Cao, L. Mechanism of the formation of contractile ring in dividing cultured animal cells. Recruitment of preexisting actin filaments into the cleavage furrow. Fukui, Y. Myosin II-independent F-actin flow contributes to cell locomotion in Dictyostelium. F-actin ring formation and the role of F-actin cables in the fission yeast Schizosaccharomyces pombe. Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division.
Winter, D. Natl Acad. USA 96 , — The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin. EMBO J. Theriot, J.
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The polymerization motor. Traffic 1 , 19—28 Sagot, I. Yeast formins regulate cell polarity by controlling the assembly of actin cables. Evangelista, M. Noguchi, T. Reorganization of actin cytoskeleton at the growing end of the cleavage furrow of Xenopus egg during cytokinesis. Cytokinesis in fission yeast Schizosaccharomyces pombe. Methods Enzymol. Download references. We thank K. Burgess, B. Feierbach, D. McCollum, A. Paoletti, R. Kessin and B. Goode for comments on the manuscript; T.
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This work was supported by research grants to F. Correspondence to Fred Chang. Reprints and Permissions. Molecular Biology of the Cell Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology We find that the lack of increase in light scattering with cell length was independent of whether excitation was performed at , , , or nm data not shown. The blocking bar, employed in all flow cytometers to exclude direct laser exposure and reflection into the detectors, also excludes light scattered below a certain critical angle.
It is likely that, as the cells grow longer, the increased light scattering due to the increased long axis of the cells will fall below the critical angle and will thus not contribute significantly to the measured scattering signal.
Actin dynamics in the contractile ring during cytokinesis in fission yeast
Similar analyses were performed on cells arrested in G 1 phase by incubating a population of a cdc10ts strain at the restrictive temperature. The cells in such a culture formed three main subpopulations Figure 3A. The vast majority of the cells were found in this population and could constitute the basis for synchronized release into the cell cycle from G 1 phase. Subpopulation 6 consisted almost exclusively of cell doublets of two cells in G 1 phase Figure 3G.
Reliable cell cycle commitment in budding yeast is ensured by signal integration | eLife
This characterizes cells in late mitosis or G 1 phase and this analysis identified cells that had not gone through cytokinesis during the temperature shift. This subpopulation is equivalent to Subpopulation 2 in the analysis of exponentially growing cells Figure 2A. Moreover, the method can be used to monitor the quality of a method of synchronization, since the subpopulations could be identified. Flow cytometric and microscopic analyses of S. The three major subpopulations are indicated in panel A and they were analyzed individually by sorting, as shown in panels C, F and I.
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SSC cytogram is necessary to obtain reliable DNA histograms of single cells and therefore to arrive at the correct cell-cycle distribution. Whereas the presence of cells with 4C DNA content in the ungated histogram panel C could suggest the presence of cells in G 2 phase that had not yet passed through cytokinesis, gating on light scattering revealed that these cells were in fact cell doublets panel E. The true DNA content distributions of mononuclear and binuclear cells were obtained after additional gating with low and high DNA-W, respectively, as described above.
After renormalization according to the percentage of cells with low and high DNA-W, the fractions of cells in the different compartments were obtained Table 1. From these fractions, and the cell cycle time, it is possible to calculate the length of the G 2 , G 1 and S phases. Table 1 shows that the population of cells with 2C DNA content in the G 1 - arrested culture contain cells in G 1 phase that have not divided, but also a small fraction of cells in G 2 phase. Flow cytometric analyses of a culture of exponentially growing S. The gates are shown in panels A and F.
The ability to separate mononuclear from binuclear cells was exploited to monitor, by flow cytometry, the passage of fission yeast cells through mitosis. Here we have employed flow cytometry to measure the frequency of binuclear cells the binuclear index, BI and compared the BI, the MI and the SI for one and the same cell culture synchronized in G 2 phase and released into the cell cycle.
After applying the gate to eliminate cell doublets Figure 5A we identified binuclear cells Figure 5B , i. We conclude that flow cytometric measurement of the BI is a convenient and simple method to estimate the time of mitotic entry. Cell-cycle kinetics of a culture arrested in G 2 phase and released into the cell cycle for 60 min.
Cell doublets were eliminated by employing the gate shown in panel A, resulting in the DNA cytogram shown in panel B. Indicated are the positions of the binuclear cells and of cells in S phase. Results are shown for one representative experiment where all parameters were measured on the very same cell sample. Previous approaches to monitor entry into S phase include the measurement of the SI or of incorporation of externally supplied bromodeoxyuridine BrdU  , .
The first method may be misleading, since there is no obligatory link between septum formation and S-phase entry and in many cases septation can finish well before S phase has started . The second approach is tedious and requires extensive strain constructions because fission yeast cells do not take up BrdU and do not express thymidine kinase, which is required for BrdU incorporation into DNA.
Here we have measured the frequency of S-phase cells by flow cytometry and found that this identification is easy and straightforward in a two-parametric analysis Figure 5 A, B. For comparison, we have measured, in the same culture, the appearance of cells that contain Mcms bound to chromatin, an obligatory event before S-phase entry . Thus, all five parameters show a timing of increase consistent with previous reports and we conclude that flow cytometry can be efficiently used to monitor mitotic entry in fission yeast cells.
Furthermore, flow cytometry allows, in contrast to analyses by microscopy, the analysis of a large number of cells in a short time. This work presents major advances in the analysis of fission yeast cells by flow cytometry.
First, we have presented a method allowing the separation of binuclear from mononuclear cells. Second, another method presented facilitates the elimination of cell doublets; such doublets are formed at a significant frequency when the cells are fixed. These two methods will greatly improve single-cell analyses of fission yeast by flow cytometry and thereby allow a much more detailed, exact and sophisticated cell-cycle analysis. Furthermore, this application opens up novel possibilities to screen for fission yeast mutants with deficiencies in cell-cycle regulation, i.
Buy Softcover. FAQ Policy. About this book This detailed book collects the main methodologies used for the analysis of the activity, localization, and regulation of the components of the Mitotic Exit Network MEN pathway during mitotic exit in Saccharomyces cerevisiae , as well as for the evaluation of the roles of these proteins in other cellular processes, such as the condensation of the rDNA, the functionality of the mitotic checkpoints, and cytokinesis. Show all. Show next xx.
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