The Stanford SHAPE Lab, directed by Prof. Sean Follmer, explores how we can interact with digital information in a more physical and tangible way. Here we identify an essential two-component signal transduction protein that controls multiple events in the Caulobacter cell cycle, including cell division, stalk synthesis, and cell cycle-specific transcription. We also seek opportunities for applying these rules to improve engineering systems. The P- and L-rings are structural components of the flagellar basal body that are positioned in the periplasmic space and outer membrane, respectively. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. 25(7):488-490. Bayas, C. A., Schrader, J. M., Lee, M. K., Shapiro, L., Moerner, W. E. CauloBrowser: A systems biology resource for Caulobacter crescentus. We are interested in studying microbial fungal pathogens, and we are developing and employing CRISPR-based genomic technologies to allow us to better understand the biology and pathogenesis of these organisms. Bacterial chromosomes are generally approximately 1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. These genes are organized in several classes which form a transcriptional regulatory hierarchy. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. Dr. Shapiro's laboratory question in developmental biology involves the mechanisms used to generate the three-dimensional organization of a cell from a one-dimensional genetic code. Nature Biotechnology (2023). View details for Web of Science ID 000304978400010, View details for PubMedCentralID PMC3370875. The Cantor Arts Center is a laboratory of learning and a center of scholarly inquiry. Dr. Weissmans laboratory is working on identifying and characterizing the progression of discrete changes, genetic and epigenetic, that leads to the generation of cancer stem cells (CSCs) from a variety of blood and solid tissue cancers. Interestingly, M. xanthus, which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells. Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Jun 2022 24. Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences. An impressively diverse array of mechanisms underlies bacterial polarity, including oscillatory systems, phospho-signaling pathways, the sensing of membrane curvature, and the integration of cell cycle regulators with polar maturation. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. We propose that polar recruitment of CtrA evolved to ensure that CtrA is degraded only in the stalked half of the predivisional cell, perhaps by localizing a proteolytic adaptor protein to the stalked pole. Electron micrographs reveal the phage to be among the largest DNA bacteriophages reported, with head dimensions of 64 by 195 nm and a flexible tail 275 nm in length. (Credit: Paul Mueller) The scientists developed the platform at the Advanced Light Source, which produces light in the X-ray region of the electromagnetic spectrum. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Analyses of cell polarity, division, and differentiation in prokaryotes have identified several regulatory proteins that exhibit dramatic changes in expression and spatial localization over the course of a cell cycle. Current areas of research in developmental biology. Graduate Student (joined @ 06/2017) Bioengineering. We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. (1-3) In the alpha-proteobacterium, Caulobacter crescentus, the CtrA global transcriptional regulator exhibits a spatially and temporally dynamic expression pattern across the cell cycle. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence. We also determined that mmpA and yaeL can complement each other in C. crescentus and E. coli, indicating functional conservation. View details for Web of Science ID A1982PG49500029, View details for Web of Science ID A1981MJ92600005. Many important biological processes occur deep inside living organisms. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. Follow @StanfordBioX, Stanford University, Stanford, California 94305, James H. Clark Center, Stanford University, Department of Developmental Biology Homepage, Stanford Interdisciplinary Life Sciences Council. View details for DOI 10.1016/j.cell.2006.05.038, View details for Web of Science ID 000239224800023. Analysis of mutations in the IHF-binding region of the hook operon demonstrated that an intact IHF-binding site is necessary for transcription in vivo. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. Jacobs, C., Domian, I. J., Maddock, J. R., Shapiro, L. The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle, Protein localization during the Caulobacter crescentus cell cycle. The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. This degradation is surprising, since SsrA RNA is both highly structured and highly abundant. See all the current Searle Scholars here. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. The spatial distribution of the membrane chemoreceptors and associated cytoplasmic chemotaxis proteins in Escherichia coli were examined as a prototypic functional aggregate in bacterial cells. Martin J. Smith. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell. Drug Discovery, Small Molecule Synthesis, University of Illinois 2009-2014. The initiation of DNA replication is under differential control in Caulobacter crescentus. