seq experiments revealed that GapR binds AT-rich DNA globally throughout the chromosome, including over a majority of Gap regions. Although GapR shares many similarities with the E. coli NAP H-NS, there are two important differences between these proteins. First, whereas H-NS represses transcription of many genes in E. coli, depletion-RNAseq experiments in which gene expression changes were measured after proteolytic removal of GapR from Caulobacter demonstrated that GapR does not control transcription. In addition, although GapR is essential in Caulobacter, hns is dispensable in E. coli. In the final Chapter I will explore efforts to elucidate the essential function of GapR. Perspectives on additional functions of GapR proposed by our colleagues, including the regulation of initiation of chromosome segregation, are also discussed. Altogether, the work in this chapter provides evidence that NAPs can perform multiple diverse functions, including those that help fine-tune cell cycle progression. These experiments highlight an integrated view of the bacterial cell cycle in which both genic as well as non-genic and physical elements of the chromosome play key roles. Further, they herald novel applications of NGS techniques as promising tools for microbiologists.

The development of live cell RNA imaging techniques will lead to the unraveling of many important biological processes. To achieve this goal, there have been three different strategies developed. They are the development of small molecule probes, nucleic acid probes, and green fluorescent protein (GFP) probes. In the following thesis, the pros and cons of each approach are discussed, followed by a proposal to resolve the limitations. In the small molecule case, a probe was developed that utilized a quenched sulforhodamine dye. It was designed so that its structure can be rationally modified from the initial lead compound. An aptamer sequence that activates the sulforhodamine probe with micro molar affinity was found by in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX), followed by fluorescence screening in E.coli. The rational modification of the structure of the initial sulforhodamine probe resulted in an overall 33-fold increase in binding affinity compared to the initial lead compound. Instead of the chemical modification of the lead compound, the small molecule's cell permeability and binding affinity to the target could be improved by linking to cell penetrating peptides (CPP). A CPP is a short peptide sequence composed of poly arginine amino acids which shows excellent cell uptake and affinity to RNA. However, the use of the CPP-linked dye in live cell imaging has been limited by strong signals in the endosome region. An attempt was made to overcome this difficulty by linking a quencher molecule to the dye-CPP via a disulfide bond, which only breaks when it enters the cytosol. For the nucleic acid probe, the major problem was its low cell permeability and low signal-to-background ratio due to the low copy number of mRNA targets within the cell. We made mutant Hammerhead ribozymes and embedded them in a non-coding region of the GFP expression vector that can be transfected to mammalian cells. This modified Hammerhead ribozyme acts as a logic gate, and the signal is amplified by the expression of GFP in the presence of the target mRNA. In vitro and in vivo results are discussed. Finally, a fragmented GFP system, the fluorescence of which could be recovered by binding to a specific RNA tag, was developed. The major problem for the GFP-mediated RNA imaging system was the low signal-to-background ratio from the GFP probe that is not bound to the RNA tag. To find the non-fluorescent GFP, the GFP was truncated from the C-terminus such that it loses its fluorescence with minimum loss of amino acids. An RNA sequence that has high affinity to this GFP was found by in vitro SELEX. The subsequent E.coli screening found an RNA sequence that reactivates the fluorescence of the GFP probe.

