The mitotic phase also known as M phase is a multistep process during which the duplicated chromosomes are aligned, separated, and move into two new, identical daughter cells. The first portion of the mitotic phase is called karyokinesis , or nuclear division. The second portion of the mitotic phase, called cytokinesis, is the physical separation of the cytoplasmic components into the two daughter cells.
Karyokinesis, also known as mitosis, is divided into a series of phases—prophase, metaphase, anaphase, and telophase—that result in the division of the cell Figure 2. The nucleolus disappears. The centrosomes begin to move to opposite poles of the cell. Microtubules that will form the mitotic spindle extend between the centrosomes, pushing them farther apart as the microtubule fibers lengthen. The sister chromatids begin to coil more tightly and become visible under a light microscope.
Each sister chromatid develops a protein structure called a kinetochore in the centromeric region Figure 3. The proteins of the kinetochore attract and bind mitotic spindle microtubules. During prometaphase , the nuclear envelope is fully broken down and chromosomes are attached to microtubules from both poles of the mitotic spindle, which begin to move them toward the middle of the cell.
Figure 3. Once a mitotic fiber attaches to a chromosome, the chromosome will be oriented until the kinetochores of sister chromatids face the opposite poles. Eventually, all the sister chromatids will be attached via their kinetochores to microtubules from opposing poles. During metaphase , all the chromosomes are aligned in a plane called the metaphase plate , or the equatorial plane, midway between the two poles of the cell. At this time, the chromosomes are maximally condensed.
During anaphase , the sister chromatids separate at the centromere. Each chromatid, now called a chromosome, is pulled rapidly toward the centrosome to which its microtubule is attached. The cell becomes visibly elongated oval shaped as the polar microtubules slide against each other at the metaphase plate where they overlap. The data therefore suggest the existence of a nutrient-modulated mechanism that measures growth during mitosis and delays completion of mitosis until sufficient growth has occurred.
This would explain why cells shifted from rich to poor carbon during metaphase undergo a prolonged mitotic delay, whereas cells shifted during anaphase do not Fig. If the delay were a consequence of a reduction in ATP or other metabolites needed for mitotic spindle events, one would expect to see delays in both metaphase and anaphase.
Rather, we suggest that a shift to poor carbon during metaphase causes a delay because the daughter bud has not yet undergone sufficient growth, whereas a shift in anaphase does not cause a delay because buds have already reached the threshold amount of growth needed to complete mitosis in poor carbon. Because a large fraction of total growth occurs in mitosis, it would make sense that mechanisms that control the extent of growth in mitosis play a significant role in cell size control.
The existence of major cell size control mechanisms in mitosis would explain why cells lacking critical regulators of the G1 size checkpoint still show robust nutrient modulation of cell size Jorgensen et al. Work in fission yeast has suggested that there are mitotic cell size control mechanisms that act independently of Cdk1 inhibitory phosphorylation, which could explain why loss of Cdk1 inhibitory phosphorylation in budding yeast causes only modest effects on cell size Wood and Nurse, An alternative model is that the duration of mitosis is controlled by a nutrient-modulated timer.
In rich medium, the timer would be set for a short duration of growth, whereas in poor medium it would be set for a longer interval. However, comparison of the data from synchronous and asynchronous cells would appear to rule out a nutrient-modulated timer model.
Synchronous cells spend a total of 51 min in metaphase and anaphase in poor carbon, whereas asynchronous cells spend 61 min, despite growing under identical nutrient conditions. The difference is most likely a result of slower growth rates in asynchronous cells see Figs. A timer model is also not consistent with the large variance in mitotic duration observed between individual cells growing under identical conditions Fig.
In both synchronous and asynchronous cells, the mean size of mother cells initiating bud emergence was only slightly smaller in poor carbon compared with rich carbon, and the difference was not statistically significant Fig. In addition, asynchronous daughter cells completed G1 phase at nearly identical sizes in rich and poor carbon Fig. This may at first seem paradoxical, because poor carbon reduces mean size nearly twofold. However, cells in poor carbon grow slowly in mitosis, complete mitosis at a reduced cell size, and spend more time in G1 phase.
As a result, they spend more time at small sizes compared with cells in rich carbon, which contributes to a smaller mean size when population averages are measured with a Coulter counter.
Previous studies suggested that growth rate changes during the cell cycle, but did not include analysis of growth during specific stages of mitosis in unperturbed single cells Goranov et al.
To extend these studies, we calculated mean growth rates at each stage of the cell cycle in rich and poor carbon Fig. When the bud first emerges, growth is relatively slow.
Entry into mitosis initiates a fast growing phase that lasts nearly the entire length of mitosis. As cells complete anaphase, the growth rate slows. A slow rate of growth persists during G1 phase. Poor carbon reduced the rate of growth in mitosis by half but caused smaller reductions in growth rate during other stages of the growth cycle. The rate of bud growth was greater in synchronized cells, most likely because of increased mother cell size Schmoller et al.
Previous studies have shown that polar bud growth is driven by Cdk1 activity; however, the signals that control bud growth at other stages of the cell cycle are unknown McCusker et al.
