The Experts below are selected from a list of 174084 Experts worldwide ranked by ideXlab platform
Philippe Pierre - One of the best experts on this subject based on the ideXlab platform.
-
novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique
The FASEB Journal, 2011Co-Authors: Craig A Goodman, Philippe Pierre, Danielle M Mabrey, Man Hing Miu, John Frey, Enrico K Schmidt, Troy A HornbergerAbstract:In this study, the principles of Surface Sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). In addition, IV-SUnSET detected food deprivation-induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV-SUnSET (IV-IHC-SUnSET). By combining IV-IHC-SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.
-
novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique
The FASEB Journal, 2011Co-Authors: Craig A Goodma, Enrico K Schmid, Philippe Pierre, Danielle M Mabrey, Joh Frey, Man Hing Miu, Troy A HornbergeAbstract:In this study, the principles of Surface Sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [3H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). In addition, IV-SUnSET detected food deprivation-induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV-SUnSET (IV-IHC-SUnSET). By combining IV-IHC-SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also suff...
-
sunset a nonradioactive method to monitor protein synthesis
Nature Methods, 2009Co-Authors: Enrico K Schmid, Giovanna Clavarino, Maurizio Ceppi, Philippe PierreAbstract:We developed a nonradioactive fluorescence-activated cell sorting-based assay, called Surface Sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.
Beat Christen - One of the best experts on this subject based on the ideXlab platform.
-
the type iv pilin pila couples Surface attachment and cell cycle initiation in caulobacter crescentus
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Luca Del Medico, Dario Cerletti, Matthias Christen, Philipp Schachle, Beat ChristenAbstract:Understanding how bacteria colonize Surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how Surface Sensing drives cell-cycle initiation in Caulobacter crescentus. We identify the type IV pilin protein PilA as the primary signaling input that couples Surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for “cell-cycle initiating pilin” peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits Surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit Surface Sensing, prevent biofilm formation and control persistent infections.
-
the type iv pilin pila couples Surface attachment and cell cycle initiation in caulobacter crescentus
bioRxiv, 2019Co-Authors: Luca Del Medico, Dario Cerletti, Matthias Christen, Beat ChristenAbstract:Understanding how bacteria colonize Surfaces and regulate cell cycle progression in response to cellular adhesion is of fundamental importance. Here, we used transposon sequencing in conjunction with FRET microscopy to uncover the molecular mechanism how Surface Sensing drives cell cycle initiation in Caulobacter crescentus. We identified the type IV pilin protein PilA as the primary signaling input that couples Surface contact to cell cycle initiation via the second messenger c-di-GMP. Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17 amino-acid transmembrane helix of PilA to activate the PleC-PleD two component signaling system, increase cellular c-di-GMP levels and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for cell cycle initiating pilin peptide. Addition of the chemically synthesized CIP peptide initiates cell cycle progression and simultaneously inhibits Surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer new strategies to inhibit Surface-Sensing, prevent biofilm formation and control persistent infections. Significance Statement Pili are hair-like appendages found on the Surface of many bacteria to promote adhesion. Here, we provide systems-level findings on a molecular signal transduction pathway that interlinks Surface Sensing with cell cycle initiation. We propose that Surface attachment induces depolymerization of pili filaments. The concomitant increase in pilin sub-units within the inner membrane function as a stimulus to activate the second messenger c-di-GMP and trigger cell cycle initiation. Further-more, we show that the provision of a 17 amino acid synthetic peptide corresponding to the membrane portion of the pilin sub-unit mimics Surface Sensing, activates cell cycle initiation and inhibits Surface attachment. Thus, synthetic peptide mimetics of pilin may represent new chemotypes to control biofilm formation and treat bacterial infections.
Courtney K Ellison - One of the best experts on this subject based on the ideXlab platform.
-
acinetobacter baylyi regulates type iv pilus synthesis by employing two extension motors and a motor protein inhibitor
Nature Communications, 2021Co-Authors: Courtney K Ellison, Triana N Dalia, Catherine A Klancher, Joshua W Shaevitz, Zemer Gitai, Ankur B DaliaAbstract:Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, Surface Sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis. Type IV pili (T4P) are retractile appendages used by bacteria for DNA uptake and other purposes. T4P extension is thought to occur through the action of a single motor protein, PilB. Here, Ellison et al. show that T4P synthesis in Acinetobacter baylyi depends not only on PilB but also on an additional, distinct motor, TfpB.