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. Berdis, A. J., Lee, I., Coward, J. K., Stephens, C., Wright, R., Shapiro, L., Benkovic, S. J. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. Hillson, N. J., Hu, P., Andersen, G. L., Shapiro, L. A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. Evidence is presented that suggests that the B and C transcripts initiate at or near the major A promoter but terminate at different termination or pause sites within the early region of the phage genome. SHAPE Lab. The first labeled Dra I fragment to appear contains the site of replication initiation. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. By. A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. Lab Objectives. These developmental decisions require global changes in genomic readout, and bacteria typically employ intricate (yet poorly understood) signaling networks that enable changes in cell function. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression. Proteins are positioned at particular sites in bacteria, including the cell pole, the incipient division plane, and the septum. The cheR, cheB and cheT genes appeared to be located in a three-gene cluster. Now Shapiro, who is also the Virginia and D. K. Ludwig Professor at the medical school, can add a Canada Gairdner International Award to her list of accolades. 1967 Brooklyn College Ph.D. 1972 Purdue University Postdoc. An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. 1996. Comparison to other organisms reveals conservation of cell cycle regulatory logic, even if regulatory proteins, themselves, are not conserved. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. Bryan, R., Purucker, M., Gomes, S. L., Alexander, W., Shapiro, L. GENETIC-ANALYSIS AND CHARACTERIZATION OF A CAULOBACTER-CRESCENTUS MUTANT DEFECTIVE IN MEMBRANE BIOGENESIS. Gahlmann, A., Ptacin, J. L., von Diezmann, A. S., Shapiro, L., Moerner, W. E. Quantitative Registration and Distribution Analysis of Multicolor 3D Super-Resolution Images of Proteins Reveals Nanoscale Spatial Organization. A., Britos, L., Shapiro, L. A spindle-like apparatus guides bacterial chromosome segregation. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. Stanford Artificial Intelligence Laboratory. Bayas, C., Wang, J., Lee, M. K., Schrader, J. M., Shapiro, L., Moerner, W. E. Herrmann, J., Jabbarpour, F., Bargar, P. G., Nomellini, J. F., Li, P., Lane, T. J., Weiss, T. M., Smit, J., Shapiro, L., Wakatsuki, S. Ultra-photostable, genetically directed fluoromodule enables STED nanoscopy and long time scale single protein tracks in live bacteria. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. Alley, M. R., Gomes, S. L., Alexander, W., Shapiro, L. TEMPORAL AND SPATIAL REGULATION OF DEVELOPMENTALLY EXPRESSED GENES IN CAULOBACTER, EXPRESSION OF POSITIONAL INFORMATION DURING CELL-DIFFERENTIATION IN CAULOBACTER. They have found a single molecular event present in all cancers studied to date that protects them from macrophages of the innate immune system. We are hiring for a range of staff and faculty positions. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. An overexpression screen for sRNA-induced growth inhibition along with sequence conservation in a related Caulobacter species led to the identification of a novel sRNA, CrfA, that is specifically induced upon carbon starvation. The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. A restriction map of the Caulobacter crescentus bacteriophage phi Cd1 genome was constructed by using the restriction endonucleases HindIII and HpaI. David J Shapiro's profile. cell, and (2) the CcrM protein is rapidly degraded prior to cell division. The subset of developmentally regulated flagellar genes that exhibit mRNA segregation has the same upstream cis-acting elements: an activator-binding site known as the ftr sequence and an IHF-binding site. How this is brought about remains one of the most fundamental questions of developmental biology. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying beta-galactosidase protein fusions to portions of the operon. CHAMPER, R., Bryan, R., Gomes, S. L., Purucker, M., Shapiro, L. ANALYSIS OF THE PLEIOTROPIC REGULATION OF FLAGELLAR AND CHEMOTAXIS GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS BY USING PLASMID COMPLEMENTATION. Our goal is to define these mechanisms using both molecular genetics and biochemistry. View details for Web of Science ID A1996VB22500010. We engineered a strain of the bacterium Caulobacter crescentus to fluoresce in the presence of micromolar levels of uranium at ambient temperatures when it is exposed to a hand-held UV lamp. A specific binding activity for the region between -81 and -122 base-pairs was shown to be temporally controlled, appearing prior to the activation of hook operon transcription. Hodgson, D., Shaw, P., Letts, V., Henry, S., Shapiro, L. ISOLATION AND GENETIC-ANALYSIS OF CAULOBACTER MUTANTS DEFECTIVE IN CELL-SHAPE AND MEMBRANE LIPID-SYNTHESIS, GENERATION OF A TN5 PROMOTER PROBE AND ITS USE IN THE STUDY OF GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS. Of Illinois 2009-2014 replication is under differential shapiro lab stanford in Caulobacter crescentus has been determined cheR... An intact IHF-binding site is necessary for transcription in vivo and a loss of methylesterase activity in vitro control! 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