1985 — 1986 |
Shapiro, Lucille |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Genetic Control of Cell Growth and Development
The goals of this Program are to define the organization and expression of identified genetic loci which function as essential components of cell growth and development. Two criteria are critical to a productive study of growth and developmental regulation. The organism of choice must be amenable to both genetic and biochemical manipulation, and in each case specific genetic loci must be identified which play a key role in the process being studied. (a) The cell membrane has an essential role in the coordination of cellular events in such diverse organisms as Caulobacter, yeast, E. coli and Dictyostelium. L. Shapiro and S. Henry will determine the mechanism by which membrane lipid and protein synthesis is involved in the temporal and spacial regulation of a set of identified structural proteins during the Caulobacter cell cycle. (b) S. Henry will analyze the structure and expression of the gene encoding the essential lipid-biosynthetic enzyme inositol-l-phosphate synthase during the yeast cell cycle, in order to understand the coordinate control of cytoplasmic and membrane-bound enzymes. (c) Chromosomal genetic loci in E. coli have been shown to encode proteins which participate in a variety of membrane-associated functions. P. Silverman will determine how the cell envelope functions in what appears to be an organizational capacity to regulate donor activity and the ilv biosynthetic pathway. (d) Motility mutants in Dictyostelium express a surprising array of membrane-mediated functions which are related to the cytoplasmic actin-myosin complex. Dr. Clarke will determine how such events as motility, axenic growth, pinocytosis, cell shape and surface substrate-cell interactions are co-regulated by the products of single genetic loci. (e) Drs. J. Chase and S. Hawley are studying DNA ligase, which is an essential enzyme in replication, repair and recombination, from an organism, Drosophila, which exhibits a full complement of developmental functions yet permits access to genetic manipulation. Their objectives are to determine how the gene encoding histone proteins form a multigene family whose expression varies as a function of cell differentiation. The goals of Drs. Emmons and Childs are to determine the consequences of this differential gene expression and to understand the organization and controlled expression of this multigene family in C. elegans.
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0.957 |
1985 — 2014 |
Shapiro, Lucille |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Regulation of Differentiation in Caulobacter
DESCRIPTION (provided by applicant): The goal of this proposal is to define the regulatory mechanisms that control the bacterial cell cycle and to understand how these mechanisms function within an integrated system. We have shown that Caulobacter exerts exquisite spatial and temporal control of its cell cycle by the use of transcriptional and proteolytic networks integrated with dynamic subcellular protein localization. Key to cell cycle control, a small number of master transcriptional regulators orchestrate cell cycle progression. A two component phospho-signaling pathway, involving the polarly-localized CckA histidine kinase, mediates the activation of the CtrA master regulator whose function is to regulate the genes involved in polar morphogenesis and the biogenesis of the cell division apparatus. Using robotic high throughput screens for genes involved in protein localization, we identified the DivL kinase for the localization of the CckA histidine kinase, and the CpaE pili protein for the localization of the PleC histidine kinase that is essential for polar morphogenesis. We will explore the mechanism of polar localization of these critical kinases and determine how it is related to their function within the cell cycle regulatory circuit. To understand the cell cycle integration of transcriptional regulation, we will define the mechanism of action of the newly identified SciP transcriptional regulator that functions as a repressor of CtrA activated genes at a specific time in the cell cycle, and the novel CrfA non-coding RNA that modifies the cell cycle regulatory circuitry in response to nutrient deprivation. Finally, the mid-cell establishment of the FtsZ cytokinetic ring is dependent on signals from the cell poles and is an integral component of the core cell cycle circuitry. Accordingly, we will explore the temporally regulated localization, assembly, and disassembly of the divisome, as a function of the cell cycle. PUBLIC HEALTH RELEVANCE: We examine the molecular mechanisms of each of the consecutive steps in the bacterial cell cycle and then elucidate how these individual events are integrated into a functional system. This approach has allowed the identification of novel mechanisms that coordinate the temporal and spatial control of cell cycle progression, leading to the identification of new antibiotic targets and ultimately the design and development of a new class of antibiotics.
