Species‐ and C‐terminal linker‐dependent variations in the dynamic behavior of FtsZ on membranes in vitro

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146365/1/mmi14081.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146365/2/mmi14081-sup-0001-FigS1-S5.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146365/3/mmi14081_am.pd

Pivotal role of VASP in Arp2/3 complex–mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility

Place-Based Learning Communities on a Rural Campus: Turning Challenges into Assets

At Humboldt State University (HSU), location is everything. Students are as drawn to our spectacular natural setting as they are to the unique majors in the natural resource sciences that the university has to offer. However, the isolation that nurtures the pristine natural beauty of the area presents a difficult reality for students who are accustomed to more densely populated environments. With the large majority of our incoming students coming from distant cities, we set out to cultivate a “home away from home” by connecting first-year students majoring in science, technology, engineering and math (STEM) to the communities and local environment of Humboldt County. To achieve this, we designed first-year place-based learning communities (PBLCs) that integrate unique aspects and interdisciplinary themes of our location throughout multiple high impact practices, including a summer experience, blocked-enrolled courses, and a first-year experience course entitled Science 100: Becoming a STEM Professional in the 21st Century. Native American culture, traditional ways of knowing, and contemporary issues faced by tribal communities are central features of our place-based curriculum because HSU is located on the ancestral land of the Wiyot people and the university services nine federally recognized American Indian tribes. Our intention is that by providing a cross-cultural, validating environment, students will: feel and be better supported in their academic pursuits; cultivate values of personal, professional and social responsibility; and increase the likelihood that they will complete their HSU degree. As we complete the fourth year of implementation, we aim to harness our experience and reflection to improve our programming and enable promising early results to be sustained

Regulation of the transcription factor CdnL promotes adaptation to nutrient stress in <i>Caulobacter</i>

In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation inCaulobacter crescentus, SpoT synthesizes the secondary messengers guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate (collectively known as (p)ppGpp), which affect transcription by binding RNA polymerase (RNAP) to down-regulate anabolic genes. (p)ppGpp also impacts the expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. InCaulobacter, a major regulator of anabolic genes is the transcription factor CdnL. If and how CdnL is controlled during the SR and why that might be functionally important are unclear. In this study, we show that CdnL is down-regulated posttranslationally during starvation in a manner dependent on SpoT and the ClpXP protease. Artificial stabilization of CdnL during starvation causes misregulation of ribosomal and metabolic genes. Functionally, we demonstrate that the combined action of SR transcriptional regulators and CdnL clearance allows for rapid adaptation to nutrient repletion. Moreover, cells that are unable to clear CdnL during starvation are outcompeted by wild-type cells when subjected to nutrient fluctuations. We hypothesize that clearance of CdnL during the SR, in conjunction with direct binding of (p)ppGpp and DksA to RNAP, is critical for altering the transcriptome in order to permit cell survival during nutrient stress

Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium

Obligate intracellular bacteria of the order Rickettsiales include important human pathogens. However, our understanding of the biology of Rickettsia species is limited by challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed methods to assess cell wall composition, growth, and morphology of Rickettsia parkeri, a human pathogen in the spotted fever group of the Rickettsia genus. Analysis of the cell wall of R. parkeri revealed unique features that distinguish it from free-living alphaproteobacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphology in live host cells and found that the fraction of the population undergoing cell division decreased over the course of infection. We further demonstrated the feasibility of localizing fluorescence fusions, for example, to the cell division protein ZapA, in live R. parkeri for the first time. To evaluate population growth kinetics, we developed an imaging-based assay that improves on the throughput and resolution of other methods. Finally, we applied these tools to quantitatively demonstrate that the actin homologue MreB is required for R. parkeri growth and rod shape. Collectively, a toolkit was developed of high-throughput, quantitative tools to understand growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria.This article is distributed by The American Societyfor Cell Biology under license from the author(s). Two months after publication itis available to the public under an Attribution–Noncommercial-Share Alike 4.0 International Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/4.0)</p

FtsEX-mediated regulation of the final stages of cell division reveals morphogenetic plasticity in <i>Caulobacter crescentus</i>

<div><p>During its life cycle, <i>Caulobacter crescentus</i> undergoes a series of coordinated shape changes, including generation of a polar stalk and reshaping of the cell envelope to produce new daughter cells through the process of cytokinesis. The mechanisms by which these morphogenetic processes are coordinated in time and space remain largely unknown. Here we demonstrate that the conserved division complex FtsEX controls both the early and late stages of cytokinesis in <i>C</i>. <i>crescentus</i>, namely initiation of constriction and final cell separation. <i>ΔftsE</i> cells display a striking phenotype: cells are chained, with skinny connections between cell bodies resulting from defects in inner membrane fusion and cell separation. Surprisingly, the thin connections in <i>ΔftsE</i> cells share morphological and molecular features with <i>C</i>. <i>crescentus</i> stalks. Our data uncover unanticipated morphogenetic plasticity in <i>C</i>. <i>crescentus</i>, with loss of FtsE causing a stalk-like program to take over at failed division sites.</p></div

Stalk-localized proteins StpX and PbpC localize to the skinny constrictions in <i>ftsE</i> mutants.

<p>(A) Localization of StpX-CFP induced for 18 h in WT, <i>ΔftsE</i> or <i>ΔftsE</i> cells depleted of AmiC for 18 h. (B) Localization of StpX-CFP induced for 2.75 h in <i>ΔftsEΔpbpC</i>. (C) Localization of Venus-PbpC induced for 2 h in WT or <i>ΔftsE</i>. (D) Localization of cross-band protein StpB-mCherry induced for 18 h in WT, <i>ΔftsE</i> or <i>ΔftsE</i> cells depleted of AmiC for 18 h. # = stalk enrichment; * = skinny connection enrichment. Scale bars = 2 μm.</p

FtsEX-mediated regulation of constriction initiation and final cell separation reveals morphogenetic plasticity in <i>C</i>. <i>crescentus</i>.

<p>(A) Cell cycle localization of FtsZ and stalk proteins in WT and <i>ΔftsE</i> cells. (B) Three putative cell separation pathways in <i>C</i>. <i>crescentus</i>.</p

Synthetic genetic interactions are observed between <i>dipM</i> and <i>amiC</i>, <i>ftsE</i>, or <i>ldpF</i> and between <i>ftsE</i> and <i>spmX</i>.

<p>Phase contrast micrographs of (A) WT, <i>ΔftsE</i>, and <i>ΔdipM</i> cells; (B) WT, <i>ΔftsE</i>, or <i>ΔldpF</i> cells depleted of DipM for 19 h; (C) WT or <i>ΔdipM</i> cells depleted of AmiC for 19 h. (D) Spot dilutions of strains in (A-C). Depletion strains were grown for 19 h without inducer before spotting on PYE agar plates also without inducer. Cells in log phase were diluted to an OD₆₀₀ of 0.05, serially diluted and spotted onto the same PYE agar plate, and incubated at 30°C for 2 days. Images were cropped as shown and re-ordered for clarity of presentation. (E) Phase contrast micrographs of <i>ΔspmX</i> and <i>ΔftsEΔspmX</i> cells. (F) Spot dilutions of strains in (E). Cells in log phase were diluted to an OD₆₀₀ of 0.05, serially diluted and spotted onto the same PYE agar plate, and incubated at 30°C for 2 days. Images were cropped as shown and re-ordered for clarity of presentation. Scale bars = 2 μm.</p