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. 2013 Feb;6(2):134-143.
doi: 10.4236/jbise.2013.62017.

Spatial perturbation with synthetic protein scaffold reveals robustness of asymmetric cell division

Jiahe Li  1 , Pengcheng Bu  2 , Kai-Yuan Chen  2 , Xiling Shen  3
Affiliations

Spatial perturbation with synthetic protein scaffold reveals robustness of asymmetric cell division

Jiahe Li et al. J Biomed Sci Eng. 2013 Feb.

Abstract

Asymmetric cell division is an important mechanism for creating diversity in a cellular population. Stem cells commonly perform asymmetric division to generate both a daughter stem cell for self-renewal and a more differentiated daughter cell to populate the tissue. During asymmetric cell division, protein cell fate determinants asymmetrically localize to the opposite poles of a dividing cell to cause distinct cell fate. However, it remains unclear whether cell fate determination is robust to fluctuations and noise during this spatial allocation process. To answer this question, we engineered Caulobacter, a bacterial model for asymmetric division, to express synthetic scaffolds with modular protein interaction domains. These scaffolds perturbed the spatial distribution of the PleC-DivJ-DivK phospho-signaling network without changing their endogenous expression levels. Surprisingly, enforcing symmetrical distribution of these cell fate determinants did not result in symmetric daughter fate or any morphological defects. Further computational analysis suggested that PleC and DivJ form a robust phospho-switch that can tolerate high amount of spatial variation. This insight may shed light on the presence of similar phospho-switches in stem cell asymmetric division regulation. Overall, our study demonstrates that synthetic protein scaffolds can provide a useful tool to probe biological systems for better understanding of their operating principles.

Keywords: Asymmetric Cell Division; Caulobacter; Protein Scaffold; Synthetic Biology.

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Figures

Figure 1
Figure 1
Overview of asymmetric cell division and spatial perturbation of cell fate proteins in C. crescentus. (a) The spatial and temporal dynamics of cell fate proteins in the bacterial cell cycle. The cell cycle begins with a swarmer cell with PleC, a phosphatase localized at the flagellum pole. After entering DNA replication, S phase, the swarmer cell differentiates into a stalked cell. As cell cycle progresses from S to G2 phase, dividing cells asymmetrically segregate DivJ, a histidine kinase and PleC to stalked and swarmer cells, respectively; (b) PleC and DivJ play opposite roles in the dephosphorylation and phosphorylation of DivK, dephosphorylated DivK triggers a signaling cascade leading to the accumulation of CtrA~P which blocks transition into S phase; (c) Schematic design of co-localizing two different cell fate proteins at the same pole. Membrane-bound proteins PleC or DivJ are fused with GBD peptide and cytoplasmic DivK is tagged with SH3 peptide. Scaffold proteins expressing SH3 and GBD domains are driven by xylose-inducible promoter and therefore can be induced upon addition of xylose to culture media; (d) Four possible scenarios and corresponding phenotypic outcomes after perturbation of cell fate proteins by scaffolds.
Figure 2
Figure 2
Design of recombinant vectors. (a) Schematic design of scaffold proteins containing fluorescent protein mCherry, one, four and eight SRC Homology 3 (SH3) domains and a single guanosine triphosphatase (GTPase)-binding domain (GBD) under the control of xylose-inducible promoter; (b) Schematic design of recombinant cell fate proteins PleC, DivJ and DivK. They are fused with GBD peptide or SH3 peptide to interact with scaffold proteins. Cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) are used for fluorescent microscopy.
Figure 3
Figure 3
Engineering modular and orthogonal interaction domains for the perturbation of cell fate determinants. (a) The localization patterns of recombinant DivJ, DivK and PleC during C. crescentus division. Fluorescent microscopy showed that their cellular localization are consistent with wild-type proteins; (b) Western blot of scaffold proteins from C. crescentus lysate after induction with 0.3% xylose for 4 hours. From left to right: (1) wild-type strain, (2) strain expressing mCherry-SH3(1)-GBD, (3) mCherry-SH3(4)-GBD and (4) mCherry-SH3(8)-GBD. Star symbol indicates nonspecific band; (c) The cellular localizatio n of protein scaffolds in C. crescentus. Fluorescent microscopy indicated cytoplasmic expression of scaffold proteins.
Figure 4
Figure 4
Perturbation of spatial localization of cell fate determinants by scaffold proteins. (a) Strains carrying DivK-SH3pep-YFP, PleC-GBDpep-CFP or DivJ-GBDpep-CFP still localize properly when each is co-expressed with individual scaffolds (mCherry-SH3(1)-GBD, mCherry-SH3(4)-GBD and mCherry-SH3(8)-GBD); (b) Co-localization of PleC with DivK at both poles in the strain PB021 which expresses PleC-GBDpep-CFP, DivK-SH3pep-YFP and mcherry-SH3(8)-GBD; (c) Scanning electron microscopy (SEM) revealed that the localization of PleC to both poles did not lead to morphological change in dividing C. crescentus as compared to control strain PB018 which expresses only PleC-GBDpep-CFP and DivK-SH3pep-YFP; (d) Co-localization of DivJ with DivK at both poles in strain PB025 which expresses DivJ-GBDpep-CFP, DivK-SH3pep-YFP and mcherry-SH3(8)-GBD; (e) SEM revealed that the localization of DivJ to both poles did not lead to morphological change in dividing C. crescentus as compared to control strain PB022 which expresses only DivJ-GBDpep-CFP and DivK-SH3pep-YFP; (f) Growth rate of different genetically modified strains. The growth curve showed that the perturbation of cell fate proteins did not significantly alter growth rate. PB018: PleC-GBDpep-CFP, DivK-SH3pep-YFP; PB021: PleC-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD; PB022: DivJ-GBDpep-CFP, DivK-SH3pep-YFP; PB025: DivJ-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD. N = 3, scale bars represent SEM.
Figure 5
Figure 5
Mathematical modeling of a robust PleC/DivJ switch. (a) Steady state analysis of DivK and DivK~P. The curves show that PleC/DivJ regulation forms a sensitive switch to convert DivK and DivK~P concentration to two distinctly different stable states; (b) and (c) are heat maps of DivK~P concentration level with PleC/DivJ regulation. Crosses represent wild type of swarmer cells (lower-right) and stalked cells (upper-left). Circles shows the cells with spatial perturbations. The solid lines indicates the arbitrary threshold that separate swarmer cell and stalked cell based on the concentration level of DivK~P; (b) Represents that PleC is recruited to the stalked cell; and (c) Shows DivJ is recruited to the swarmer cell. Heatmaps show that DivK~P levels in both of stalked and swarmer cells are changed with spatial perturbations, but the cell fates are not converted without strong enough effects; (d) The ratio of fluorescent intensity of recombinant DivJ and PleC between stalked cells and swarmer cells. In strain PB021 (PleC-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD), the average ratio of mis-localized PleC in stalked cells and correctly localized PleC in swarmer cells was 0.55:1, SEM = 0.05. In strain PB025 (DivJ-GBDpep-CFP, DivK-SH3pep-YFP, mCherry-SH3(8)-GBD), the average ratio of correctly localized DivJ in stalked cells to mis-localized DivJ in swarmer cells was 2.2:1, SEM = 0.22.
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