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A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide.

Shokhirev MN, Almaden J, Davis-Turak J, Birnbaum HA, Russell TM, Vargas JA, Hoffmann A - Mol. Syst. Biol. (2015)

Bottom Line: B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically.Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells.We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Signaling Systems Laboratory, UCSD, La Jolla, CA, USA San Diego Center for Systems Biology, UCSD, La Jolla, CA, USA Bioinformatics and Systems Biology Graduate Program, UCSD, La Jolla, CA, USA.

No MeSH data available.


Time-lapse microscopy reveals two distinct generation-dependent growth patterns for B cellsA Overview of the time-lapse microscopy experimental and analysis pipeline. B cells were purified from mouse spleen, stimulated with TLR9 agonist CpG, imaged on an environmentally controlled microscope for 6 days, and tracked using a semi-automated tracking tool to quantify generation-dependent cell statistics.B Generational cell counts relative to initial count.C The observed fraction of cells dividing or dying in each generation. Error bars = 1/n, where n = 85, 55, 93, 103, 125, 90, 45 for generations 0–6, respectively.D Growth trajectories of generation 0 cells that grew by more than 350 μm3 or ended with a volume of at least 800 μm3 (blue) and trajectories of generation 0 cells that did not end with a large volume (black).E, F Cell size trajectories as a function of % lifetime for growers (E) and non-growers (F) in each generation (colors as in B). Error bars are SD, with n = 34, 44, 60, 54, 40, 15, 2 growing cells, 51, 11, 33, 49, 85, 75, 43 non-growing cells in generations 0–6, respectively.
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fig02: Time-lapse microscopy reveals two distinct generation-dependent growth patterns for B cellsA Overview of the time-lapse microscopy experimental and analysis pipeline. B cells were purified from mouse spleen, stimulated with TLR9 agonist CpG, imaged on an environmentally controlled microscope for 6 days, and tracked using a semi-automated tracking tool to quantify generation-dependent cell statistics.B Generational cell counts relative to initial count.C The observed fraction of cells dividing or dying in each generation. Error bars = 1/n, where n = 85, 55, 93, 103, 125, 90, 45 for generations 0–6, respectively.D Growth trajectories of generation 0 cells that grew by more than 350 μm3 or ended with a volume of at least 800 μm3 (blue) and trajectories of generation 0 cells that did not end with a large volume (black).E, F Cell size trajectories as a function of % lifetime for growers (E) and non-growers (F) in each generation (colors as in B). Error bars are SD, with n = 34, 44, 60, 54, 40, 15, 2 growing cells, 51, 11, 33, 49, 85, 75, 43 non-growing cells in generations 0–6, respectively.

Mentions: In order to obtain cell lineage information that accounts for the population response, we tracked 1,295 live primary B cells using a time-lapse microscopy pipeline (Fig2A). We developed a semi-automated image analysis method, combining the advantages of computational automation and human input to minimize errors (see Materials and Methods). Analysis of wild-type B cells responding to high CpG stimulation confirmed the expected population expansion followed by a contraction period (Fig2B). After cells that died from mechanical death in the initial phase (Hawkins et al, 2007a) were filtered out (Supplementary Fig S1A and B), we found that approximately 38% of the starting, ‘generation 0’, cells divided; then, 85% of generation 1 cells divided with subsequent generations, showing a steady decrease in this fraction such that only 9% of cells divided in generation 6 (Fig2C). To quantify the cellular response, we classified cell size trajectories into two categories: (i) cells that grew by at least 350 μm3 (representing at least two standard deviations on average, Supplementary Fig S1C) or reached a final size of at least 800 μm3 (based on the bimodal size distribution (Supplementary Fig S1D) and to ensure that large generation 1+ cells are included), dubbed ‘growers’ and (ii) cells that did not meet these criteria, dubbed ‘non-growers’ (Fig2D). To test the sensitivity of the growth threshold, we repeated the quantification with a 25% lower and higher growth threshold, revealing that few cells exhibited ambiguous growth (Supplementary Fig S1E). Averaging the growth trajectories of ‘growers’ (Fig2E) and ‘non-growers’ (Fig2F) in each generation normalized by percent cell lifespan revealed that progenitors (generation 0) that grew exhibited a growth delay followed by rapid growth to approximately fivefold their starting size, while generation 1+ growers did not exhibit the delay phase, and started growing immediately after mitosis. Furthermore, ‘grower’ cells generally grew to the same size on average in all generations except prior to their final division. While ‘non-growers’ by definition did not exhibit significant growth (as defined above), they nevertheless typically exhibited some growth.


