scholarly journals Analytics and visualization tools to characterize single-cell stochasticity using bacterial single-cell movie cytometry data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Athanasios D. Balomenos ◽  
Victoria Stefanou ◽  
Elias S. Manolakos
Keyword(s):  
Cell Division ◽  
Cell Growth ◽  
Single Cell ◽  
Bacterial Communities ◽  
Information Transfer ◽  
Community Organization ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Bioimage Analysis

Abstract Background Time-lapse microscopy live-cell imaging is essential for studying the evolution of bacterial communities at single-cell resolution. It allows capturing detailed information about the morphology, gene expression, and spatial characteristics of individual cells at every time instance of the imaging experiment. The image analysis of bacterial "single-cell movies" (videos) generates big data in the form of multidimensional time series of measured bacterial attributes. If properly analyzed, these datasets can help us decipher the bacterial communities' growth dynamics and identify the sources and potential functional role of intra- and inter-subpopulation heterogeneity. Recent research has highlighted the importance of investigating the role of biological "noise" in gene regulation, cell growth, cell division, etc. Single-cell analytics of complex single-cell movie datasets, capturing the interaction of multiple micro-colonies with thousands of cells, can shed light on essential phenomena for human health, such as the competition of pathogens and benign microbiome cells, the emergence of dormant cells (“persisters”), the formation of biofilms under different stress conditions, etc. However, highly accurate and automated bacterial bioimage analysis and single-cell analytics methods remain elusive, even though they are required before we can routinely exploit the plethora of data that single-cell movies generate. Results We present visualization and single-cell analytics using R (ViSCAR), a set of methods and corresponding functions, to visually explore and correlate single-cell attributes generated from the image processing of complex bacterial single-cell movies. They can be used to model and visualize the spatiotemporal evolution of attributes at different levels of the microbial community organization (i.e., cell population, colony, generation, etc.), to discover possible epigenetic information transfer across cell generations, infer mathematical and statistical models describing various stochastic phenomena (e.g., cell growth, cell division), and even identify and auto-correct errors introduced unavoidably during the bioimage analysis of a dense movie with thousands of overcrowded cells in the microscope's field of view. Conclusions ViSCAR empowers researchers to capture and characterize the stochasticity, uncover the mechanisms leading to cellular phenotypes of interest, and decipher a large heterogeneous microbial communities' dynamic behavior. ViSCAR source code is available from GitLab at https://gitlab.com/ManolakosLab/viscar.

scholarly journals Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Han Wang ◽  
Gloria M. Conover ◽  
Song-I Han ◽  
James C. Sacchettini ◽  
Arum Han
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Culture Media ◽  
Low Cost ◽  
Bacterial Pathogen ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

scholarly journals Cell-to-cell heterogeneity in Escherichia coli stress response originates from pulsatile expression and growth

2020 ◽  
Author(s):  
Nadia M. V. Sampaio ◽  
Caroline M. Blassick ◽  
Jean-Baptiste Lugagne ◽  
Mary J. Dunlop
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Stress Response ◽  
Single Cell ◽  
Phenotypic Variability ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Heterogeneity ◽  
Temporal Expression ◽  
Functional Consequences

AbstractCell-to-cell heterogeneity in gene expression and growth can have critical functional consequences, such as determining whether individual bacteria survive or die following stress. Although phenotypic variability is well documented, the dynamics that underlie it are often unknown. This information is critical because dramatically different outcomes can arise from gradual versus rapid changes in expression and growth. Using single-cell time-lapse microscopy, we measured the temporal expression of a suite of stress response reporters in Escherichia coli, while simultaneously monitoring growth rate. In conditions without stress, we found widespread examples of pulsatile expression. Single-cell growth rates were often anti-correlated with gene expression, with changes in growth preceding changes in expression. These pulsatile dynamics have functional consequences, which we demonstrate by measuring survival after challenging cells with the antibiotic ciprofloxacin. Our results suggest that pulsatile expression and growth dynamics are common in stress response networks and can have direct consequences for survival.

scholarly journals Single-cell growth inference of Corynebacterium glutamicum reveals asymptotically linear growth

2020 ◽  
Author(s):  
Joris Messelink ◽  
Fabian Meyer ◽  
Marc Bramkamp ◽  
Chase P. Broedersz
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
Corynebacterium Glutamicum ◽  
Cell Size ◽  
Linear Growth ◽  
Growth Dynamics ◽  
Asymptotically Linear ◽  
Inference Method ◽  
Growth Modes ◽  
Regulation Mechanisms

