Name: Comparing Budding Curves, Flow Cytometry Data, and Experimental Data
Uploaded: 2023-06-09T13:00:00
Description: Watch this Scientific Journal Video about Comparing Budding Curves, Flow Cytometry Data, and Experimental Data at JoVE.com

comparing budding curves, flow cytometry data, and experimental data

Alinhamento de dados de séries temporais sincronizados para comparações entre experimentos

Time-series measurements made on synchronized populations of cells as they progress through the cell cycle is a standard method for investigating the mechanisms that control cell-cycle progression1,2,3,4,5,6,7,8. The ability to make comparisons across synchrony/release time-series experiments is vital to our understanding of these dynamic processes. The use of replicate experiments to corroborate findings can increase the confidence in the reproducibility of the conclusions. Furthermore, comparisons between environmental conditions, across mutants, and even between species can uncover many new insights into cell-cycle regulation. However, interexperimental variability in the recovery from synchrony and in the speed of cell-cycle progression impairs the ability to make time-point-to-time-point comparisons across replicates or between experiments with altered cell-cycle timing. Due to these challenges, replicates are often not included for the full time series (e.g., Spellman et al.4). When replicates for the entire time series are gathered, the data cannot be analyzed in aggregate, but rather a single replicate is used for analysis, and other replicates are often relegated to supplemental figures (e.g., Orlando et al.8). Furthermore, comparisons between experiments with different recovery or cell-cycle progression characteristics are difficult. The measurements of smaller intervals between an event of interest and a cell-cycle landmark (e.g., bud emergence, S-phase entry, or anaphase onset) can help reduce errors if these landmark events are tracked1,2,3,9,10,11,12. However, subtle but important differences may remain undetected or obscured using these ad hoc methods. Finally, single-cell analyses allow for analyzing cell-cycle progression without relying on synchronization or alignment13, though large-scale measurements in single-cell studies can be challenging and costly.
To overcome these difficulties, we developed the Characterizing Loss of Cell Cycle Synchrony (CLOCCS) model to aid the analysis of time-series measurements made on synchronized populations14,15. CLOCCS is a flexible mathematical model that describes the distribution of synchronized cells across cell-cycle phases as they are released from synchrony and progress through the cell cycle. The branching process framework enables the model to account for the asymmetric qualities of mother and daughter cells after division, as observed in S. cerevisiae, while still being useful for organisms that divide by fission, such as S. pombe. The model can take inputs from a diverse set of measurement types to specify the cell-cycle phase. It can ingest budding cell-cycle phase data, which includes measurements of the percent budded cells over time, allowing for the estimation of the number of cells outside of the unbudded G1 phase14,15. The model can also ingest flow cytometric data that measures the DNA content, thus enabling the assessment of landmark transitions from G1 to S, S to G2, and M to G115. Fluorescent morphological markers can also be used to identify the cell-cycle phase. The fluorescent labeling of myosin rings, nuclei, and spindle pole bodies (SPBs) can be used to determine the cell-cycle phase, and these were incorporated into the CLOCCS model11; however, these measurements will not be described in this protocol. Additionally, the septation index was used as an input for modeling data from S. pombe14. Thus, the model can be used for cell-cycle analyses in a variety of organisms and can be further expanded.
CLOCCS is a parametric model that allows for the full Bayesian inference of multiple parameters from the input data (e.g., budding percentage, DNA content). These parameters include the recovery time from synchrony, the length of the cell-cycle period (estimated separately for mother and daughter cells), and the average cell-cycle position of the cells at each time point. These parameters represent the behavior of the average cell in the population, enabling the researcher to map each time point to a cell-cycle position expressed as a lifeline point. The conversion to lifeline points depends on the CLOCCS parameters lambda (&#955;) and mu0 (&#181;0)14,15. The parameter &#955; corresponds to the average cell-cycle period of the mother cells. However, due to the mother-daughter delay14,15, this is not the average cell-cycle period of the full population that includes both the mother and daughter cells. CLOCCS additionally infers the parameter delta (&#948;), which corresponds to the mother-daughter delay and, thus, allows for the calculation of the average cell-cycle period of the full population. Finally, because each experiment begins after release from cell-cycle synchronization, the time required to recover from the synchronization method is represented by the CLOCCS parameter &#181;0. CLOCCS fits a model to the input cell-cycle phase data and then infers these parameters using a random walk Markov chain Monte Carlo algorithm14,15. By mapping multiple experiments to a common cell-cycle lifeline time scale, direct phase-specific comparisons can be made between replicates or experiments where the recovery time or cell-cycle periods are not identical8,14,15.
As synchronized populations lose synchrony at some rate over the course of the time series14,15,16,17, variability in the rate of synchrony loss can also impede quantitative comparisons across experiments. By identifying the location of populations and the variance in their distributions, CLOCCS accounts for differences in rates of synchrony loss. This powerful tool allows for specific and detailed comparisons across experiments, thus providing the ability to directly make relevant comparisons not only between replicates but also between environmental conditions, mutants, and even species that have dramatically different cell-cycle timing14,15.
This paper describes a method using CLOCCS to estimate parameters by fitting data from synchrony/release time-series experiments, map the data to a common lifeline scale, and then make relevant comparisons between replicates or experiments. Lifeline alignment allows for direct phase-specific comparisons across these experiments, which allows for the aggregation and comparison of replicates and for making more relevant comparisons across experiments with different recovery timings and cell-cycle periods.

