scholarly journals A localized adaptor protein performs distinct functions at the Caulobacter cell poles

2021 ◽  
Vol 118 (13) ◽  
pp. e2024705118
Author(s):  
Jiarui Wang ◽  
W. E. Moerner ◽  
Lucy Shapiro
Keyword(s):  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Regulatory Protein ◽  
Adaptor Protein ◽  
Signaling Proteins ◽  
Swarmer Cell ◽  
Binding Partner ◽  
Cell Pole ◽  
Daughter Cells ◽  
Asymmetrically Distributed

Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Positioning different regulatory and signaling proteins at the opposing ends of the predivisional cell produces molecularly distinct daughter cells. Here, we report a strategy deployed by the asymmetrically dividing bacterium Caulobacter crescentus where a regulatory protein is programmed to perform distinct functions at the opposing cell poles. We find that the CtrA proteolysis adaptor protein PopA assumes distinct oligomeric states at the two cell poles through asymmetrically distributed c-di-GMP: dimeric at the stalked pole and monomeric at the swarmer pole. Different polar organizing proteins at each cell pole recruit PopA where it interacts with and mediates the function of two molecular machines: the ClpXP degradation machinery at the stalked pole and the flagellar basal body at the swarmer pole. We discovered a binding partner of PopA at the swarmer cell pole that together with PopA regulates the length of the flagella filament. Our work demonstrates how a second messenger provides spatiotemporal cues to change the physical behavior of an effector protein, thereby facilitating asymmetry.

scholarly journals A circuit of protein-protein regulatory interactions enables polarity establishment in a bacterium

2018 ◽  
Author(s):  
Wei Zhao ◽  
Samuel W. Duvall ◽  
Kimberly A. Kowallis ◽  
Dylan T. Tomares ◽  
Haley N. Petitjean ◽  
...  
Keyword(s):  
Cell Division ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Scaffold Protein ◽  
Cell Polarization ◽  
Negative Regulator ◽  
Scaffolding Proteins ◽  
Negative Bacterium ◽  
Regulatory Interactions ◽  
Daughter Cells

AbstractAsymmetric cell division generates specialized daughter cells that play a variety of roles including tissue morphogenesis in eukaryotes and pathogenesis in bacteria. In the gram-negative bacteriumCaulobacter crescentus, asymmetric localization of two biochemically distinct signaling hubs at opposite cell poles provides the foundation for asymmetric cell division. Through a set of genetic, synthetic biology and biochemical approaches we have characterized the regulatory interactions between three scaffolding proteins. These studies have revealed that the scaffold protein PodJ functions as a central mediator for organizing the new cell signaling hub, including promoting bipolarization of the central developmental scaffold protein PopZ. In addition, we identified that the old pole scaffold SpmX serves as a negative regulator of PodJ subcellular accumulation. These two scaffold-scaffold regulatory interactions serve as the core of an integrated cell polarization circuit that is layered on top of the cell-cycle circuitry to coordinate cell differentiation and asymmetric cell division.

scholarly journals Chromosome Methylation and Measurement of Faithful, Once and Only Once per Cell Cycle Chromosome Replication inCaulobacter crescentus

1999 ◽  
Vol 181 (7) ◽  
pp. 1984-1993 ◽  
Author(s):  
Gregory T. Marczynski
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Cell Division ◽  
Dna Synthesis ◽  
Replication Origin ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Chromosome Replication ◽  
Cell Stage ◽  
Swarmer Cell

ABSTRACT Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Ω-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Ω-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Ω-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Ω-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the “once and only once per cell cycle” rule of chromosome replication inC. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.

scholarly journals Scaffold-scaffold interactions regulate cell polarity in a bacterium

2020 ◽  
Author(s):  
Wei Zhao ◽  
Samuel W. Duvall ◽  
Kimberly A. Kowallis ◽  
Chao Zhang ◽  
Dylan T. Tomares ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Polarity ◽  
Chromosome Segregation ◽  
Time Course ◽  
Asymmetric Cell Division ◽  
Caulobacter Crescentus ◽  
Unknown Mechanism ◽  
Cell Pole ◽  
Signaling Complex ◽  
Client Proteins