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1 |
1985 |
Shapiro, Lucille |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training Program in Biomedical Reserach |
0.957 |
1986 — 1987 |
Shapiro, Lucille |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training Program in Microbiology For Infectious Diseases @ Columbia Univ New York Morningside |
0.907 |
1988 |
Shapiro, Lucille |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Infectious Diseases--Microbiology @ Columbia Univ New York Morningside |
0.907 |
1994 — 1997 |
Shapiro, Lucille |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development Control of Dna Replication in Caulobacter
Our goal is to understand the mechanisms that control the timing of the initiation of DNA replication as a function of the cell cycle. We propose to study this crucial event in Caulobacter crescentus, an organism whose developmental cell cycle exhibits inherent asymmetry. The newly replicated chromosomes differ in replication potential: the chromosome that partitions to the progeny stalked cell immediately initiates DNA replication whereas the progeny swarmer cell chromosome does not initiate replication until later in the cell cycle. Caulobacter is uniquely suited to a study of the factors that control DNA replication because it has a single chromosome that initiates replication from an identified origin once per cell cycle, in a cell type that can be easily obtained from synchronized populations. We have recently isolated mini-chromosomes driven solely by the cloned origin that correctly initiate replication coincident with the bona fide chromosomal origin. The proposed research has four main objectives. The first is to define the replication origin sequences and cognate factors that mediate the differential initiation of DNA replication. To do this we will identify, both in vivo and in vitro, factors that bind to regions of the origin that are essential for replication initiation or for the timing of replication. We will also determine if a promoter, shown to reside within the origin and to be selectively expressed from replication-competent chromosome, contributes to the control of replication initiation. We will also attempt to directly visualize origin-specific transcripts at one pole of the predivisional cell using in situ hybridization. The second objective is to isolate and characterize mutants defective in DNA replication and/or chromosome segregation. The third is to determine the mechanisms that control the cell cycle expression of identified enzymes and factors that are involved in DNA replication, such as DnaA, the beta subunit of DNA polymerase III, and gyrase. The fourth objective is to determine the mechanisms that regulate the cell cycle expression of a newly identified DNA methyltransferase, and examine the role of this methylation system in the control of replication initiation and cell differentiation. These studies will address the underlying mechanisms that control polarity during the bacterial cell cycle.
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1 |
1998 — 2013 |
Shapiro, Lucille |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Developmental Control of Dna Replication in Caulobacter
[unreadable] DESCRIPTION (provided by applicant): Our goal is to identify the mechanisms that integrate temporal and spatial signals to coordinate the initiation of DNA replication, chromosomal origin movement to the cell poles, and the mid-cell assembly of the cell division ring during a bacterial cell cycle. A small number of critical master transcriptional regulators, DnaA, GcrA, and CtrA, control cell cycle progression in Caulobacter. These three proteins are part of a regulatory circuit that together control the expression of genes required for chromosome replication and cytokinesis. We have discovered that DnaA controls both replication initiation and the transcription of cell cycle-regulated genes. DnaA turns on the transcription of GcrA, which, in turn, turns on the transcription of CtrA. GcrA and CtrA oscillate out of phase during the cell cycle to control approximately 150 temporally-regulated genes for polar morphogenesis, DNA methylation, and cell division. DnaA is a critical lynchpin in the regulatory cascade and we will now analyze the temporal control of DnaA availability and activity. We will characterize the genes controlled, in turn, by GcrA and identify a new factor that appears to negatively control the expression of a set of 5 replication enzymes. Caulobacter coordinates the transcription of ctrA with the progression of DNA replication using the differential methylation state of the replicating chromosome. We will now explore the role of DNA methylation in the control of transcription of multiple replication genes all of which have methylation sites in their promoters. Finally, an important question is how chromosome replication and segregation is coordinated with cell division. We have a discovered a unique mechanism that links the MreB actin-dependent movement of the newly replicated origin to the opposite cell pole and the mid-cell positioning of the FtsZ division ring. A ParA-family ATPase, MipZ, in complex with ParB, binds to the origin region and moves with the replicated origin as it transits the length of the cell. Because MipZ is an inhibitor of FtsZ polymerization, the Z-ring can form only at mid-cell once the origins and the accompanying MipZ complex are safely secured at the two cell poles. We will now define the mechanisms that coordinate these events. Defining the regulatory circuitry that drives the bacterial cell cycle has revealed methyltranferases as circuit nodes and as such, targets for antibiotic discovery. Based on this work, we have succeeded in designing a new small molecule antibiotic that is currently in clinical trials. [unreadable] [unreadable] [unreadable]
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1 |
2005 — 2008 |
Shapiro, Lucille |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
3d Chromosome/Replisome Positioning in Bacterial Cells
