Kress, H.

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Kress, HS Holger

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Spatiotemporal control of cellular microenvironments with photonics

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 Spatiotemporal control of cellular microenvironments withphotonicsCitation for published version (APA):Kress, H. (2010). Spatiotemporal control of cellular microenvironments with photonics. In Spring Meeting 2010 ofthe Society for General Microbiology (SGM), 29 March -1 April 2010, Edingburgh, UK (pp. 16-17)Document status and date:Published: 01/01/2010Document Version:Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers)Please check the document version of this publication:• A submitted manuscript is the version of the article upon submission and before peer-review. There can beimportant differences between the submitted version and the official published version of record. Peopleinterested in the research are advised to contact the author for the final version of the publication, or visit theDOI to the publisher's website.• The final author version and the galley proof are versions of the publication after peer review.• The final published version features the final layout of the paper including the volume, issue and pagenumbers.Link to publicationGeneral rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            • Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying the publication in the public portal.If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, pleasefollow below link for the End User Agreement:www.tue.nl/taverneTake down policyIf you believe that this document breaches copyright please contact us at:openaccess@tue.nlproviding details and we will investigate your claim.Download date: 16. Nov. 2023SGM Spring 2010 MeetingEdinburgh International  Conference Centre29 March–1 April 2010Systems, Mechanisms and Micro-organismsABSTRACTSDELIVERING MODERN MICROBIAL SCIENCE16 sgmconferencessoc ie t y  fo r    gene ra l  m ic rob io logyEDINBURGH 29 MARCH–1 APRIL 2010WWW.SGMEICC2010.ORG.UKS E S S I O N S ED02 CONT.Stochastic computer simulations of the molecular interaction networks can be used to understand molecular events leading to the emergence of population heterogeneity and generate the hypotheses leading experimental work. I will present the stochastic kinetic model of the two component system signalling (TCS) showing different modes of phenotypic switching in this signal transduction network. The TCSs are involved in the regulation of adaptive responses to environmental cues and host-pathogen interaction. Therefore, the stochastic effects in the TCSs may potentially lead to the emergence of many phenotypic switching phenomena observed in the bacterial populations. I will also present computer simulations demonstrating propagation of the gene expression and signalling noise to the level of metabolic networks and discuss the consequences of these stochastic events for the understanding of the epigenetic changes in global physiological state of the bacterial cell.Differential	control	of	bacteria	gene	expressionEduardo A. GroismanWashington University School of Medicine, USAAbstract not receivedIndividuality	of	bacterial	responses	to	antimicrobialsJohn D. McKinney, Ph.D.Laboratory of Bacteriology, Global Health Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, SwitzerlandBacteria behave as individuals. Mutation and genetic exchange are important drivers of bacterial individuation, but these events are rare. At higher frequencies, genetically identical cells display metastable variation in growth rate, response kinetics, stress resistance, and other quantifiable phenotypes. These between-cell differences arise from non-genetic sources, such as stochastic gene expression and asymmetric partitioning of components at cell division. Phenotypic variation at the single-cell level generates phenotypic diversity at the population level. This diversity is critical for bacterial persistence in fluctuating environments. It ensures that some individuals will survive a potentially lethal stress that would otherwise extinguish the population. For example, the refractoriness of bacterial infections to antibiotic therapy has been ascribed to spontaneous variants (‘persisters’) that survive and regrow despite prolonged antibiotic exposure. The persisters are not antibiotic resistant mutants and it is unclear why they tolerate antibiotics that kill their genetically identical siblings. Our studies focus on the mechanistic basis of the reversible persister switch in the clinically important micro-organism Mycobacterium tuberculosis. We use automated time-lapse microscopy, microfluidics, and