AbstractThe localization of two biochemically distinct signaling hubs at opposite cell poles provides the foundation for asymmetric cell division in Caulobacter crescentus. Here we identify an interaction between the scaffolds PodJ and PopZ that regulates the assembly of the new cell pole signaling complex. Time-course imaging of a mCherry-sfGFP-PopZ fluorescent timer throughout the cell cycle revealed that existing PopZ resides at the old cell pole while newly translated PopZ accumulates at the new cell pole. Our studies suggest that interactions between PodJ and PopZ promotes the sequestration of older PopZ and robust accumulation of newl PopZ at the new cell pole. Elimination of the PodJ-PopZ interaction impacts PopZ client proteins, leading to chromosome segregation defects in one-third of cells. Additionally, this PopZ-PodJ interaction is crucial for anchoring PodJ and preventing PodJ extracellular loss at the old cell pole through unknown mechanism. Therefore, segregation of PopZ protein at the old pole and recruitment of newly translated PopZ at the new pole via the PodJ scaffold ensures stringent inheritance and maintenance of the polarity axis within dividing C. crescentus cells.

scholarly journals Environmental Conditions Modulate the Transcriptomic Response of Both Caulobacter crescentus Morphotypes to Cu Stress

2021 ◽  
Vol 9 (6) ◽  
pp. 1116
Author(s):  
Laurens Maertens ◽  
Pauline Cherry ◽  
Françoise Tilquin ◽  
Rob Van Houdt ◽  
Jean-Yves Matroule
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Stress Responses ◽  
Caulobacter Crescentus ◽  
Oxidative Stress Response ◽  
Transport Systems ◽  
Transcriptomic Response ◽  
Swarmer Cell ◽  
Cu Stress ◽  
Cellular Environment

Bacteria encounter elevated copper (Cu) concentrations in multiple environments, varying from mining wastes to antimicrobial applications of copper. As the role of the environment in the bacterial response to Cu ion exposure remains elusive, we used a tagRNA-seq approach to elucidate the disparate responses of two morphotypes of Caulobacter crescentus NA1000 to moderate Cu stress in a complex rich (PYE) medium and a defined poor (M2G) medium. The transcriptome was more responsive in M2G, where we observed an extensive oxidative stress response and reconfiguration of the proteome, as well as the induction of metal resistance clusters. In PYE, little evidence was found for an oxidative stress response, but several transport systems were differentially expressed, and an increased need for histidine was apparent. These results show that the Cu stress response is strongly dependent on the cellular environment. In addition, induction of the extracytoplasmic function sigma factor SigF and its regulon was shared by the Cu stress responses in both media, and its central role was confirmed by the phenotypic screening of a sigF::Tn5 mutant. In both media, stalked cells were more responsive to Cu stress than swarmer cells, and a stronger basal expression of several cell protection systems was noted, indicating that the swarmer cell is inherently more Cu resistant. Our approach also allowed for detecting several new transcription start sites, putatively indicating small regulatory RNAs, and additional levels of Cu-responsive regulation.

scholarly journals Phase Separation and Mechanical Forces in Regulating Asymmetric Cell Division of Neural Stem Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10267
Author(s):  
Yiqing Zhang ◽  
Heyang Wei ◽  
Wenyu Wen
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Phase Separation ◽  
Cell Division ◽  
Neural Stem Cells ◽  
Asymmetric Cell Division ◽  
Stem Cell Population ◽  
Mechanical Forces ◽  
Major Mechanism ◽  
Asymmetrically Distributed

Asymmetric cell division (ACD) of neural stem cells and progenitors not only renews the stem cell population but also ensures the normal development of the nervous system, producing various types of neurons with different shapes and functions in the brain. One major mechanism to achieve ACD is the asymmetric localization and uneven segregation of intracellular proteins and organelles into sibling cells. Recent studies have demonstrated that liquid-liquid phase separation (LLPS) provides a potential mechanism for the formation of membrane-less biomolecular condensates that are asymmetrically distributed on limited membrane regions. Moreover, mechanical forces have emerged as pivotal regulators of asymmetric neural stem cell division by generating sibling cell size asymmetry. In this review, we will summarize recent discoveries of ACD mechanisms driven by LLPS and mechanical forces.