Cell division is a major developmental event in the life cycle of a bacterial cell. Caulobacter crescentus division is asymmetric, producing daughter cells that differ in morphology and polar features: a sessile stalked cell and a motile swarmer cell that subsequently differentiates into a stalked cell. In this work we investigate the assembly of the Caulobacter cell division machinery (the divisome) using genetics, biochemistry, and microscopy. In Caulobacter, the cell division process requires a set of approximately twenty-three proteins localizing from the cytoplasm to the outer membrane. To understand divisome assembly as a function of the cell cycle, we generated fluorescent fusions to analyze the temporal regulation of 19 representative divisome and division-site localized proteins. In Chapter 2, we identified a series of stages and transitions in divisome assembly and the associated events yielding a comprehensive temporal picture of the process. The assembly interdependency for divisome formation in Caulobacter appears to involve cooperative rather than sequential recruitment, suggesting that it is a multiprotein subcomplex model. In Chapter 3, we describe our investigation of the Tol-Pal complex where we demonstrated that it plays a vital role for membrane integrity maintenance and that it is essential for viability. Cryo-electron microscope images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Thus, the Caulobacter trans-envelope Tol-Pal complex is a key component of cell envelope structure and function, mediating outer membrane constriction at the final step of cell division, as well as the positioning of a protein localization factor. In Chapter 4, we describe our examination of the FtsZ binding protein, ZapA. FtsZ is the most highly conserved divisome protein that polymerizes into a contractile ring near midcell, defining the future site of cell division. We showed that ZapA is required to maintain a normal cell length, and promotes Z ring assembly. The biochemical and functional studies suggest that Caulobacter ZapA is a positive regulator of Z-ring assembly. In summary, we have addressed three major stages in developments of the divisome in Caulobacter: Z-ring assembly, divisome maturation and outer membrane invagination. These experiments have provided a new understanding of how the Caulobacter cell temporally executes the cell division program to propagate reliably and how Caulobacter cell division is performed.

Fluorescence microscopy is a widely used tool in biological studies, allowing specific labeling and imaging of proteins and structures within living cells. As reviewed in Chapter 1, conventional fluorescence microscopy is unable to distinguish structures below ~250 nm in size due to the diffraction limit of light. Over the last two decades, the field of super-resolution microscopy has overcome this diffraction limit, allowing fluorescence imaging of structures just a few tens of nanometers in size. This thesis discusses one super-resolution technique, stimulated emission depletion (STED) microscopy, and its application to various cellular and molecular biophysics questions. In Chapter 2, the bespoke STED microscope used for much of this thesis is described in detail. Some guiding principles behind STED microscopes and their design are discussed. We next discuss the major improvements to the STED microscope: fast scanning and two-color. Fast scanning protects fluorophores from photobleaching during imaging, improving image quality and expanding the range of available fluorophores for STED imaging. Two-color imaging allows the examination of two different protein species, a universal requirement in many biological studies. In Chapter 3, we apply STED microscopy to study how the bacteria Caulobacter crescentus assembles a 2D paracrystalline surface layer (S-layer) composed of the protein RsaA. We show that S-layer assembly is localized to highly curved regions of the cell, and that this localized assembly depends upon the presence of a pre-existing crystalline S-layer. Together, our results suggest the topology of the cell surface combined with continuous protein crystallization localizes S-layer assembly. In Chapter 5, 2-color STED microscopy is used to examine protein interactions important for the 3D organization of the genome in eukaryotic cells. We quantify clustering of the DNA binding protein CCCTC-binding factor (CTCF), as well as the molecular coupling between CTCF and cohesin, two proteins hypothesized to play a major role in organizing topologically associating domains (TADs). We show that cohesin plays a regulatory role in CTCF clustering by perturbing its activity and monitoring cluster size. Finally, we discuss several applications of STED imaging to support larger studies. Chapter 4 discusses applying a genetically encoded fluoromodule for STED imaging in live bacteria cells. In the first half of Chapter 6, STED reveals the localization of the centriole protein Cby1 to the transition zone, supporting a role for Cby1 in recruiting Ahi1, another transition zone protein. Finally, the second half of Chapter 6 uses two-color STED microscopy to demonstrate that deletion of the intracellular cargo transport protein IFT25 causes simultaneous defects in the cilium cytoskeleton as well as ciliary membrane morphology

