Live imaging of theDictyosteliumcell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

Abstract

The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.

DNA double-strand break repair pathway choice inDictyostelium

“…Until recently, DNA-PKcs was thought to be conserved only in vertebrates. However, we and others identified core components of the NHEJ pathway in the amoeba Dictyostelium, including a functional orthologue of DNA-PKcs (Block and LeesMiller, 2005;Hsu et al, 2006;Hudson et al, 2005;Muramoto and Chubb, 2008). Other DNA repair factors absent in conventional invertebrate model organisms used to study DNA repair are also conserved in Dictyostelium, including components of the Fanconi anaemia (Zhang et al, 2009) and single-strand break repair pathways (Rajawat et al, 2007).…”

“…By contrast, dnapkcs is not required for cells to phosphorylate H2AX or tolerate DSBs during vegetative cell growth, suggesting that other repair pathways predominate, at least as regards cell viability following DNA damage (Hudson et al, 2005). However, Ku and DNA-PKcs have recently been implicated in recovery from DSBs and progression of vegetative Dictyostelium through mitosis, suggesting that NHEJ does function in some respect at this stage of the life cycle (Block and Lees-Miller, 2005;Hsu et al, 2006;Hudson et al, 2005;Muramoto and Chubb, 2008). Here, we address these questions by further characterising the relative contributions of HR and NHEJ to DSB repair in vegetative Dictyostelium.…”

Digital nature of the immediate-early transcriptional response

“…S2A in the supplementary material). Nor was there an obvious cell cycle bias in responding cells, as assessed using the marker RFP-PCNA (Muramoto and Chubb, 2008). At 10 hours of development, all cells had the diffuse nuclear PCNA distribution of G2.…”

“…Cells were viewed on a previously described inverted fluorescence microscope using protocols optimised for long-term high-resolution 3D imaging of photosensitive samples (Muramoto and Chubb, 2008). Three dimensional stacks (43 slices) were captured at multiple positions every 3.5 minutes with 250 nm z-steps and 50 msecond exposures.…”

Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity

“…In mESCs, treatments prolonging cell cycles do not perceptibly alter the expression of pluripotency genes such as Nanog (Li et al, 2012;Li and Kirschner, 2014). However, although early embryonic cell cycles can be highly synchronous, many eukaryotic cycles are highly heterogeneous (Brooks, 1981;Di Talia et al, 2007;Muramoto and Chubb, 2008) and, with different signalling associated with different cycle stages, cycle variability potentially provides a driver of gene expression heterogeneity. The heterogeneity of the ESC cycle has not been determined.…”