Presentation Information
[2ASPR-11]Quantitative analysis of cell cycle regulators governing differential cell cycle speed in stem cells versus transit-amplifying cells in mouse small intestinal epithelium
○Shuji Matsuguchi1 (1. RIKEN BDR (Japan))
Keywords:
mouse intestine,cell cycle,multiplexed imaging,high-sensitivity reporer
[Purpose]
Previous studies have revealed that stem cells and transit-amplifying (TA) cells have differential cell cycle speed in small intestinal epithelium; cell cycle duration is ~24 hours in stem cells, while 12~18 hours in TA cells. It has been long believed that stem cells maintain genomic stability by slower cycling, while TA cells contribute to producing a number of differentiated cells by faster cycling. However, the molecular mechanism underlying this differential cell cycle speed is largely unknown. In general, the total cell cycle duration is determined by G1 phase, which has the biggest variability among cells. Thus, we first focused on G1 phase, where cell cycle regulators including G1 cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors (CKIs), and Rb protein function to control the G1/S transition. In this study, we aim to quantitatively analyze these key cell cycle regulators to understand the molecular mechanisms underlying the differential cell cycle speed in stem cells and TA cells in mouse small intestinal epithelium.
[Methods]
To quantitatively measure cell cycle regulators in mouse small intestine, we developed a protocol to perform multiplexed immunohistochemistry for cryo-sectioned tissues, termed Cryo-4i (Matsuguchi, et al., STAR Protocols, in press). This iterative staining technique allows us to analyze multiple protein targets from same fixed sample at the single cell level while maintaining tissue architecture. In addition, to obtain the temporal dynamics of cell cycle regulators, we used CRISPR-Cas9 system to tag endogenous CKI with a fluorescent protein in cultured cells, enabling live-cell imaging of the reporter cells.
[Results]
We first found that differential expression patterns of G1 cyclins, CKIs, and Rb phospho-isoforms in mouse intestinal epithelium in vivo. Furthermore, using Cryo-4i, we were able to measure the expression balance between G1 cyclins and CKIs to determine Rb phosphorylation. Interestingly, p27 is highly expressed at the bottom of the crypt including stem cells but is downregulated at the TA zone. Therefore, to reveal the temporal dynamics of p27, we introduced a GFP tag into the C-terminus CDKN1B (gene encoding p27). Caco-2 cell line with the endogenous tagging of p27 allowed us to precisely measure the p27 expression levels in live cells at the single cell level.
[Consideration]
Our in vivo data suggests that differential expression patterns of G1 cyclins, CKIs, and Rb phospho-isoforms account for the slower cell cycle in stem cells and faster cell cycle in TA cells. In particular, our results indicate that the balance between cyclins and CKIs determine Rb phosphorylation status and thus the cell cycle speed in mouse small intestinal epithelium.
[Conclusion]
In this study, we successfully established a protocol for multiplexed immunohistochemistry for cryo-sectioned tissues, termed Cryo-4i. Cryo-4i enables analysis of multiple protein targets in the same tissues at the single cell level, leading to our hypothesis that the balance between cyclins and cyclin-dependent kinase inhibitors (CKIs) determine Rb phosphorylation status and thus the cell cycle speed in vivo tissues. Moreover, using the CRISPR-Cas9 based tagging system, we were able to visualize the endogenous p27 expression levels with a fluorescent protein in live cells.
Previous studies have revealed that stem cells and transit-amplifying (TA) cells have differential cell cycle speed in small intestinal epithelium; cell cycle duration is ~24 hours in stem cells, while 12~18 hours in TA cells. It has been long believed that stem cells maintain genomic stability by slower cycling, while TA cells contribute to producing a number of differentiated cells by faster cycling. However, the molecular mechanism underlying this differential cell cycle speed is largely unknown. In general, the total cell cycle duration is determined by G1 phase, which has the biggest variability among cells. Thus, we first focused on G1 phase, where cell cycle regulators including G1 cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors (CKIs), and Rb protein function to control the G1/S transition. In this study, we aim to quantitatively analyze these key cell cycle regulators to understand the molecular mechanisms underlying the differential cell cycle speed in stem cells and TA cells in mouse small intestinal epithelium.
[Methods]
To quantitatively measure cell cycle regulators in mouse small intestine, we developed a protocol to perform multiplexed immunohistochemistry for cryo-sectioned tissues, termed Cryo-4i (Matsuguchi, et al., STAR Protocols, in press). This iterative staining technique allows us to analyze multiple protein targets from same fixed sample at the single cell level while maintaining tissue architecture. In addition, to obtain the temporal dynamics of cell cycle regulators, we used CRISPR-Cas9 system to tag endogenous CKI with a fluorescent protein in cultured cells, enabling live-cell imaging of the reporter cells.
[Results]
We first found that differential expression patterns of G1 cyclins, CKIs, and Rb phospho-isoforms in mouse intestinal epithelium in vivo. Furthermore, using Cryo-4i, we were able to measure the expression balance between G1 cyclins and CKIs to determine Rb phosphorylation. Interestingly, p27 is highly expressed at the bottom of the crypt including stem cells but is downregulated at the TA zone. Therefore, to reveal the temporal dynamics of p27, we introduced a GFP tag into the C-terminus CDKN1B (gene encoding p27). Caco-2 cell line with the endogenous tagging of p27 allowed us to precisely measure the p27 expression levels in live cells at the single cell level.
[Consideration]
Our in vivo data suggests that differential expression patterns of G1 cyclins, CKIs, and Rb phospho-isoforms account for the slower cell cycle in stem cells and faster cell cycle in TA cells. In particular, our results indicate that the balance between cyclins and CKIs determine Rb phosphorylation status and thus the cell cycle speed in mouse small intestinal epithelium.
[Conclusion]
In this study, we successfully established a protocol for multiplexed immunohistochemistry for cryo-sectioned tissues, termed Cryo-4i. Cryo-4i enables analysis of multiple protein targets in the same tissues at the single cell level, leading to our hypothesis that the balance between cyclins and cyclin-dependent kinase inhibitors (CKIs) determine Rb phosphorylation status and thus the cell cycle speed in vivo tissues. Moreover, using the CRISPR-Cas9 based tagging system, we were able to visualize the endogenous p27 expression levels with a fluorescent protein in live cells.
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