[unreadable] DESCRIPTION (provided by applicant): This is a program of collaborative research between engineers and physicists at Stanford University together with specialists in advanced microscopy to determine the three dimensional (3D) configuration of the chromosome and the replisome in the bacterial cell as the cell cycle progresses. Specific aims are: 1. To precisely define the dynamic 3D organization of the chromosome within the non-replicating bacterial cell (the Caulobacter swarmer cell) and during the cell cycle while the chromosome is being replicated. To do this, we will perform high-resolution 3D imaging by EM tomography and soft X-ray tomography to locate and map chromosomal loci in the cell. We will also perform time-lapse fluorescent microscopy tracking of the same loci in living cells. 2. To identify the proteins that mediate the spatial deployment of both replicating and non-replicating chromosomes by (a) determining the effect of mutations in proteins known to be involved in chromosome organization, (b) carrying out an automated high throughput screen for mutants that mislocalize discrete chromosomal loci, (c) determining the effect of cell structure by examining the chromosome organization in long filamentous cells, (d) modeling and analyzing the chromosome movement taking account of the hydrodynamic properties of the cytoplasm, and (e) determining if the actin-like MreB protein directly or indirectly binds to DNA at different times in the cell cycle using chromosome immunoprecipitation assays. 3. To define the spatial deployment of the replisome (replication factory) in the Caulobacter celt during its assembly at the cell pole and its movement during DNA replication. To do this, we will (a) use high resolution (20-100 nm) imaging by EM tomography and soft X-ray tomography, (b) use total internal reflection (TIR) microscopy to determine if the moving replisome follows an axial or a spiral path on its way to the cell division plane, and (c) use tomographic imaging to determine if the replisome co-positions with and follows the path of the MreB spiral that is deployed along the long axis of the cell. [unreadable] [unreadable]
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1 |
2008 — 2011 |
Moerner, William E [⬀] Rao, Jianghong (co-PI) [⬀] Rao, Jianghong (co-PI) [⬀] Shapiro, Lucille |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Actively Controlled and Targeted Single-Molecule Probes For Cellular Imaging
DESCRIPTION (provided by applicant): Actively Controlled and Targeted Single-Molecule Probes for Cellular Imaging Recent advances in microscopic imaging techniques with single fluorescent molecules have led to superresolution information, that is, the locations and shapes of objects in cells have been determined with resolution beyond the standard diffraction limit. These methods may be collectively termed Single-Molecule Active Control Microscopy (SMACM), because single emitting molecules are used as nanometer-scale light sources, and these emitters must be actively turned on and off to be sure that only a few molecules are emitting at any given time. Photoactivatable fluorescent protein fusions have been used for SMACM, but these emitters are large and may perturb the biological system. Though some emitters such as quantum dots provide high photostability, many additional properties are simultaneously required for advanced single-molecule imaging in cells, such as ease of functionalization, control of photophysics and photochemistry, and ease of targeting to specific cellular structures. Organic synthesis can make a huge array of "small" molecules with multiple tailored functionalities, and the present application makes use of this high degree of flexibility to develop new, targeted single-molecule emitters with active control capabilities This research will attack the problem of 3-D superresolution imaging with three interconnected thrusts which combine the skills of four investigators expert in organic synthesis, single-molecule imaging, chemistry for cellular targeting, and regulatory protein localization in bacterial cells. First, organic synthesis will generate new fluorophores with "turn-on" capability, where chemical reactivity is used to generate emission only when two protofluorophores are allowed to react, or where secondary photochemical illumination creates a fluorescent molecule in situ. Secondary illumination will also be used to photoswitch molecules on and off for additional control. The utility of the turn-on concept is that fluorescence can more easily be generated only where needed;hence backgrounds are lower. The second thrust involves selective targeting of the fluorescent labels to proteins and RNA in the cell. This will be accomplished by N-terminal cysteine labeling and RNA aptamer generation, respectively. Finally, to validate and challenge the fluorophore development, the new emitters will be used at the single-molecule level to image specific subwavelength structures, both in eukaryotic and in tiny bacterial cells. The results of this research will be to greatly extend the availability of high-resolution probes for cellular imaging at the single-molecule level, thus enabling a much deeper understanding of cellular functions. By providing a large new array of controllable and targeted single-molecule emitters, the ability of the researcher to noninvasively look inside cells will be extended into the nanoscale regime of the single-molecule emitters themselves. Public Health Relevance: The understanding of biological systems is intimately connected with unraveling disease mechanisms, and to understand the operation of the cell, optical imaging has long been an essential method by virtue of its generally noninvasive character, its capacity to assess from a distance, and its ability to observe time- dependent dynamical processes. In the cell, many small molecular machines operate one at a time, therefore scientists are now routinely observing individual single molecules, one by one, to examine the behavior of each without averaging over many inequivalent copies. To observe single molecules in cells at the spatial scale of a few tens of nm, new actively controllable and targetable emitting labels are required, and this proposed research combines the skills of four investigators to design, synthesize, and optimize a large and novel class of molecules for labeling individual proteins and RNA in living cells.