Autor em destaque: Alinhamento de dados de séries temporais sincronizadas usando o modelo de caracterização da perda de sincronia do ciclo celular para comparações entre experimentos  

Alinhamento de dados sincronizados de séries temporais usando o modelo de caracterização de perda de sincronia do ciclo celular para comparações entre experimentos

A major challenge of analyzing time-series experiments is that they often differ in the length of recovery from synchrony and the cell cycle period. So measurements from different experiments cannot readily be compared or analyzed in aggregate without alignment. Clocks lifeline alignment is a method for phase specific and biologically relevant comparisons between experiments and for aggregating multiple replicate experiments both of which were previously difficult or impossible.Previously phase specific and biologically relevant comparisons were made by tracking landmark events. However, using these ad hoc methods, subtle but important differences remain undetected. Comparison between time series experiments is particularly difficult between experiments with significant differences in timing such as in mutant populations or growth conditions that affect the synchrony recovery time or the cell cycle period.Clocks lifeline alignment allows for phase specific comparison in these cases. We have used clocks to align time series transcriptomic and proteomic data, which allowed for the direct comparison between the dynamics of the mRNA and the corresponding protein. Furthermore, we align time series experiments across different species, providing critical insights into cell cycle evolutionary changes.

Watch this Scientific Journal Video about Comparing Budding Curves, Flow Cytometry Data, and Experimental Data at JoVE.com

Comparing Budding Curves, Flow Cytometry Data, and Experimental Data  

Comparando curvas de brotamento, dados de citometria de fluxo e dados experimentais

Plote as curvas de brotamento antes do alinhamento usando a função de utilitário Python inserindo o comando em uma nova célula. Em seguida, plote as curvas de brotamento após o alinhamento. Usando a função de utilitário Python.Use a comparação de gráfico de linha de plotagem fornecida no frasco de utilitários Python para executar comparações de gráfico de linha no quadro de dados original, alinhado ou alinhado e interpolado. Ao digitar o comando em uma nova célula, importe um arquivo de lista de genes CSV ou TSV para o bloco de anotações usando o comando em uma nova célula. Em seguida, use a comparação de mapa de calor de gráfico de função fornecida no arquivo de utilitários Python para executar uma comparação de mapa de calor no quadro de dados alinhado interpolado e alinhado à fase digitando o comando em uma nova célula.Uma comparação dos dados transcriptomáticos alinhados e não alinhados mostrou que, antes do alinhamento, o primeiro pico de expressão dos experimentos de microarranjos apareceu alinhado com o segundo pico do experimento RNA-seq. No entanto, após o alinhamento, os primeiros picos do ciclo celular de cada conjunto de dados são alinhados adequadamente. A comparação dos dados de fase do ciclo celular entre experimentos com períodos variados exibiu diferenças visíveis de período nas curvas de brotamento não alinhadas.Enquanto o alinhamento dos relógios fez com que as três curvas parecessem notavelmente semelhantes, tornando possíveis comparações de dados experimentais, os dados de fase do ciclo celular para cada ponto comparável da linha de vida não foram idênticos entre as duas condições. A comparação dos dados transcriptômicos entre experimentos com períodos variados mostrou que, antes do alinhamento, a dinâmica de transcrição do CDC20 não se sobrepunha. Mas, após o alinhamento, os picos ocorreram na mesma fase do ciclo celular, mas as formas das curvas eram diferentes.Os genes foram plotados como mapas de calor na mesma ordem para as três condições para desalinhadas e alinhadas.

Research

JoVE Journal

Biology

Comparing Budding Curves, Flow Cytometry Data, and Experimental Data

Alignment of Synchronized Time-Series Data Using the Characterizing Loss of Cell Cycle Synchrony Model for Cross-Experiment Comparisons

Investigating the cell cycle often depends on synchronizing cell populations to measure various parameters in a time series as the cells traverse the cell ...