-
acinetobacter baylyi regulates type iv pilus synthesis by employing two extension motors and a motor protein inhibitor
bioRxiv, 2021Co-Authors: Courtney K Ellison, Triana N Dalia, Catherine A Klancher, Joshua W Shaevitz, Zemer Gitai, Ankur B DaliaAbstract:Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, Surface Sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, yet little is known about the mechanisms controlling these dynamics or the extent to which their regulation is conserved across bacterial species. Here, we develop Acinetobacter baylyi as a new model to study T4P by employing a recently developed pilus labeling method. Our findings overturn the current dogma that T4P extension occurs through the action of a single, highly conserved motor, PilB, by showing that T4P synthesis in A. baylyi is dependent on an additional, phylogenetically distinct motor, TfpB. Furthermore, we uncover an inhibitor of T4P extension that specifically binds to and inhibits PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to inhibit T4P synthesis.
-
Surface Sensing stimulates cellular differentiation in caulobacter crescentus
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Rhett A Snyder, Courtney K Ellison, Geoffrey B Severin, Gregory B Whitfield, Christopher M WatersAbstract:Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium Caulobacter crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a Surface, regulate this cell cycle remain unclear. Here, we find that Surface Sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that Surface contact stimulates cell-cycle progression by demonstrating that Surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that Surface contact and Sensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a Surface-associated lifestyle.
-
flagellar mutants have reduced pilus synthesis in caulobacter crescentus
Journal of Bacteriology, 2019Co-Authors: Courtney K Ellison, Yves V Brun, Douglas B RuschAbstract:Surface appendages, such as flagella and type IV pili, mediate a broad range of bacterial behaviors, including motility, attachment, and Surface Sensing. While many species harbor both flagella and type IV pili, little is known about how or if their syntheses are coupled. Here, we show that deletions of genes encoding different flagellum machinery components result in a reduction of pilus synthesis in Caulobacter crescentus First, we show that different flagellar mutants exhibit different levels of sensitivity to a pilus-dependent phage and that fewer cells within populations of flagellar mutants make pili. Furthermore, we find that single cells within flagellar mutant populations produce fewer pili per cell. We demonstrate that these gene deletions result in reduced transcription of pilus-associated genes and have a slight but significant effect on general transcription profiles. Finally, we show that the decrease in pilus production is due to a reduction in the pool of pilin subunits that are polymerized into pilus fibers. These data demonstrate that mutations in flagellar gene components not only affect motility but also can have considerable and unexpected consequences for other aspects of cell biology.IMPORTANCE Most bacterial species synthesize Surface-exposed appendages that are important for environmental interactions and survival under diverse conditions. It is often assumed that these appendages act independently of each other and that mutations in either system can be used to assess functionality in specific processes. However, we show that mutations in flagellar genes can impact the production of type IV pili, as well as alter general RNA transcriptional profiles compared to a wild-type strain. These data demonstrate that seemingly simple mutations can broadly affect cell-regulatory networks.
-
feedback regulation of caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor hfia
Molecular Microbiology, 2018Co-Authors: Cécile Berne, Courtney K Ellison, Geoffrey B Severin, Christopher M Waters, Aretha Fiebig, Radhika Agarwal, Robert I Morton, Yves V BrunAbstract:To permanently attach to Surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by Surface Sensing, in which swarmer cells rapidly synthesize holdfast in direct response to Surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, Surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to Surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus Surface Sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.
Yves V U - One of the best experts on this subject based on the ideXlab platform.
-
a bifunctional atpase drives tad pilus extension and retraction
Science Advances, 2019Co-Authors: Courtney K Elliso, Nicolas Iais, Anku Dalia, Yves V U, Jennife L Chlebek, Katherine R Hummels, Gaёl Panis, Patrick H ViollieAbstract:A widespread class of prokaryotic motors powered by secretion motor adenosine triphosphatases (ATPases) drives the dynamic extension and retraction of extracellular fibers, such as type IV pili (T4P). Among these, the tight adherence (tad) pili are critical for Surface Sensing and biofilm formation. As for most other motors belonging to this class, how tad pili retract despite lacking a dedicated retraction motor ATPase has remained a mystery. Here, we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through adenosine 5′-triphosphate (ATP) hydrolysis. We show that mutations within CpaF result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction.