The first optical detection of a single molecule (SM) at cryogenic temperatures 30 years ago laid the groundwork for the routine detection of SMs today at biologically relevant temperatures, thus uncovering hidden heterogeneity that might be obscured by ensemble techniques. In addition to enabling studies of the intricate photochemistry and photophysics of fluorescent labels at the SM level, SM fluorescence has also proven useful for the imaging and tracking of cellular structures and biomolecules in a non-invasive manner with high sensitivity. The ability to genetically express fluorescent protein fusions in live cells has allowed specific labeling, and thus imaging and tracking, of dynamic processes and structures in cells. This dissertation describes applications of SM-based single-particle tracking (SPT) and super-resolution (SR) microscopy for the study of spatial organization and dynamics of bacterial proteins in two and three spatial dimensions. In an SPT experiment, the position of a SM emitter at very low concentration is measured over time to generate a trajectory, allowing for observation and quantification of labeled biomolecule dynamics at the SM level. In a SR microscopy experiment, the diffraction-limited (DL) resolution of a conventional fluorescence microscope (~200 nm in xy) is circumvented by temporally separating the emission of many SM emitters decorating a structure through control of their emissive state. A "super-resolved" image, with a factor of ~5-10 resolution improvement over a conventional DL fluorescence image, is generated by estimating the positions of many non-moving SM emitters over many frames and building up an image reconstruction in a pointillist manner. Chapter 1 of this dissertation provides an introduction to fluorescence, SM imaging, SM-based SR microscopy, and SPT. Chapter 1 also gives a brief introduction to Caulobacter crescentus, the bacterium used as the model organism in the SM studies in Chapters 4 and 5. Chapter 2 describes the experimental methods used to perform quantitative SM imaging of bacterial cells. The combination of SM imaging with point spread function (PSF) engineering has enabled the accurate and precise localization of SMs in three dimensions (3D) by the intentional introduction of specifically chosen aberrations in the emission path of an SM microscope. Throughout this dissertation, the double-helix (DH) PSF, a rotating PSF composed of two lobes whose angle encodes axial position, was used to estimate 3D SM positions. Chapter 2 describes the implementation of the DH-PSF via optical Fourier processing, and Chapter 3 describes the robust and comprehensible two-color Easy-DHPSF v2 software for localizing molecules in 3D and for registering localizations from two spectral channels into the same coordinate system with nanoscale accuracy. The resolution improvement gained from SM-based techniques is particularly useful for bacteria, the sizes of which are on the order of the DL. 3D SM-based SR and SPT have enabled the observation of structures and dynamics at length scales below the DL. Caulobacter is a useful biological target where understanding of the mechanisms for asymmetric cell division need to be explored and quantified. Central to Caulobacter's asymmetric division is the dynamic spatiotemporal regulation of gene expression and protein localization. Chapters 4 and 5 describes research performed in collaboration with Prof. Lucy Shapiro's laboratory (Department of Developmental Biology, Stanford School of Medicine) to study gene expression and signaling proteins in Caulobacter. Chapter 4 describes work studying the spatial organization and dynamics of ribosomes and a RNA-degrading enzyme RNase E using 3D SR microscopy and SPT. Results showed that the organization and dynamics of RNase E and ribosomes are closely related to the transcriptional activity of the cell. Finally, Chapter 5 describes SPT studies of the membrane-bound histidine kinase and stalked cell fate determinant DivJ in an effort to probe the physical properties of the Caulobacter stalked pole. Preliminary SPT results suggest that disrupting the physical properties and interactions at the stalked pole has an influence on DivJ diffusion and signaling.