scholarly journals Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesis

2017 ◽  
Author(s):  
Duvernoy Marie-Cécilia ◽  
Mora Thierry ◽  
Ardré Maxime ◽  
Croquette Vincent ◽  
Bensimon David ◽  
...  
Keyword(s):  
Bacterial Adhesion ◽  
Time Lapse ◽  
Adhesion Forces ◽  
Time Resolved ◽  
Cell Contacts ◽  
E Coli ◽  
Daughter Cells ◽  
Substrate Adhesion ◽  
Asymmetrically Distributed ◽  
Cell Cell

Bacterial biofilms are spatially structured communities, within which bacteria can differentiate depending on environmental conditions. During biofilm formation, bacteria attach to a surface and use cell-cell contacts to convey the signals required for the coordination of biofilm morphogenesis. How bacteria can maintain both substrate adhesions and cell-cell contacts during the expansion of a microcolony is still a critical yet poorly understood phenomenon. Here, we describe the development of time-resolved methods to measure substrate adhesion at the single cell level during the formation of E. coli and P. aeruginosa microcolonies. We show that bacterial adhesion is asymmetrically distributed along the cell body. Higher adhesion forces at old poles put the daughter cells under tension and force them to slide along each other. These rearrangements increase cell-cell contacts and the circularity of the colony. We propose a mechanical model based on the microscopic details of adhesive links, which recapitulates microcolony morphogenesis and quantitatively predicts bacterial adhesion from simple time lapse movies. These results explain how the distribution of adhesion forces at the subcellular level directs the shape of bacterial colonies, which ultimately dictates the circulation of secreted signals.

scholarly journals The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2

2018 ◽  
Author(s):  
Nina Kozlova ◽  
Daniela Mennerich ◽  
Anatoly Samoylenko ◽  
Elitsa Y. Dimova ◽  
Peppi Koivunen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Survival Function ◽  
Hypoxia Inducible Factor ◽  
Adaptor Protein ◽  
Prolyl Hydroxylase ◽  
Binding Partner ◽  
And Migration ◽  
Hif Prolyl Hydroxylase ◽  
Promote Breast Cancer

SummaryThe EGFR-adaptor protein CIN85 has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill-defined. Here, we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SH3 domains of CIN85 interact with the proline-arginine rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF-degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9 edited cells affected growth and migration properties as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2, and uncovered an essential survival function in tumor cells linking growth factor adaptors with hypoxia signaling.

scholarly journals Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251684
Author(s):  
Patricia R. Nano ◽  
Taylor K. Johnson ◽  
Takamasa Kudo ◽  
Nancie A. Mooney ◽  
Jun Ni ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Signaling Proteins ◽  
Gtpase Activating Protein ◽  
Mutagenesis Screen ◽  
Binding Partner ◽  
Spinal Cord Development ◽  
Terminal Domain ◽  
Structure Activity ◽  
Truncation Mutant ◽  
Gli Transcription Factors

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.

scholarly journals Developmentally regulated GTPases: structure, function and roles in disease

2021 ◽  
Author(s):  
Christian A. E. Westrip ◽  
Qinqin Zhuang ◽  
Charlotte Hall ◽  
Charlotte D. Eaton ◽  
Mathew L. Coleman
Keyword(s):  
Cell Proliferation ◽  
Structural Biology ◽  
Regulatory Protein ◽  
Growth Control ◽  
Developmentally Regulated ◽  
Binding Partner ◽  
Cellular Processes ◽  
Gtp Binding ◽  
Evolutionarily Conserved ◽  
Cell Growth Control

AbstractGTPases are a large superfamily of evolutionarily conserved proteins involved in a variety of fundamental cellular processes. The developmentally regulated GTP-binding protein (DRG) subfamily of GTPases consists of two highly conserved paralogs, DRG1 and DRG2, both of which have been implicated in the regulation of cell proliferation, translation and microtubules. Furthermore, DRG1 and 2 proteins both have a conserved binding partner, DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively, that prevents them from being degraded. Similar to DRGs, the DFRP proteins have also been studied in the context of cell growth control and translation. Despite these proteins having been implicated in several fundamental cellular processes they remain relatively poorly characterized, however. In this review, we provide an overview of the structural biology and biochemistry of DRG GTPases and discuss current understanding of DRGs and DFRPs in normal physiology, as well as their emerging roles in diseases such as cancer.

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