-
a bifunctional atpase drives tad pilus extension and retraction
bioRxiv, 2019Co-Authors: Courtney K Elliso, Nicolas Iais, Anku Dalia, Yves V U, Jennife L Chlebek, Katherine R Hummels, Gaёl Panis, Patrick H ViollieAbstract:Abstract Molecular motors convert chemical energy directly into mechanical work1 and are found in all domains of life2. These motors are critical to intracellular transport3, motility4,5, macromolecular protein assembly3,6, and many essential processes7. A wide-spread class of related bacterial motors drive the dynamic activity of extracellular fibers, such as type IV pili (T4P), that are extended and retracted using so-called secretion motor ATPases. Among these, the tight adherence (tad) pili are critical for Surface Sensing, Surface attachment, and biofilm formation8–10. How tad pili undergo dynamic cycles of extension and retraction8 despite lacking a dedicated retraction motor ATPase has remained a mystery. Here we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through ATP hydrolysis. Specifically, we show that mutations within the ATP hydrolysis active site of Caulobacter crescentus CpaF result in a correlated reduction in the rates of extension and retraction. Moreover, a decrease in the rate of ATP hydrolysis directly scales with a decrease in the force of retraction and reduced dynamics in these CpaF mutants. This mechanism of motor protein bifunctionality extends to another genus of tad-bearing bacteria. In contrast, the T4aP subclass of pili possess dedicated extension and retraction motor ATPase paralogs. We show that these processes are uncoupled using a slow ATP hydrolysis mutation in the extension ATPase of competence T4aP of Vibrio cholerae that decreases the rate of extension but has no effect on the rate of retraction. Thus, a single motor ATPase is able to drive the bidirectional processes of pilus fiber extension and retraction.
-
obstruction of pilus retraction stimulates bacterial Surface Sensing
bioRxiv, 2017Co-Authors: Courtney K Elliso, Rebecca S Dillard, David T Kysela, Cheri M Hampto, Elizabeth R Wrigh, Nicolas Iais, Anku Dalia, Yves V UAbstract:Surface association provides numerous fitness advantages to bacteria. Thus, it is critical for bacteria to recognize Surface contact and to consequently initiate physiological changes required for a Surface-associated lifestyle (1). Ubiquitous microbial appendages called pili are involved in Sensing Surfaces and mediating downstream Surface-associated behaviors (2-6). The mechanism by which pili mediate Surface Sensing remains unknown, largely due to the difficulty to visualize their dynamic nature and to directly modulate their activity without genetic modification. Here, we show that Caulobacter crescentus pili undergo dynamic cycles of extension and retraction that cease within seconds of Surface contact, and this arrest of pilus activity coincides with Surface-stimulated holdfast synthesis. By physically blocking pili, we show that imposing resistance to pilus retraction is sufficient to stimulate holdfast synthesis in the absence of Surface contact. Thus, resistance to type IV pilus retraction upon Surface attachment is used for Surface Sensing.
Yves V Brun - One of the best experts on this subject based on the ideXlab platform.
-
flagellar mutants have reduced pilus synthesis in caulobacter crescentus
Journal of Bacteriology, 2019Co-Authors: Courtney K Ellison, Yves V Brun, Douglas B RuschAbstract:Surface appendages, such as flagella and type IV pili, mediate a broad range of bacterial behaviors, including motility, attachment, and Surface Sensing. While many species harbor both flagella and type IV pili, little is known about how or if their syntheses are coupled. Here, we show that deletions of genes encoding different flagellum machinery components result in a reduction of pilus synthesis in Caulobacter crescentus First, we show that different flagellar mutants exhibit different levels of sensitivity to a pilus-dependent phage and that fewer cells within populations of flagellar mutants make pili. Furthermore, we find that single cells within flagellar mutant populations produce fewer pili per cell. We demonstrate that these gene deletions result in reduced transcription of pilus-associated genes and have a slight but significant effect on general transcription profiles. Finally, we show that the decrease in pilus production is due to a reduction in the pool of pilin subunits that are polymerized into pilus fibers. These data demonstrate that mutations in flagellar gene components not only affect motility but also can have considerable and unexpected consequences for other aspects of cell biology.IMPORTANCE Most bacterial species synthesize Surface-exposed appendages that are important for environmental interactions and survival under diverse conditions. It is often assumed that these appendages act independently of each other and that mutations in either system can be used to assess functionality in specific processes. However, we show that mutations in flagellar genes can impact the production of type IV pili, as well as alter general RNA transcriptional profiles compared to a wild-type strain. These data demonstrate that seemingly simple mutations can broadly affect cell-regulatory networks.
-
feedback regulation of caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor hfia
Molecular Microbiology, 2018Co-Authors: Cécile Berne, Courtney K Ellison, Geoffrey B Severin, Christopher M Waters, Aretha Fiebig, Radhika Agarwal, Robert I Morton, Yves V BrunAbstract:To permanently attach to Surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by Surface Sensing, in which swarmer cells rapidly synthesize holdfast in direct response to Surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, Surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to Surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus Surface Sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.