The H + H2 reaction and its isotopic variants have served for many years as a benchmark reaction system for studying bimolecular reaction dynamics. It is well-established that the minimum energy path for the H + H2 reaction is where the three atoms line up to form a collinear transition state. This path, commonly known as the direct recoil mechanism or Spiral mechanism, is very classical in nature and shows its signature in the angular distribution of the products which is a universal probe to the collision dynamics of a chemical reaction. The signature of the direct recoil mechanism is generally a single backscattered peak for low impact parameter or head-on collisions in the differential cross sections (DCSs) of the product states and a more sideways/forward scattered peak for high impact parameter or glancing collisions. While this behavior has been seen repeatedly in state-of-the-art experiments as well as high dimensional QM and classical theories, there have also been exceptions when the H + H2 system did not behave so classically and provided us with new insights to this reaction and to chemical reactions in general. This dissertation primarily focuses on two studies which bring forward new insights to this simplest chemical reaction: (i) scattering dynamics of H + D2 ⟶HD (v'=1, j') + D reaction at 1.97 eV, and (ii) probing the dynamics of H + D2 reaction beyond the energy of the conical intersection of H3 surface at a collision energy of 3.26 eV. Using the PHOTOLOC (photoinitiated reactions analyzed via law of cosines) technique developed by Zare and coworkers, 4 we have performed state-to-state measurements of the H + D2(v, j=0-2)⟶ HD(v', j') + D reaction at varying collision energies and investigated how energetics play a crucial role in the dynamics of this reaction. What has been observed for the HD product states is that the scattering dynamics do not show signatures of the conventional minimum energy path, vide supra. Instead of showing the conventional backward/sideways scattered single peak, the experimental DCSs showed multiple oscillatory structures. These structures are well reproduced by time-independent quantum mechanical (TIQM) calculations, but, somewhat surprisingly, not by the quasiclassical approach. Analysis of the classical results reveals that for these HD (v', j') states, several classical scattering mechanisms occur simultaneously. However, no clear evidence of these mechanisms is obvious in the quasiclassical state-to-state DCSs because their outcomes overlap in the range of scattering angles where they could be observable. It turns out that, analogous to the observations in a double-slit experiment, quantum interferences between the various mechanisms change and govern the angular distribution of the HD products. Owing to its quantum nature, this effect cannot be described in terms of the classical motion of the nuclei, and the QCT method fails. These mechanisms are characterized by different values of the total angular momentum J, which makes it possible to investigate theoretically the effect of 'closing' or 'opening' one of the slits in the two-slit experiment and determine the origin of the interferences. What is interesting is that while these different classical mechanisms and the interferences arising from them start to show for HD (v'=1, j') at energies (1.97 eV) way below the conical intersection (CI) of the H3 surface (2.74 eV), it is only at collision energies much higher than the CI that the higher vibrational manifolds of HD (v'=3, 4; j') show similar behavior. This can be explained with a total energy perspective and its distribution between the kinetic, internal and potential energies required to cross the barrier. At Ecoll = 1.97 eV, while the HD(v'=1, j') state has enough total energy available for the reaction to go through pathways with higher energy barriers, the relatively higher internal energies for the v' = 3 and 4 manifolds leaves less energy for the reaction and therefore the only feasible pathway for these states is the minimum energy path. At higher energies, for example, at 3.26 eV, there is enough energy available for even the higher vibrational states to undergo multiple mechanisms.