AbstractIn many bacteria, protein mass production is thought to be rate limiting for growth, implying exponential growth at the single cell level. To maintain cell-size homeostasis in proliferating populations of exponentially growing bacteria, tight growth and division mechanisms are required. However, it remains unclear whether these considerations set universal physical limits to bacterial growth. Here, we characterize the growth dynamics of the actinobacterium Corynebacterium glutamicum - a promising candidate for uncovering novel growth modes. This bacterium exhibits apical cell wall synthesis and division site selection systems appear to be absent, as reflected by a broad distribution of division asymmetries. We develop a novel growth inference method that averages out measurement noise and single-cell variability to obtain elongation rate curves as a function of birth length. Using this approach, we find that C. glutamicum exhibits asymptotically linear single-cell growth. To explain this growth mode, we model elongation as being rate-limited by the apical growth mechanism mediated by cell wall transglycosylases. This model accurately reproduces the observed elongation rate curves, and we further validate the model with growth measurements on a transglycosylase deficient ΔrodA mutant. Finally, with simulations we show that asymptotically linear growth yields a narrower distribution of cell lengths, suggesting that this growth mode can act as a substitute for tight division length and division symmetry regulation.SignificanceRegulation of growth and cell size is crucial for the optimization of bacterial cellular function. So far, single bacterial cells have been found to grow exponentially, which implies the need for tight regulation mechanisms to maintain cell size throughout growth and division cycles. Here, we characterize the growth behavior of the apically growing bacterium Corynebacterium glutamicum, by developing a novel and broadly applicable inference method for single-cell growth dynamics. We find that this bacterium grows asymptotically linearly, enabling it to maintain a narrow distribution of cell sizes, despite having a large variability of single-cell growth features. Our results imply a novel interplay between mode of growth and division regulation mechanisms, which may extend to other bacteria with non-exponential growth modes.

scholarly journals Single-cell growth inference of Corynebacterium glutamicum reveals asymptoticallylinear growth

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Joris Jan Boudewijn Messelink ◽  
Fabian Meyer ◽  
Marc Bramkamp ◽  
Chase P Broedersz
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
Corynebacterium Glutamicum ◽  
Cell Size ◽  
Linear Growth ◽  
Growth Dynamics ◽  
Growth Mode ◽  
Bacterial Cells ◽  
Asymptotically Linear ◽  
Regulation Of Growth

Regulation of growth and cell size is crucial for the optimization of bacterial cellular function. So far, single bacterial cells have been found to grow predominantly exponentially, which implies the need for tight regulation to maintain cell size homeostasis. Here, we characterize the growth behavior of the apically growing bacterium Corynebacterium glutamicum using a novel broadly applicable inference method for single-cell growth dynamics. Using this approach, we find that C. glutamicum exhibits asymptotically linear single-cell growth. To explain this growth mode, we model elongation as being rate-limited by the apical growth mechanism. Our model accurately reproduces the inferred cell growth dynamics and is validated with elongation measurements on a transglycosylase deficient ΔrodA mutant. Finally, with simulations we show that the distribution of cell lengths is narrower for linear than exponential growth, suggesting that this asymptotically linear growth mode can act as a substitute for tight division length and division symmetry regulation.

scholarly journals The Ribbon-Helix-Helix Domain Protein CdrS Regulates the Tubulin Homolog ftsZ2 To Control Cell Division in Archaea

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Cynthia L. Darnell ◽  
Jenny Zheng ◽  
Sean Wilson ◽  
Ryan M. Bertoli ◽  
Alexandre W. Bisson-Filho ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Transcriptional Control ◽  
Control Cell ◽  
Time Lapse ◽  
Live Cells ◽  
Central Function ◽  
Individual Organism ◽  
Healthy Cell

ABSTRACT Precise control of the cell cycle is central to the physiology of all cells. In prior work we demonstrated that archaeal cells maintain a constant size; however, the regulatory mechanisms underlying the cell cycle remain unexplored in this domain of life. Here, we use genetics, functional genomics, and quantitative imaging to identify and characterize the novel CdrSL gene regulatory network in a model species of archaea. We demonstrate the central role of these ribbon-helix-helix family transcription factors in the regulation of cell division through specific transcriptional control of the gene encoding FtsZ2, a putative tubulin homolog. Using time-lapse fluorescence microscopy in live cells cultivated in microfluidics devices, we further demonstrate that FtsZ2 is required for cell division but not elongation. The cdrS-ftsZ2 locus is highly conserved throughout the archaeal domain, and the central function of CdrS in regulating cell division is conserved across hypersaline adapted archaea. We propose that the CdrSL-FtsZ2 transcriptional network coordinates cell division timing with cell growth in archaea. IMPORTANCE Healthy cell growth and division are critical for individual organism survival and species long-term viability. However, it remains unknown how cells of the domain Archaea maintain a healthy cell cycle. Understanding the archaeal cell cycle is of paramount evolutionary importance given that an archaeal cell was the host of the endosymbiotic event that gave rise to eukaryotes. Here, we identify and characterize novel molecular players needed for regulating cell division in archaea. These molecules dictate the timing of cell septation but are dispensable for growth between divisions. Timing is accomplished through transcriptional control of the cell division ring. Our results shed light on mechanisms underlying the archaeal cell cycle, which has thus far remained elusive.

scholarly journals Size control in mammalian cells involves modulation of both growth rate and cell cycle duration