microelectromechanical systems to analyse bacterial responses to antibiotics at the single-cell level. Our studies show that the conventional interpretation of the persister phenomenon – viz., that persistence is due to pre-existing subpopulations of dormant cells – is incorrect. Instead, we find no correlation between the growth rates of individual cells and their probability of survival during antibiotic exposure. We also find that the survival probability of closely related sister cells is strongly and positively correlated. These observations suggest that the (unknown) factors that determine whether a cell lives or dies are metastable within individual cell lineages.O2	be	a	pathogen:	signalling	of	host	microenvironments	to	promote	Shigella	virulenceChristoph M. TangImperial College London, SW7 2AZSee p. 173EUKARYOTIC CELL BIOLOGYSpatiotemporal	control	of	cellular	microenvironments	with	photonicsHolger KressDept of Mechanical Engineering, Yale University, USALiving cells respond to biochemical and mechanical stimuli. A quantitative understanding of the interplay between signaling and cell mechanics requires experimental methods to stimulate cells flexibly with biochemical and mechanical cues. We use optical tweezers in combination with functionalized microparticles to control the mechanical and biochemical microenvironment of individual cells. Our research focuses on investigating phagocytosis and chemotaxis in single immune cells. I will discuss the mechanics of macrophages and bacterial pathogens during the early stages of phagocytic uptake. Our studies revealed that macrophage filopodia act as tentacles which pull bound objects with discrete steps and a load-dependent velocity towards the cell for further phagocytosis. In addition, I will present a novel method for flexible biochemical cell stimulation. This method is based on optically manipulated microsources, which 17 sgmconferencessoc ie t y  fo r    gene ra l  m ic rob io logyEDINBURGH 29 MARCH–1 APRIL 2010WWW.SGMEICC2010.ORG.UKS E S S I O N S ED02 CONT.steadily release molecules. The technique enables control over biochemical cellular microenvironments down to length scales of one micrometer and timescales from an hour down to seconds. We demonstrated this technique by guiding and perturbing the migration of single human neutrophils.Towards	systems	analysis	of	small	GTPase	activities	in	early	HIV	infectionGary Warnes1 & MATTHIAS T. DITTMAR21Flow Cytometry Core Facility, 2Centre for Immunology & Infectious Disease, Blizard Institute of Cell & Molecular Science,London E1 2ATViruses are obligatory cellular parasites, with their replication crucially depending on host cell functions. They have evolved strategies to modulate host cell cytoskeleton dynamics and activate signal transduction pathways to facilitate entry into new target cells and release of progeny virus from infected cells. Therefore viruses are ideal tools to study signalling and cytoskeleton networks in an integrative way by comparing the dynamics of such virus exploited networks with the dynamics induced by natural ligand interaction. Changes in cytoskeletal structure are tightly regulated by the small GTPases like RhoA, Rac1 and Cdc42.Using dominant-negative and constitutive active forms of those GTPases we identified RhoA and Rac1 as factors contributing to changes in microtubule stability and reduced susceptibility to virus infection, thus identifying microtubules as potential cellular target for virus-cell fusion (Malinowsky et al., 2008). In order to visualize and quantify changes in signalling we have recently established a FRET based assay to detect activation of RhoA, Rac1 and Cdc42 using Raichu-FRET probes described by M Matsuda’s group in 2003 (Yoshizaki et al., 2003).The Gβ	protein	Gpb1	and	the	co-repressor	TupA	bind	to	Protein	Kinase	A	Tpk2	to	act	as	antagonistic	molecular	switchesof	fungal	morphological	changesThamarai K. Janganan1, Gongyou Chen1,3, Daliang Chen1, Marcia E. da Silva Ferreira2, Everaldo R. Marques2,Gustavo H. Goldman2, Adrian R. Walmsley1 & M. INÊS BORGES-WALMSLEY11School of Biological & Biomedical Sciences, Durham University, South Road, Durham DH1 3LE; 2Depto de Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, CEP 14040-903, Ribeirao Preto, Sao Paulo, Brazil;.3Dept of Agriculture & Biology, Shanghai Jiaotong University, 800 Dongchuan Road, 200240, Shanghai, ChinaParacoccidioides brasiliensis is one of a group of six phylogenetically related ascomycete fungi that are adapted for survival in mammalian hosts and which cause millions of infections every year. In P. brasiliensis the transition from the mycelium to the pathogenic yeast form of is controlled by the