Transcription is considered the first step in gene expression. During the transcription initiation phase, a cell decides where and when transcription will occur on its template DNA using a complex network of regulation. Understanding the initiation process is therefore fundamental to any broader understanding of gene regulation. Despite being an active research area for past few decades, fundamental questions regarding mechanistic aspects of initiation remain unanswered. Here, we present findings related to the transcription initiation processes in prokaryotes and eukaryotes, based on single-molecule, real-time observations of RNA polymerase (RNAP) activity using optical tweezers. The only protein complex required to initiate transcription in bacteria is the RNAP holoenzyme itself, which is composed of RNAP core enzyme and a σ factor. The RNAP holoenzyme is able to make multiple contacts with promoter DNA sequences. These contacts modulate both the frequency of transcription, and duration of subsequent steps in the transcription initiation process. Here, we describe a novel single-molecule optical trapping assay that enables us to map the contacts of the Escherichia coli (E. coli) RNAP holoenzyme on the promoter DNA. Using this assay, we were able to observe the initiation process in real time. Through the study, my colleague and I were able to identify strong contacts between the RNAP holoenzyme and promoter DNA at various promoter sequence elements. By monitoring the initiation process from holoenzyme binding to promoter escape, we were also able to observe the remodeling of key contacts between the RNAP holoenzyme and promoter DNA. In contrast to bacterial transcription initiation, the eukaryotic transcription initiation is less studied. A major difference between prokaryotic and eukaryotic transcription initiation is that more proteins are essential to initiate transcription in eukaryotes. To form a minimal, transcription-competent pre-initiation complex (PIC), five general transcription factors (GTFs) are required, in addition to a 12-subunit RNAPII (pol II). Previous biochemical and structural studies have shown that the initiation of pol II transcription proceeds in the following stages: 1) assembly of pol II with GTFs and promoter DNA in a "closed" complex, within which the DNA still remains fully annealed; 2) unwinding about 15 bp of the promoter DNA to form an "open" complex; 3) scanning the downstream DNA sequence for a transcription start site; 4) synthesis of a short RNA transcript, believed to be about 10 nucleotides; 5) and promoter escape. In my thesis, I present findings based on real-time observations of initiation using a reconstituted 32-protein, 1.5 megadalton PIC derived from Saccharomyces cerevisiae. Measurements were made on detailed aspects of the motions of transcription factor IIH (TFIIH), as well as those of pol II, throughout the initiation process. In addition to characterizing the physical properties of TFIIH, including velocity and processivity, our findings establish TFIIH as the motor responsible for start-site scanning. Contrary to expectations, scanning driven by TFIIH entailed the rapid opening of a large transcription bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended transcription bubble, followed by promoter escape.

Single fluorescent molecules and particles can be localized in space with precision on the order of tens of nanometers (i.e. "super localized") using state-of-the-art microscopy techniques. The ability to probe complex environments at the sub-diffraction size scale has proven invaluable in revealing fundamental heterogeneity and improving overall understanding across the applied physical sciences. Super-localization microscopy is at the heart of both single-molecule super-resolution microscopy and single-particle tracking. The work presented in this dissertation concerns the application of super-localization microscopy to problems of biophysical interest, as well as theoretical and experimental advances in the methodology of this class of techniques. While the most common methods of super-localized position estimation ensure high localization precision, they might not always ensure high accuracy. In particular, the anisotropy of single-molecule dipole emission can result in mislocalizations of hundreds of nanometers, depending on the orientation of the molecule and its distance from the focal plane. In this dissertation I discuss different ways to correct this potential source of error. On the one hand, a theory based on a wobbling-in-a-cone model is presented that shows how this error is mitigated by molecular rotational mobility. On the other hand, for the worst-case scenario of a rotationally fixed emitter, an experimental approach based on Fourier optics is also discussed that allows for estimation of molecular orientation and enables active correction of mislocalization effects. The last third of this dissertation discusses applications of super-localization microscopy to three-dimensional tracking of fluorescently labeled genetic loci in budding yeast. In order to localize loci in the axial dimension, I used a Fourier optics approach to engineer the point spread function of the microscope into a Double-Helix Point Spread Function (DH-PSF). With this method, many single copies of a specific locus were analyzed, each with 3D spatial precision on the order of 10 nm at a rate of 10 Hz. A two-color implementation of the microscope allowed measurement of the correlations of 3D motion between pairs of loci under variable transcriptional pressure. I also discuss the importance of properly accounting for the inescapable effects of static and dynamic tracking errors caused by finite photon statistics and motion blur, respectively. These errors affect the statistics of the estimated motion and distort common metrics for characterizing stochastic motion such as the mean-squared displacement (MSD) and velocity autocorrelation (VAC). Analytical expressions for the MSD and VAC in the presence of these errors are given, along with applications to chromosomal locus tracking.