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1 |
2008 |
Shapiro, Lucille |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
High Pressure Freezer
[unreadable] DESCRIPTION (provided by applicant): This proposal requests funds to purchase a Leica EM PACT2-RTS high pressure freezer for rapid, cryo- fixation of biological samples. The high pressure freezer will be a shared resource, located in a well- established, multi-user, light and electron microscopy facility at Stanford University: the Cell Sciences Imaging Facility. This facility is accessible to the entire Stanford campus as well as surrounding institutions such as UC Santa Cruz and UC San Francisco. [unreadable] [unreadable] High pressure freezing has become the gold standard for fixation of biological electron microscopy samples. In the numerous studies were it has now been applied, this method has extended our understanding of the structural and molecular organization of cells and tissues. High pressure freezing provides much improved fixation, both in terms of quality and quantity, over all other fixation techniques and, when used in conjunction with a rapid transfer system, provides increased temporal resolution for correlative light to electron microscopy studies. For many studies and experimental systems high pressure cryo-fixation is not just desirable but necessary and essential for proper preservation of cellular fine structure and antigenicity. [unreadable] [unreadable] The high pressure freezer will support NIH funded projects from eight major users. These projects investigate a wide range of NIH supported topics, including, 1) mechano-electrical transduction and microtubule cytoskeleton organization in nematode touch receptor neurons (Goodman); 2) microtubule cytoskeleton organization and regulation (Stearns); 3) molecular basis of T and B lymphocyte recognition and immuno-synapse formation (Davis); 4,5) molecular mechanisms of viral replication, assembly and egress from infected cells (Arvin, Kirkegaard); 6) development and function of the neural circuitry in Zebra fish, rodents and humans (Smith); 7) development of cell polarity in yeast (Pringle); 8) 3D organization of the bacterial chromosome and replisome (Shapiro, McAdams). [unreadable] [unreadable] These studies investigate critical structural questions in a variety of model organisms and human tissues and cover areas of research with implications for diverse aspects of human health and disease, ranging from cancer and viral pathogenesis to understanding the molecular basis of immuno-responses and sensory biology. All require levels of sample preservation that approach the in vivo state for ultrastructural study of subcellular structures and molecular complexes. Each and every one of these projects demands a level of fixation that approaches the native, hydrated state which is possible only by application of high pressure freezing. [unreadable] [unreadable]
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1 |
2012 — 2015 |
Moerner, William E [⬀] Shapiro, Lucille |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Subcellular Architecture of Regulatory Protein Complexes At the Bacterial Pole
DESCRIPTION (provided by applicant): 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.
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1 |
2016 — 2020 |
Shapiro, Lucille |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Integration of Regulatory Networks and Subcellular Architecture to Control the Caulobacter Cell Cycle
? DESCRIPTION (provided by applicant): The fundamental basis for the generation of cellular diversity in all organisms is the asymmetric deployment of structural and regulatory proteins to the cell poles prior to cell division and the consequent differential readout of the genome in the two daughter cells. The bacterium Caulobacter crescentus provides an elegant system in which to decipher the complete molecular circuitry that controls the asymmetry that underlies cell differentiation. As Caulobacter moves through its cell cycle, cell differentiation is accompanied by the polar localization of distinct complements of phospho-signaling proteins. The goals of our research program turn on three important questions: 1- How does a polar matrix nanodomain function to dynamically re-wire polar phosphosignaling pathways to drive cell differentiation and asymmetric cell division? We are approaching this question through reconstitution of the polar environment using liposomes and microfabricated solid substrates, three dimensional superresolution imaging modalities and single molecule tracking in living cells, and the creation of optogenetic mutants that enable instantaneous light-induced reconfiguration of the cell pole composition, thereby allowing us to directly observe the consequences of re-wiring a spatially-restricted signaling cascade in a living cell. 2- How do cell-type specific signaling pathways beget cell type-specific gene expression? We are defining the exquisite complexity of the complete genetic circuitry that uses both transcriptional and translation control to drive cell cyce progression culminating in daughter cells of different cell fate. 3- How does chromosome organization, replication and segregation along the long axis of the cell serve as a timer of cell cycle-regulated transcription using epigenetic mechanisms? Our goal is to integrate these spatiotemporal regulatory paradigms to establish the logic that is applicable to the dissection of asymmetry in all living systems.
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