Visualizing the metazoan proliferation-terminal differentiation decisionin vivo

Abstract

During organismal development, differential regulation of the cell cycle is critical to many cell biological processes, including cell fate specification and differentiation. While the mechanisms of cell cycle regulation are well studied, how control of the cell cycle is linked to differentiated cellular behavior remains poorly understood, mostly due to our inability to directly and precisely measure cell cycle state. In order to characterize cell cycle state live, we adapted a cyclindependent kinase (CDK) biosensor for in vivo use in the roundworm nematode, Caenorhabditis elegans. The CDK biosensor measures the cytoplasmic-to-nuclear localization of a portion of human DNA Helicase B (DHB) fused to a fluorescent protein to assess cell cycle state. The dynamic localization of DHB results from phosphorylation of the biosensor by CDKs, thereby allowing for quantitative assessment of cell cycle state. We demonstrate here the use of this biosensor to quantify lineage-specific differences between cycling cells and to examine the proliferation-differentiation decision. Unlike other live cell imaging tools (e.g., FUCCI), we show that DHB can be used to distinguish between actively cycling cells in the G1 phase of the cell cycle and terminally differentiated cells exited in G0. Thus, we provide here a new resource to study the control and timing of the metazoan cell cycle during cell fate specification and differentiation.

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasionin vivo

“…Interestingly, 27.9% (12/43) of animals treated with swsn-4(RNAi) were absent of SMs on either the left or the right side, whereas 100% (30/30) control animals had SMs on both sides, which may indicate a defect in either specification or migration of SMs ( Fig S5 B ). We then employed a cyclin-dependent kinase (CDK) sensor, which uses a fragment of mammalian DNA Helicase B (DHB) fused to two copies of mKate2 (35, 67), to quantify cell cycle state. In cells with low CDK activity that are quiescent or post-mitotic, the ratiometric CDK sensor is strongly nuclear localized (35,66,67).…”

“…We then employed a cyclin-dependent kinase (CDK) sensor, which uses a fragment of mammalian DNA Helicase B (DHB) fused to two copies of mKate2 (35, 67), to quantify cell cycle state. In cells with low CDK activity that are quiescent or post-mitotic, the ratiometric CDK sensor is strongly nuclear localized (35,66,67). In cycling cells with increasing CDK activity, the CDK sensor progressively translocates from the nucleus to the cytosoplasm, with a ratio approaching 1.0 in S phase and >1 in cells in G 2 (35).…”

“…This finding parallels similar studies in cancer (30–33) and C. elegans mesoblast development (34). Examination using a DNA Helicase B (DHB)-based CDK activity sensor (35) revealed assembly-specific contributions to AC invasion: whereas BAF promotes AC invasion in a cell cycle-dependent manner, PBAF contributes to invasion in a cell cycle-independent manner. Finally, we utilized the auxin-inducible degron (AID) system combined with PBAF RNAi to achieve strong PBAF subunit depletion in the AC, which resulted in loss of both AC invasion and adhesion to the BM.…”

“…We generated two anti-GFP nanobody constructs, using conserved cis-regulatory elements from the cdh-3 and egl-43 promoters (22,24,38,65,66) and introduced them to a strain containing an endogenous GFPtagged allele of swsn-4 as well as background AC and BM reporters (Fig 3 (35,67), to quantify cell cycle state. In cells with low CDK activity that are quiescent or post-mitotic, the ratiometric CDK sensor is strongly nuclear localized (35,66,67). In cycling cells with increasing CDK activity, the CDK sensor progressively translocates from the nucleus to the cytosoplasm, with a ratio approaching 1.0 in S phase and >1 in cells in G 2 (35).…”

“…In cells with low CDK activity that are quiescent or post-mitotic, the ratiometric CDK sensor is strongly nuclear localized (35,66,67). In cycling cells with increasing CDK activity, the CDK sensor progressively translocates from the nucleus to the cytosoplasm, with a ratio approaching 1.0 in S phase and >1 in cells in G 2 (35). Altogether, our data demonstrated that in the AC, the SWSN-4 core ATPase of the SWI/SNF complex contributes to invasion in a dose-dependent manner: moderate loss of expression produced non-invasive single ACs while extreme loss of expression led to a non-invasive hyperproliferative state.…”