cAMP-signalling pathway and during the transition there is a change in the expression of the Gβ-protein Gpb1 that interacts with adenylate cyclase. We have exploited the fact that the cAMP-signaling-pathway of S. cerevisiae does not include a Gβ-protein, so as to use this organism to probe the functional role of Gpb1. We present data that indicates that Gpb1 and the transcriptional regulator TupA both bind to the PKA protein Tpk2; through which they act as antagonistic molecular switches of cell morphology, with TupA and Gpb1 inducing and repressing filamentous growth, respectively. In S. cerevisiae, PbTupA-induced filamentation occurs at two levels, with the early production of numerous pseudohyphae, a hyperfilamentious state, followed by the formation of an invasive projection and aerial stalk. In contrast, overexpressing ScTup1 represses the filamentous growth of S. cerevisiae, suggesting that an organisms Tup protein determines the morphological outcome. Conversely, Gpb1, which binds to PbTpk2, but not ScTpk2, could over-ride the effect of PbTupA to repress filamentous growth in S. cerevisiae. Our findings define a mechanism for controlling the morphological switch that underpins the virulence of dimorphic fungi.The	responses	of	fungal	pathogens	to	environmental	insultsAlistair J.P. BrownAberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZDThe success of Candida albicans as a human pathogen is enhanced by a battery of fungal virulence factors (such as cellular morphogenesis, adhesins and secreted hydrolytic enzymes) and fitness attributes (such as stress responses and metabolic adaptation). The stress responses help C. albicans combat host immune defences, which attack the pathogen with a battery of environmental insults that include reactive oxygen and nitrogen species. These stress responses appear to be regulated spatially and temporally during disease establishment and progression. We (and others) have begun to dissect the molecular mechanisms by which C. albicans responds to these oxidative and nitrosative stresses. Our approach is to combine mathematical modelling with molecular biology and genomics with a view to generating models that help us to predict the behaviour of C. albicans within the relatively complex and varied microenvironments of the host.

 Spatiotemporal control of cellular microenvironments withphotonicsCitation for published version (APA):Kress, H. (2010). Spatiotemporal control of cellular microenvironments with photonics. In Spring Meeting 2010 ofthe Society for General Microbiology (SGM), 29 March -1 April 2010, Edingburgh, UK (pp. 16-17)Document status and date:Published: 01/01/2010Document Version:Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers)Please check the document version of this publication:• A submitted manuscript is the version of the article upon submission and before peer-review. There can beimportant differences between the submitted version and the official published version of record. Peopleinterested in the research are advised to contact the author for the final version of the publication, or visit theDOI to the publisher's website.• The final author version and the galley proof are versions of the publication after peer review.• The final published version features the final layout of the paper including the volume, issue and pagenumbers.Link to publicationGeneral rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            • Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            • You may not further distribute the material or use it for any profit-making activity or commercial gain            • You may freely distribute the URL identifying the publication in the public portal.If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, pleasefollow below link for the End User Agreement:www.tue.nl/taverneTake down policyIf you believe that this document breaches copyright please contact us at:openaccess@tue.nlproviding details and we will investigate your claim.Download date: 09. Jul. 2024SGM Spring 2010 MeetingEdinburgh International  Conference Centre29 March–1 April 2010Systems, Mechanisms and Micro-organismsABSTRACTSDELIVERING MODERN MICROBIAL SCIENCE16 sgmconferencessoc ie t y  fo r    gene ra l  m ic rob io logyEDINBURGH 29 MARCH–1 APRIL 2010WWW.SGMEICC2010.ORG.UKS E S S I O N S ED02 CONT.Stochastic computer simulations of the molecular interaction networks can be used to understand molecular events leading to the emergence of population heterogeneity and generate the hypotheses leading experimental work. I will present the stochastic kinetic model of the two component system signalling (TCS) showing different modes of phenotypic switching in this signal transduction