A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide.

Shokhirev MN, Almaden J, Davis-Turak J, Birnbaum HA, Russell TM, Vargas JA, Hoffmann A - Mol. Syst. Biol. (2015)

Time-lapse microscopy reveals two distinct generation-dependent growth patterns for B cellsA Overview of the time-lapse microscopy experimental and analysis pipeline. B cells were purified from mouse spleen, stimulated with TLR9 agonist CpG, imaged on an environmentally controlled microscope for 6 days, and tracked using a semi-automated tracking tool to quantify generation-dependent cell statistics.B Generational cell counts relative to initial count.C The observed fraction of cells dividing or dying in each generation. Error bars = 1/n, where n = 85, 55, 93, 103, 125, 90, 45 for generations 0–6, respectively.D Growth trajectories of generation 0 cells that grew by more than 350 μm3 or ended with a volume of at least 800 μm3 (blue) and trajectories of generation 0 cells that did not end with a large volume (black).E, F Cell size trajectories as a function of % lifetime for growers (E) and non-growers (F) in each generation (colors as in B). Error bars are SD, with n = 34, 44, 60, 54, 40, 15, 2 growing cells, 51, 11, 33, 49, 85, 75, 43 non-growing cells in generations 0–6, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358656&req=5

fig02: Time-lapse microscopy reveals two distinct generation-dependent growth patterns for B cellsA Overview of the time-lapse microscopy experimental and analysis pipeline. B cells were purified from mouse spleen, stimulated with TLR9 agonist CpG, imaged on an environmentally controlled microscope for 6 days, and tracked using a semi-automated tracking tool to quantify generation-dependent cell statistics.B Generational cell counts relative to initial count.C The observed fraction of cells dividing or dying in each generation. Error bars = 1/n, where n = 85, 55, 93, 103, 125, 90, 45 for generations 0–6, respectively.D Growth trajectories of generation 0 cells that grew by more than 350 μm3 or ended with a volume of at least 800 μm3 (blue) and trajectories of generation 0 cells that did not end with a large volume (black).E, F Cell size trajectories as a function of % lifetime for growers (E) and non-growers (F) in each generation (colors as in B). Error bars are SD, with n = 34, 44, 60, 54, 40, 15, 2 growing cells, 51, 11, 33, 49, 85, 75, 43 non-growing cells in generations 0–6, respectively.
Mentions: In order to obtain cell lineage information that accounts for the population response, we tracked 1,295 live primary B cells using a time-lapse microscopy pipeline (Fig2A). We developed a semi-automated image analysis method, combining the advantages of computational automation and human input to minimize errors (see Materials and Methods). Analysis of wild-type B cells responding to high CpG stimulation confirmed the expected population expansion followed by a contraction period (Fig2B). After cells that died from mechanical death in the initial phase (Hawkins et al, 2007a) were filtered out (Supplementary Fig S1A and B), we found that approximately 38% of the starting, ‘generation 0’, cells divided; then, 85% of generation 1 cells divided with subsequent generations, showing a steady decrease in this fraction such that only 9% of cells divided in generation 6 (Fig2C). To quantify the cellular response, we classified cell size trajectories into two categories: (i) cells that grew by at least 350 μm3 (representing at least two standard deviations on average, Supplementary Fig S1C) or reached a final size of at least 800 μm3 (based on the bimodal size distribution (Supplementary Fig S1D) and to ensure that large generation 1+ cells are included), dubbed ‘growers’ and (ii) cells that did not meet these criteria, dubbed ‘non-growers’ (Fig2D). To test the sensitivity of the growth threshold, we repeated the quantification with a 25% lower and higher growth threshold, revealing that few cells exhibited ambiguous growth (Supplementary Fig S1E). Averaging the growth trajectories of ‘growers’ (Fig2E) and ‘non-growers’ (Fig2F) in each generation normalized by percent cell lifespan revealed that progenitors (generation 0) that grew exhibited a growth delay followed by rapid growth to approximately fivefold their starting size, while generation 1+ growers did not exhibit the delay phase, and started growing immediately after mitosis. Furthermore, ‘grower’ cells generally grew to the same size on average in all generations except prior to their final division. While ‘non-growers’ by definition did not exhibit significant growth (as defined above), they nevertheless typically exhibited some growth.

Bottom Line: B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically.Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells.We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Signaling Systems Laboratory, UCSD, La Jolla, CA, USA San Diego Center for Systems Biology, UCSD, La Jolla, CA, USA Bioinformatics and Systems Biology Graduate Program, UCSD, La Jolla, CA, USA.

No MeSH data available.