2017 ◽  
Author(s):  
Clotilde Cadart ◽  
Sylvain Monnier ◽  
Jacopo Grilli ◽  
Rafaele Attia ◽  
Emmanuel Terriac ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Cell Division ◽  
Cell Growth ◽  
Single Cell ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Size Control ◽  
Cycle Duration ◽  
Mathematical Framework

SummaryDespite decades of research, it remains unclear how mammalian cell growth varies with cell size and across the cell division cycle to maintain size control. Answers have been limited by the difficulty of directly measuring growth at the single cell level. Here we report direct measurement of single cell volumes over complete cell division cycles. The volume added across the cell cycle was independent of cell birth size, a size homeostasis behavior called “adder”. Single-cell growth curves revealed that the homeostatic behavior relied on adaptation of G1 duration as well as growth rate modulations. We developed a general mathematical framework that characterizes size homeostasis behaviors. Applying it on datasets ranging from bacteria to mammalian cells revealed that a near-adder is the most common type of size control, but only mammalian cells achieve it using modulation of both cell growth rate and cell-cycle progression.

scholarly journals High proliferation and delamination during skin epidermal stratification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mareike Damen ◽  
Lisa Wirtz ◽  
Ekaterina Soroka ◽  
Houda Khatif ◽  
Christian Kukat ◽  
...  
Keyword(s):  
Cell Division ◽  
Asymmetric Cell Division ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Tissue Growth ◽  
Epidermal Differentiation ◽  
Second Phase ◽  
Open Questions ◽  
Epithelial Barriers ◽  
Time Lapse Imaging

AbstractThe development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis leads to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

scholarly journals The ribbon-helix-helix domain proteins CdrS and CdrL regulate cell division in archaea

2020 ◽  
Author(s):  
Cynthia L. Darnell ◽  
Jenny Zheng ◽  
Sean Wilson ◽  
Ryan M. Bertoli ◽  
Alexandre W. Bisson-Filho ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Transcriptional Control ◽  
Quantitative Imaging ◽  
Time Lapse ◽  
Live Cells ◽  
Central Function ◽  
Individual Organism ◽  
Healthy Cell

AbstractPrecise control of the cell cycle is central to the physiology of all cells. In prior work we demonstrated that archaeal cells maintain a constant size; however, the regulatory mechanisms underlying the cell cycle remain unexplored in this domain of life. Here we use genetics, functional genomics, and quantitative imaging to identify and characterize the novel CdrSL gene regulatory network in a model species of archaea. We demonstrate the central role of these ribbon-helix-helix family transcription factors in the regulation of cell division through specific transcriptional control of the gene encoding FtsZ2, a putative tubulin homolog. Using time lapse fluorescence microscopy in live cells cultivated in microfluidics devices, we further demonstrate that FtsZ2 is required for cell division but not elongation. The cdrS-ftsZ2 locus is highly conserved throughout the archaeal domain, and the central function of CdrS in regulating cell division is conserved across hypersaline adapted archaea. We propose that the CdrSL-FtsZ2 transcriptional network coordinates cell division timing with cell growth in archaea.ImportanceHealthy cell growth and division are critical for individual organism survival and species long-term viability. However, it remains unknown how cells of the domain Archaea maintain a healthy cell cycle. Understanding archaeal cell cycle is of paramount evolutionary importance given that an archaeal cell was the host of the endosymbiotic event that gave rise to eukaryotes. Here we identify and characterize novel molecular players needed for regulating cell division in archaea. These molecules dictate the timing of cell septation, but are dispensable for growth between divisions. Timing is accomplished through transcriptional control of the cell division ring. Our results shed light on mechanisms underlying the archaeal cell cycle, which has thus far remained elusive.

Aging, cellular senescence and cancer: the role of genomic instability, cellular homeostasis and telomeres

2019 ◽  
pp. 227-237
Author(s):  
John C. Lucchesi
Keyword(s):  
Cell Division ◽  
Cell Growth ◽  
Genomic Instability ◽  
Normal Cell ◽  
Human Tissues ◽  
Telomeric Dna ◽  
Over Expression ◽  
Causative Factors ◽  
Cell Growth And Proliferation

Aging hallmarks are causative factors of oncogenesis. Genomic instability results from the accumulation of errors that occur during DNA replication or from exposure to endogenous or environmental insults. The genome contains genes responsible for normal cell division and differentiation (oncogenes), and genes that regulate cell division and limit cell growth and proliferation (tumor suppressor genes). Over-expression of oncogenes or inactivation of tumor suppressors results in cancer. During aging, alterations in proteostasis result in the disruption of metabolic pathways that connect with environmental factors. Telomeres are terminal regions of chromosomes that protect the DNA from attack by exonucleases, prevent end-to-end fusions and prevent the shortening of the DNA molecules at each replication cycle. Using RNA as a template, telomerase synthesizes telomeric DNA. Telomerase is absent in most adult human tissues, resulting in a progressive shortening of all telomeres and causing cells to senesce. Cancer cells must activate telomerase to gain “immortality.”

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