Neurons are highly polarized cells with their axon often extending over large distances away from the cell bodies (up to 1 meter in axonal length with a cell body less than 50 micrometer in diameter). Given that the majority of neuronal proteins and materials are synthesized in the cell body, such a long axon precludes effective diffusion of soma-produced proteins to their presynaptic destinations at the axonal terminals. As a result, neurons rely extensively on active axonal transport to deliver newly synthesized synaptic proteins, ion channels, lipids, and mitochondria to their axonal destinations via anterograde transport. On the other hand, retrograde axonal transport is responsible for carrying molecules and organelles destined for degradation from the axonal terminals back to the cell body. A highly efficient and tightly regulated machinery is thus required for a robust long-range transport of materials to ensure the neurons' proper growth, maintenance and survival. This thesis is a quantitative study of the underlying mechanism of axonal transport, with a specific focus on the retrograde axonal transport machinery in neurons. In our experimental setup, the axonal transport of cargos can be directly visualized in real-time using a neuronal microfluidic platform and fluorescence microscopy technique. We observe that cargos have high tendency to slow down their transport speed when crossing various obstacles along the axon such as non-moving cargos and stationary mitochondria. Single molecule study of retrograde nerve growth factor transport reveals that mechanical tug-of-war and intracellular motor regulation are complementary features of the near-unidirectional endosome directionality. Specifically, a stochastic mechanical simulation suggests that the endosomes are driven on average by 5-6 active dyneins and 1-2 down-regulated kinesins. This result is further supported by a study of the dynamics of endosomes detaching under load in axons, showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. Lastly, we present a quantitative characterization of the complex behavior of light-sensitive cryptochrome 2 (CRY2) protein under blue light. The results contribute to the understanding of the light-inducible CRY2 system and can be used as a guide to establish new optogenetic strategies to probe cellular processes in live cells.

The Enriched Xenon Observatory (EXO) is a series of experiments seeking to measure the neutrino mass through observation of neutrinoless double beta decay (0nbb). The next generation of 0nbb experiments aims to probe Majorana neutrino masses at or below 10 meV. To reach this sensitivity, ton-scale detectors are needed with lower radioactive backgrounds than the best ones operating today. The EXO collaboration is developing a novel strategy for a virtually background-free search for the 0nbb of Xe-136, based around detecting individual Ba-136 ions resulting from such decays. This dissertation details the efforts to develop a barium tagging technique which uses resonance ionization spectroscopy (RIS) to selectively and efficiently ionize barium atoms for injection and detection in an ion trap. A simple radionuclide-driven single-ion source has been developed to push the technology to high efficiency with a small number of ions.

Group II chaperonins are cellular protein-folding nanomachines with a stacked double-ring structure and two built-in lids. Each of the rings consists of eight or nine subunits, defining a cavity that can be closed with the lid. Chaperonins adopt distinct open and closed structures. The open structure allows substrates to bind, the closed structure encloses and folds the substrates, and ATP-regulated conformational changes between the open and closed structures are critical for the timing of the enzymatic cycle. These conformational changes are collective movements of multiple subunits and therefore highly depend on the cooperativity between the subunits. Although ensemble-averaged ATPase rates have indicated the existence of positive and negative cooperativities, the molecular mechanisms remain unknown. The present study provides more detailed understanding of the cooperativities in two group II chaperonins, TRiC and MmCpn, by measuring the stoichiometry of the various forms of nucleotides at the single-molecule level. To measure the nucleotide stoichiometry, each ATP was labeled with a Cy3 fluorescent dye and the Cy3-nucleotide-bound chaperonins were trapped in an Anti-Brownian ELectrokinetic trap (ABEL trap) for detailed study. The ABEL trap can localize a single fluorescently labeled protein for seconds in free solution, allowing single-molecule study of chaperonins without immobilization-induced artifacts. Trapped chaperonins containing multiple Cy3-nucleotides produce