scholarly journals Optogenetic tools for public goods control in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Neydis Moreno Morales ◽  
Michael T Patel ◽  
Cameron J Stewart ◽  
Kieran Sweeney ◽  
Megan Nicole McClean
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Community Development ◽  
Cooperative Interaction ◽  
Interspecies Interactions ◽  
Spatial Control ◽  
Space And Time ◽  
Diverse Communities ◽  
Spatiotemporal Control ◽  
Inhibitory Interactions ◽  
Control Of Expression

Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light-control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in Saccharomyces cerevisiae to perturb both intercellular and interspecies interactions.

scholarly journals Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes

2019 ◽  
Author(s):  
Pooja Suresh ◽  
Alexandra F. Long ◽  
Sophie Dumont
Keyword(s):  
Cell Division ◽  
Local Structure ◽  
Mammalian Cells ◽  
Chromosome Movement ◽  
Space And Time ◽  
Tightly Coupled ◽  
Transient Forces ◽  
Spatiotemporal Control

AbstractThe spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to coordinate chromosome movement. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals that the spindle retains local architecture in its center on the seconds timescale. Upon pulling, sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers freely pivot around poles but not around chromosomes, retaining their orientation within 3 µm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement of the kinetochore-fiber in the spindle center. This could help the spindle protect local structure near chromosomes from transient forces while allowing its remodeling over longer timescales, thereby supporting robust chromosome attachments and movements.

scholarly journals Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Pooja Suresh ◽  
Alexandra F Long ◽  
Sophie Dumont
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Space And Time ◽  
Tightly Coupled ◽  
Transient Forces ◽  
Spatiotemporal Control ◽  
Chromosome Movements

The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 μm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.

scholarly journals Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae

mSphere ◽  
2021 ◽  
Vol 6 (4) ◽  
Author(s):  
Neydis Moreno Morales ◽  
Michael T. Patel ◽  
Cameron J. Stewart ◽  
Kieran Sweeney ◽  
Megan N. McClean
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Community Development ◽  
Public Goods ◽  
Microbial Communities ◽  
Microbial Ecology ◽  
Intercellular Interactions ◽  
Recent Advances

Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner.

scholarly journals Characterization of the Promoter of PRS1 inSaccharomyces cerevisiae Identifies Three Regions Potentially Involved in Control of Expression

2001 ◽  
Vol 183 (2) ◽  
pp. 795-799 ◽  
Author(s):  
Yolanda Hernando ◽  
Andrew T. Carter ◽  
Stefan Sickinger ◽  
Michael Schweizer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Galactosidase Activity ◽  
Shift Analysis ◽  
Gel Shift ◽  
Control Of Expression

ABSTRACT The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at −179 bp. Measurement of β-galactosidase activity of the successively deleted PRS1promoter linked to lacZ and integrated at theura3 locus defined three DNA regions involved in the control of PRS1 expression. Gel shift analysis confirmed the data.

scholarly journals Engineering E. coli for magnetic control and the spatial localization of functions

2020 ◽  
Author(s):  
Mary Aubry ◽  
Wei-An Wang ◽  
Yohan Guyodo ◽  
Eugénia Delacou ◽  
Jean Michel Guignier ◽  
...  
Keyword(s):  
Magnetic Properties ◽  
Spatial Modulation ◽  
Adhesion Properties ◽  
Spatial Control ◽  
E Coli ◽  
Magnetic Forces ◽  
Magnetic Capture ◽  
Diagnostic Agents ◽  
Delivery Vehicles ◽  
Spatiotemporal Control

AbstractThe fast-developing field of synthetic biology enables broad applications of programmed microorganisms including the development of whole-cell biosensors, delivery vehicles for therapeutics, or diagnostic agents. However, the lack of spatial control required for localizing microbial functions could limit their use and induce their dilution leading to ineffective action or dissemination. To overcome this limitation, the integration of magnetic properties into living systems enables a contact-less and orthogonal method for spatiotemporal control. Here, we generated a magnetic-sensing Escherichia coli by driving the formation of iron-rich bodies into bacteria. We found that these bacteria could be spatially controlled by magnetic forces and sustained cell growth and division, by transmitting asymmetrically their magnetic properties to one daughter cell. We combined the spatial control of bacteria with genetically encoded-adhesion properties to achieve the magnetic capture of specific target bacteria as well as the spatial modulation of human cell invasions.

scholarly journals A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Jidapas (My) An-adirekkun ◽  
Cameron J. Stewart ◽  
Stephanie H. Geller ◽  
Michael T. Patel ◽  
Justin Melendez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Biological Networks ◽  
Modular Assembly ◽  
Expression Control ◽  
Control Gene Expression ◽  
Gene Expression Control ◽  
Rapid Generation ◽  
Spatiotemporal Control ◽  
Different Levels

AbstractOptogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control and integrate it with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae.

scholarly journals Different Regulations of ROM2 and LRG1 Expression by Ccr4, Pop2, and Dhh1 in the Saccharomyces cerevisiae Cell Wall Integrity Pathway

mSphere ◽  
2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Xia Li ◽  
Tetsuro Ohmori ◽  
Kaoru Irie ◽  
Yuichi Kimura ◽  
Yasuyuki Suda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Rna Binding ◽  
Budding Yeast ◽  
Mrna Level ◽  
Small Gtpase ◽  
Cell Wall Integrity ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Spatiotemporal Control

ABSTRACT We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex. Ccr4, a component of the Ccr4-Not cytoplasmic deadenylase complex, is known to be required for the cell wall integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae. However, it is not fully understood how Ccr4 and other components of the Ccr4-Not complex regulate the CWI pathway. Previously, we showed that Ccr4 functions in the CWI pathway together with Khd1 RNA binding protein. Ccr4 and Khd1 modulate a signal from Rho1 small GTPase in the CWI pathway by regulating the expression of ROM2 mRNA and LRG1 mRNA, encoding a guanine nucleotide exchange factor (GEF) and a GTPase-activating protein (GAP) for Rho1, respectively. Here we examined the possible involvement of the POP2 gene encoding a subunit of the Ccr4-Not complex and the DHH1 gene encoding a DEAD box RNA helicase that associates with the Ccr4-Not complex in the regulation of ROM2 and LRG1 expression. Neither ROM2 mRNA level nor Rom2 function was impaired by pop2Δ or dhh1Δ mutation. The LRG1 mRNA level was increased in pop2Δ and dhh1Δ mutants, as well as the ccr4Δ mutant, and the growth defects caused by pop2Δ and dhh1Δ mutations were suppressed by lrg1Δ mutation. Our results suggest that LRG1 expression is regulated by Ccr4 together with Pop2 and Dhh1 and that ROM2 expression is regulated by Khd1 and Ccr4, but not by Pop2 and Dhh1. Thus, Rho1 activity in the CWI pathway is precisely controlled by modulation of the mRNA levels for Rho1-GEF Rom2 and Rho1-GAP Lrg1. IMPORTANCE We find here that Ccr4, Pop2, and Dhh1 modulate the levels of mRNAs for specific Rho1 regulators, Rom2 and Lrg1. In budding yeast, Rho1 activity is tightly regulated both temporally and spatially. It is anticipated that Ccr4, Pop2, and Dhh1 may contribute to the precise spatiotemporal control of Rho1 activity by regulating expression of its regulators temporally and spatially. Our finding on the roles of the components of the Ccr4-Not complex in yeast would give important information for understanding the roles of the evolutionary conserved Ccr4-Not complex.

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