Using structural biology and biochemical findings we proposed a mechanistic basis for TCS pathway coupling in which the DivL pseudokinase is repurposed as a sensor rather than participant in phosphotransduction. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Moerner, W. E., Biteen, J., Conley, N. R., Lee, H., Lord, S. J., Thompson, M. A., Shapiro, L., Liu, N., Samuel, R., Twieg, R. J. Dutka P, Metskas LA, Hurt RA, Salahshoor H, Wang TY, Malounda D, Lu GJ, Chou TS, Shapiro MG*, Jensen GJ*. Research in our laboratory is focused on understanding how regulatory information encoded by the genome is integrated with the transcriptional machinery and chromatin context to allow for emergence of form and function during human embryogenesis and evolution, and how perturbations in this process lead to disease. Dahlberg, P. D., Sartor, A. M., Wang, J., Saurabh, S., Shapiro, L., Moerner, W. E. Integration of cell cycle signals by multi-PAS domain kinases. View details for Web of Science ID A1994MQ78200018, View details for Web of Science ID A1994NV05900013, View details for Web of Science ID A1993MH32400028. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle. Differential protein localization can control DNA replication, chromosome segregation, and cytokinesis and is responsible for generating daughter cells with different fates upon cell division. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. The dynamic range of a bacterial species' natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A series of simple, in situ immunoassays have been developed which can be used in screening for translation products of genes cloned in vitro recombination experiments with either phage or plasmid vectors. B.S. The two-component signaling protein CtrA activates or represses the expression of one-quarter of the cell-cycle-regulated genes in Caulobacter crescentus, integrating DNA replication, morphogenesis, and cell division. View details for DOI 10.1073/pnas.2001849117, View details for Web of Science ID 000513023200098, View details for Web of Science ID 000513023201258, View details for Web of Science ID 000513023201267. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. M.D. Electrical Engineering, MIT Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. View details for DOI 10.1038/sj.emboj.7600927, View details for Web of Science ID 000234952500008, View details for PubMedCentralID PMC1383511. Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Eng. Dahlberg, P. D., Saurabh, S., Sartor, A. M., Wang, J., Mitchell, P. G., Chiu, W., Shapiro, L., Moerner, W. E. Cryogenic Superresolution Fluorescence Correlated with Cryogenic Electron Tomography: Combining Specific Labeling and High Resolution. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. WebMikhail G. Shapiro, PhD Professor of Chemical Engineering & Medical Engineering Investigator, Howard Hughes Medical Institute Physics for Medicine Lab and Sorbonne We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. Natera advances molecular diagnostics with integrity and scientific rigor, and supports integration of information provided by our tests into health care decision making. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. Recent discoveries have revealed that progression through the cell cycle and communication between cellular compartments are mediated by two-component signal transduction systems and signaling pathways involving transcription factor activation by proteolytic processing. Each of these transcripts proved to be a de novo transcript since (a) each could be pulse labeled during the initial 20 s of the reaction and (b) each transcript contained a triphosphate at its 5' terminus. x@caltech.edu, x=mikhail, Scientists, Postdoc Scholars & Graduate Students The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. WebGraduate student, Sarah Cohen laboratory, Romberg Tiburon Center for Environmental Studies, 3152 Paradise Drive, Tiburon, CA 94920, (415) 338-3754 daniellendesmet gmail.com In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrM-mediated methylation has important cellular functions. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression. article|readcube, Making engineered cells dance to ultrasound, Researchers Make it Possible for Ultrasound to Reveal Gene Expression in the Body, Vilcek Foundation Prize Awarded to Mikhail Shapiro, CCE Postdoc Receives NIH Pathway to Independence Award, Mikhail Shapiro Wins Roger Tsien Award for Excellence in Chemical Biology, Program Brings Area High School Students, Teachers into Caltech Labs, Switching Brain Circuits On and Off Without Surgery, Mikhail Shapiro Selected as Camille Dreyfus Teacher-Scholar, Taking MRI Technology down to Micrometer Scales, Scientists Design Bacteria to Reflect Sonar Signals for Ultrasound Imaging, Biologists Give Bacteria Thermostat Controls, Designing Ultrasound Tools with Lego-Like Proteins, Newly Named Pew Scholar to Image Gut Bacteria with Sound Waves, Partnership with Heritage Medical Research Institute Will Augment Translational Medicine Research, Abedi Receives Fellowship for New Americans, Caltech Researchers Receive NIH BRAIN Funding, New Method Could Improve Ultrasound Imaging, x@caltech.edu; x=mikhail View details for DOI 10.1016/S0022-2836(02)01042-2. Get the latest news about the lab, our science and discoveries. View details for Web of Science ID 000083885400003. We have identified a single amino acid substitution in the Caulobacter structural maintenance of chromosomes (SMC) protein that disrupts chromosome segregation and cell division. The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. We also study the normal roles of such signals in stem-cell physiology and their abnormal roles in the formation and expansion of cancer stem cells. 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. alarid@oncology.wisc.edu We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. Evidence suggests that the protein product of some fla and che genes is targeted to the incipient swarmer cell pole. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. View details for Web of Science ID 000185536700040. WebLucy Shapiro is a Professor in the Department of Developmental Biology at Stanford University School of Medicine where she holds the Virginia and D. K. Ludwig Chair in Cancer Research and is the Director of the Beckman Center for Molecular and Genetic Medicine. The DNA sequence of 40% of the 16S rRNA gene, the entire 16S/23S intergenic spacer region, and portions of the 23S rRNA gene were determined. Perhaps the sequential morphogenic changes exhibited by Caulobacter are dependent on the initial synthesis of this organelle. On the basis of these interactions, a trans-acting hierarchy of flagellar and chemotaxis gene expression is proposed. Lasker, K., Abraham, A., Childers, W., Shapiro, L. Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Agricultural Biotechnology, Seoul National University x@caltech.edu, x=spolidor, Andrea Torres Awards: NIH Pioneer, Mark, IEEE Hertz, Vilcek, Saville, Tsien, Dreyfus, Van Ness, Packard, Sontag, Pew, DARPA YFA, BWF-CASI, Miller, Hertz, Soros, LSRF, TR35 We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. We report here that flagellar rotation requires the FliL protein. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci. Transcription of flagellar genes in Caulobacter crecentus is programmed to occur during the predivisional stage of the cell cycle. Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Genetic analysis of these mutants resulted in the identification of at least eight che genes located at six different positions on the Caulobacter crescentus chromosome. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. x@caltech.edu, x=rzhang5, Undergraduate Students These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Temporal and spatial regulation have emerged as the central themes, with the abundance, activity and subcellular location of key structural and regulatory proteins changing over the course of the cell cycle. The gene is located 2 kb from the origin of replication. Ph.D. Yoo S, Mittelstein DR, Hurt RC, Lacroix JJ, Shapiro MG*. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope. Ph.D. Aerospace Engineering, Georgia Tech How this is brought about remains one of the most fundamental questions of developmental biology. Perez, D., Dahlberg, P. D., Wang, J., Sartor, A. M., Borden, J. S., Shapiro, L., Moerner, W. E. ATP-responsive biomolecular condensates tune bacterial kinase signaling. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics. The ffs gene is transcribed throughout the cell-cycle and is transcribed at similar rates in mutant (ffs36) and wild-type strains, but in the mutant the 4.5 S RNA is unstable leading to lower 4.5 S RNA levels. It will bring together the resources and expertise of the national lab, the university and Silicon Valley to accelerate the deployment of batteries and other energy storage