step-wise-decreasing intensity traces corresponding to the photobleaching of individual dyes, allowing extraction of nucleotide number distributions. The distributions show that the eukaryotic chaperonin TRiC/CCT, consisting of two copies of eight different subunits, hydrolyzes either zero or eight ATPs over a wide range of ATP concentrations. Although the ensemble-averaged results agree with standard cooperative ATP binding models, the single-molecule distributions do not: at low ATP concentrations, the preference for each chaperonin to hydrolyze eight ATPs is too strong to be explained by ATP binding alone and requires modeling the cooperativity in hydrolysis as well. In addition, the time-dependent ADP number distribution suggests simultaneous release of all eight ADPs governed by a one-step reaction that is the rate-limiting step of the ATPase cycle. Since TRiC is a hetero-oligomer, the fact that only half of the subunits hydrolyze ATP can be a consequence of the heterogeneous ATP binding affinities, the allosteric regulation or a combination of both. But strikingly, MmCpn, a homo-16-mer archaeal chaperonin, also hydrolyzes about eight ATPs at saturating ATP concentrations, suggesting that allosteric regulation heavily contributes to the determination of hydrolysis-active subunits. However, unlike TRiC, the number of ATPs hydrolyzed by each MmCpn quickly decreases when the ATP concentration decreases, consistent with a standard cooperative binding model. In addition, these distributions become less stable when the sample incubation time is shortened, suggesting sequential hydrolysis of the bound ATPs and temporary closure of the chaperonin containing a mixture of hydrolyzed and unhydrolyzed ATPs. This method of counting fluorescent dyes on ABEL-trapped proteins can be also extended to measure the nucleotide or substrate stoichiometry in other multi-subunit enzymes. With the native activities maximally preserved, the obtained single-molecule information will greatly improve our understanding of enzymatic mechanisms.

Starting from an exact diagrammatic theory for density correlations in dense, atomic fluids, we derive a set of graphical approximations to this theory that are consistent with a set of physical assumptions that define an overdamped limit of the dynamics of the system. The results of a simple one loop approximation to this theory are then compared with data from molecular dynamics simulations for a number of correlation functions of a simple Lennard-Jones fluid at a single, high density and a range of temperatures. For correlation functions that have most of their decay over times for which the overdamped theory is valid, the one loop approximation gives accurate results, except for coherent correlation functions at small wavevector, for which the overdamped theory is not expected to be accurate. Although the temperature range we studied included only temperatures at or above the liquid's triple point, it is our hope that the overdamped theory can ultimately be used to characterize the dynamics of supercooled liquids. This will certainly require going beyond the one loop approximation.

The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. There is a need for new fluorophores that can be used for single-molecule imaging in biological media, because imaging in cells and in organisms require emitters that are bright and photostable, red-shifted to avoid pumping cellular autofluorescence, and chemically and photophysically tunable. To this end, we have designed and characterized fluorescent probes based on a class of nonlinear-optical chromophores termed DCDHFs. This dissertation describes various physical and optical studies on these emitters, from sensing local environment to photoactivation. Chapter 1 is a general introduction to fluorescence and single-molecule spectroscopy and imaging. Single-molecule experiments in living cells are discussed and probes used for such experiments are summarized and compared. Chapter 2 explores the basic photophysics of the DCDHF fluorophores and some general methods of measuring relevant spectroscopic parameters, including photostability. Chapter 3 discusses the various approaches we have taken to modify particular properties by changing the fluorophore's structure. We have redesigned the DCDHF fluorophore into a photoactivatable fluorogen--a chromophore that is nonfluorescent until converted to a fluorescent form using light--described in Chapter 4. Finally, a different, chemical route to fluorescence activation is presented in Chapter 5. The remainder of the Dissertation is the Appendix and a full Bibliography. The Appendix includes a table of photophysical parameter for DCDHF fluorophore, various protocols used in the experiments discussed, MatLab codes, and NMR spectra.