Using time-lapse fluorescence microscopy to study gene regulation

Zou, Fan; Bai, Lu

doi:10.1016/j.ymeth.2018.12.010

Cited by 17 publications

(11 citation statements)

References 59 publications

(70 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1B and D). GFP intensity increases sharply after ϳ30 min of induction and reaches steady state after ϳ3 h. The increase in the mCherry intensity slightly lags behind that of GFP due to a lower maturation rate (15). The mCherry expression levels in Fig.…”

Section: Resultsmentioning

confidence: 88%

“…We constructed two diploid strains containing a single copy of MET3pr-GFP inserted into either silenced (NTS1 in rDNA) or euchromatic (ATG36 open reading frame [ORF]) regions. We monitored the GFP expression during the MET3pr activation by combining time-lapse fluorescence microscopy with a microfluid device (see Materials and Methods) (15). As a control, these strains also contain a MET3pr-mCherry reporter at CDC20 in euchromatin.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Existence, Transition, and Propagation of Intermediate Silencing States in Ribosomal DNA

Zou

Chen

et al. 2019

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

The MET3 promoter (MET3pr) inserted into the silenced chromosome in budding yeast can overcome Sir2-dependent silencing upon induction and activate transcription in every single cell among a population. Despite the fact that MET3pr is turned on in all the cells, its activity still shows very high cell-to-cell variability. To understand the nature of such “gene expression noise,” we followed the dynamics of the MET3pr-GFP expression inserted into ribosomal DNA (rDNA) using time-lapse microscopy. We found that the noisy “on” state is comprised of multiple substable states with discrete expression levels. These intermediate states stochastically transition between each other, with “up” transitions among different activated states occurring exclusively near the mitotic exit and “down” transitions occurring throughout the rest of the cell cycle. Such cell cycle dependence likely reflects the dynamic activity of the rDNA-specific RENT complex, as MET3pr-GFP expression in a telomeric locus does not have the same cell cycle dependence. The MET3pr-GFP expression in rDNA is highly correlated in mother and daughter cells after cell division, indicating that the silenced state in the mother cell is inherited in daughter cells. These states are disrupted by a brief repression and reset upon a second activation. Potential mechanisms behind these observations are further discussed.

show abstract

Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Existence, Transition, and Propagation of Intermediate Silencing States in Ribosomal DNA

Zou

Chen

et al. 2019

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…For these purposes, several models of flow chambers that can be mounted on a microscope stage and used with video capture systems have been developed, enabling real-time observation of microbial adhesion, particularly when used with transparent surfaces. The employment of fluorescent probes coupled with confocal laser scanning microscopy (CLSM) makes flow chambers especially appreciated for in situ gene expression studies [ 51 ].…”

Section: Biofilm Platformsmentioning

confidence: 99%

A Selection of Platforms to Evaluate Surface Adhesion and Biofilm Formation in Controlled Hydrodynamic Conditions

Gomes

Mergulhão

2021

Microorganisms

View full text Add to dashboard Cite

The early colonization of surfaces and subsequent biofilm development have severe impacts in environmental, industrial, and biomedical settings since they entail high costs and health risks. To develop more effective biofilm control strategies, there is a need to obtain laboratory biofilms that resemble those found in natural or man-made settings. Since microbial adhesion and biofilm formation are strongly affected by hydrodynamics, the knowledge of flow characteristics in different marine, food processing, and medical device locations is essential. Once the hydrodynamic conditions are known, platforms for cell adhesion and biofilm formation should be selected and operated, in order to obtain reproducible biofilms that mimic those found in target scenarios. This review focuses on the most widely used platforms that enable the study of initial microbial adhesion and biofilm formation under controlled hydrodynamic conditions - modified Robbins devices, flow chambers, rotating biofilm devices, microplates, and microfluidic devices - and where numerical simulations have been used to define relevant flow characteristics, namely the shear stress and shear rate.

show abstract

“…Typically, the fluorescent or bioluminescent protein is expressed from the control region of interest, either on plasmids or integrated in the genome, and protein synthesis is quantified over time by time-lapse microscopy, fluorescence cytometry, or fluorescence or luminescence readers. Fluorescent markers such as green fluorescent protein (GFP) are the most widely used indicators for dynamic in vivo gene expression studies [25][26][27] because of their high photon yield that is compatible with time-elapsed studies in single cells [28][29][30]. Additionally, GFP activity does not depend on additional cellular cofactors, and engineered versions with different colors are available for the simultaneous visualization of gene expression from several loci in the same cell [31].…”

Section: Technical Approaches To Capture Gene Expression In Vivomentioning

confidence: 99%

Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell

Pascual-Ahuir

Fita-Torró

Proft

2020

IJMS

View full text Add to dashboard Cite

The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.

show abstract

Using time-lapse fluorescence microscopy to study gene regulation

Cited by 17 publications

References 59 publications

Existence, Transition, and Propagation of Intermediate Silencing States in Ribosomal DNA

Existence, Transition, and Propagation of Intermediate Silencing States in Ribosomal DNA

A Selection of Platforms to Evaluate Surface Adhesion and Biofilm Formation in Controlled Hydrodynamic Conditions

Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell

Contact Info

Product

Resources

About