network. The TCSs are involved in the regulation of adaptive responses to environmental cues and host-pathogen interaction. Therefore, the stochastic effects in the TCSs may potentially lead to the emergence of many phenotypic switching phenomena observed in the bacterial populations. I will also present computer simulations demonstrating propagation of the gene expression and signalling noise to the level of metabolic networks and discuss the consequences of these stochastic events for the understanding of the epigenetic changes in global physiological state of the bacterial cell.Differential	control	of	bacteria	gene	expressionEduardo A. GroismanWashington University School of Medicine, USAAbstract not receivedIndividuality	of	bacterial	responses	to	antimicrobialsJohn D. McKinney, Ph.D.Laboratory of Bacteriology, Global Health Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, SwitzerlandBacteria behave as individuals. Mutation and genetic exchange are important drivers of bacterial individuation, but these events are rare. At higher frequencies, genetically identical cells display metastable variation in growth rate, response kinetics, stress resistance, and other quantifiable phenotypes. These between-cell differences arise from non-genetic sources, such as stochastic gene expression and asymmetric partitioning of components at cell division. Phenotypic variation at the single-cell level generates phenotypic diversity at the population level. This diversity is critical for bacterial persistence in fluctuating environments. It ensures that some individuals will survive a potentially lethal stress that would otherwise extinguish the population. For example, the refractoriness of bacterial infections to antibiotic therapy has been ascribed to spontaneous variants (‘persisters’) that survive and regrow despite prolonged antibiotic exposure. The persisters are not antibiotic resistant mutants and it is unclear why they tolerate antibiotics that kill their genetically identical siblings. Our studies focus on the mechanistic basis of the reversible persister switch in the clinically important micro-organism Mycobacterium tuberculosis. We use automated time-lapse microscopy, microfluidics, and microelectromechanical systems to analyse bacterial responses to antibiotics at the single-cell level. Our studies show that the conventional interpretation of the persister phenomenon – viz., that persistence is due to pre-existing subpopulations of dormant cells – is incorrect. Instead, we find no correlation between the growth rates of individual cells and their probability of survival during antibiotic exposure. We also find that the survival probability of closely related sister cells is strongly and positively correlated. These observations suggest that the (unknown) factors that determine whether a cell lives or dies are metastable within individual cell lineages.O2	be	a	pathogen:	signalling	of	host	microenvironments	to	promote	Shigella	virulenceChristoph M. TangImperial College London, SW7 2AZSee p. 173EUKARYOTIC CELL BIOLOGYSpatiotemporal	control	of	cellular	microenvironments	with	photonicsHolger KressDept of Mechanical Engineering, Yale University, USALiving cells respond to biochemical and mechanical stimuli. A quantitative understanding of the interplay between signaling and cell mechanics requires experimental methods to stimulate cells flexibly with biochemical and mechanical cues. We use optical tweezers in combination with functionalized microparticles to control the mechanical and biochemical microenvironment of individual cells. Our research focuses on investigating phagocytosis and chemotaxis in single immune cells. I will discuss the mechanics of macrophages and bacterial pathogens during the early stages of phagocytic uptake. Our studies revealed that macrophage filopodia act as tentacles which pull bound objects with discrete steps and a load-dependent velocity towards the cell for further phagocytosis. In addition, I will present a novel method for flexible biochemical cell stimulation. This method is based on optically manipulated microsources, which 17 sgmconferencessoc ie t y  fo r    gene ra l  m ic rob io logyEDINBURGH 29 MARCH–1 APRIL 2010WWW.SGMEICC2010.ORG.UKS E S S I O N S ED02 CONT.steadily release molecules. The technique enables control over biochemical cellular microenvironments down to length scales of one micrometer and timescales from an hour down to seconds. We demonstrated this technique by guiding and perturbing the migration of single human neutrophils.Towards	systems	analysis	of	small	GTPase	activities	in	early	HIV	infectionGary Warnes1 & MATTHIAS T. DITTMAR21Flow Cytometry Core Facility, 2Centre for Immunology & Infectious Disease, Blizard Institute of Cell & Molecular Science,London E1 2ATViruses are obligatory cellular parasites, with their replication crucially depending on host cell functions. They have evolved strategies to modulate host cell cytoskeleton dynamics and activate signal transduction pathways to facilitate entry into new target cells and release of progeny virus from infected cells. Therefore viruses are ideal tools to study signalling and cytoskeleton networks in an integrative way by comparing the dynamics of such virus exploited networks with the dynamics induced by natural ligand interaction. Changes in cytoskeletal structure are tightly regulated by the small GTPases like RhoA, Rac1 and Cdc42.Using dominant-negative and constitutive active forms of those GTPases we identified RhoA and Rac1 as factors contributing to changes in microtubule stability and reduced susceptibility to virus infection, thus identifying microtubules as potential cellular target for virus-cell fusion (Malinowsky et al., 2008). In order to visualize and quantify changes in signalling we have recently established a FRET based assay to detect activation of RhoA, Rac1 and Cdc42 using Raichu-FRET probes described by M Matsuda’s group in 2003 (Yoshizaki et al., 2003).The Gβ	protein	Gpb1	and	the	co-repressor	TupA	bind	to	Protein	Kinase	A	Tpk2	to	act	as	antagonistic	molecular	switchesof	fungal	morphological	changesThamarai K. Janganan1, Gongyou Chen1,3, Daliang Chen1, Marcia E. da Silva Ferreira2, Everaldo R. Marques2,Gustavo H. Goldman2, Adrian R. Walmsley1 & M. INÊS BORGES-WALMSLEY11School of Biological & Biomedical Sciences, Durham University, South Road, Durham DH1 3LE; 2Depto de Ciencias Farmaceuticas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, CEP 14040-903, Ribeirao Preto, Sao Paulo, Brazil;.3Dept of Agriculture & Biology, Shanghai Jiaotong University, 800 Dongchuan Road, 200240, Shanghai, ChinaParacoccidioides brasiliensis is one of a group of six phylogenetically related ascomycete fungi that are adapted for survival in mammalian hosts and which cause millions of infections every year. In P. brasiliensis the transition from the mycelium to the pathogenic yeast form of is controlled by the cAMP-signalling pathway and during the transition there is a change in the expression of the Gβ-protein Gpb1 that interacts with adenylate cyclase. We have exploited the fact that the cAMP-signaling-pathway of S. cerevisiae does not include a Gβ-protein, so as to use this organism to probe the functional role of Gpb1. We present data that indicates that Gpb1 and the transcriptional regulator TupA both bind to the PKA protein Tpk2; through which they act as antagonistic molecular switches of cell morphology, with TupA and Gpb1 inducing and repressing filamentous growth, respectively. In S. cerevisiae, PbTupA-induced filamentation occurs at two levels, with the early production of numerous pseudohyphae, a hyperfilamentious state, followed by the formation of an invasive projection and aerial stalk. In contrast, overexpressing ScTup1 represses the filamentous growth of S. cerevisiae, suggesting that an organisms Tup protein determines the morphological outcome. Conversely, Gpb1, which binds to PbTpk2, but not ScTpk2, could over-ride the effect of PbTupA to repress filamentous growth in S. cerevisiae. Our findings define a mechanism for controlling the morphological switch that underpins the virulence of dimorphic fungi.The	responses	of	fungal	pathogens	to	environmental	insultsAlistair J.P. BrownAberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZDThe success of Candida albicans as a human pathogen is enhanced by a battery of fungal virulence factors (such as cellular morphogenesis, adhesins and secreted hydrolytic enzymes) and fitness attributes (such as stress responses and metabolic adaptation). The stress responses help C. albicans combat host immune defences, which attack the pathogen with a battery of environmental insults that include reactive oxygen and nitrogen species. These stress responses appear to be regulated spatially and temporally during disease establishment and progression. We (and others) have begun to dissect the molecular mechanisms by which C. albicans responds to these oxidative and nitrosative stresses. Our approach is to combine mathematical modelling with molecular biology and genomics with a view to generating models that help us to predict the behaviour of C. albicans within the relatively complex and varied microenvironments of the host.

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Spatiotemporal control of cellular microenvironments with photonics

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