Moreover, the mechanisms and function of growth rate modulation during the cell cycle are unknown. The discovery that most growth in volume occurs during a rapid growth phase in mitosis provides more evidence that cell size homeostasis requires tight control over the interval of mitotic growth.
Differences in cell growth and size between synchronous and asynchronous cells point to a strong influence of mother cell size on growth rate and daughter cell size. For example, synchronized cells initiate bud growth at a larger mother cell size compared with unsynchronized cells, and their daughter buds grow faster and complete mitosis at a larger size Figs.
Previous studies observed a similar correlation between mother cell size and daughter cell size Johnston et al. These observations raised the possibility that the difference in daughter cell size at completion of mitosis in rich and poor carbon could be caused by differences in mother cell size as a result of nutrient modulation of cell size in G1 phase.
To further analyze the effects of mother cell size, we plotted the relationship between mother cell size and growth rate of the daughter bud in mitosis. Growth rate was positively correlated with mother cell size in both rich and poor carbon Fig. Thus, daughters of large mothers grew faster than daughters of small mothers, consistent with the idea that mother cell size influences biosynthetic capacity.
However, carbon source had a stronger influence on growth rate than mother cell size. This can be seen by the fact that mothers of similar size in rich and poor carbon had daughter buds that grew at different rates, which was true across the entire range of mother cell sizes. We also plotted daughter cell size at completion of cytokinesis versus mother cell size Fig. Daughter cell size was positively correlated with mother cell size in both conditions.
However, the influence of carbon source was again much stronger. Mother cells growing in poor carbon that were the same size as mother cells in rich carbon consistently produced much smaller daughter cells. Together, these data indicate that effects of carbon source on daughter cell size cannot be due solely to differences in mother cell size.
The effects of mother cell size could explain why synchronized cells in rich carbon completed late G1 phase at a larger size than their counterparts in poor carbon Fig. Note that synchronized cells in both rich and poor carbon appear to overshoot the size at which asynchronous cells complete late G1 phase, which could be caused by increased mother cell size in the synchronized cells. The large mothers in synchronized cells in rich carbon drive a high rate of growth, which could lead to greater overshooting of the threshold amount of growth required for G1 progression.
Asynchronous cells in both rich and poor carbon have smaller mother cells and are born at smaller sizes relative to synchronized cells. In this case, compensatory growth in G1 appears to become more important to bring the daughter cell up to a minimal threshold size before cell cycle entry.
Previous studies found that cell size at the end of G1 phase is correlated with growth rate during G1 phase Johnston et al. The correlation holds true when comparing cells growing in the same carbon source and when comparing cells growing in different carbon sources.
To determine whether a similar relationship exists for growth during mitosis, we plotted daughter cell size at cytokinesis as a function of daughter bud growth rate during mitosis for cells growing in rich or poor carbon Fig. Daughter cell size was positively correlated with growth rate under both conditions. Thus, cell size at all key cell cycle transitions is correlated with growth rate. Because cell size is proportional to growth rate, faster growing cells should always give rise to larger daughter cells.
Moreover, because growth rate is proportional to size, larger mother cells should have a higher growth rate, leading to ever larger daughter cells.
In this case, what limits cell size? The plot of daughter bud size at cytokinesis as a function of mother cell size revealed that daughter cell size indeed increases with mother cell size, but the ratio of mother size to daughter size is not constant across the range of mother cell sizes Fig.
In other words, small mothers produce daughters of nearly equal size, whereas very large mothers produce daughters that are nearly half the size of the mother Johnston et al. This relationship would correct large variations in mother cell size, which could be the result of growth during cell cycle delays induced by other checkpoints, such as the spindle checkpoint or DNA damage checkpoints.
The strong correlation between growth rate and cell size is difficult to reconcile with simple cell size checkpoint models in which a threshold volume must be reached to pass the checkpoint. If a specific volume must be reached to pass a checkpoint, the rate at which the cell reaches that volume should not influence the final volume at which the cell passes the checkpoint.
One way to reconcile the idea of a set threshold volume with growth rate dependence would be to imagine that cell size checkpoint thresholds are noisy and imperfect.
In this view, faster growing cells will overshoot the threshold size more than slow growing cells, leading to increased size. However, this model would not explain nutrient modulation of cell size.
Thus, another model could be that cells measure their growth rate and set cell size thresholds to match growth rate Jorgensen et al. Alternatively, the same signals that set the growth rate could also set the cell size threshold. Most of the differences in cell cycle duration between species and cells are found in the duration of specific cell cycle phases.
DNA replication, for example, generally proceeds faster the simpler the organisms. One reason for this trend is simply that prokaryotes have smaller genomes and not as much DNA to be replicated. Across species and organismal complexity, embryonic cells have an increased need for rapidity in the cell cycle because they need to multiply for the development of the embryo. Early embryonic cell cycles often omit G1 and G2 and quickly proceed through successive rounds of S phase and mitosis.
For these cells, the main concern is not the regulation of the cell cycle which occurs largely in G1 and G2 , but rather in the speed of cell proliferation. In this section, we will discuss the breakdown of the durations of mitosis, G1, S phase, and G2 for the general 24 hour cell cycle found in most cells.
As we discussed in the previous section, the lengths of G1 and G2 vary in cells based on the individual cell's level of preparedness for proceeding in the cell cycle.
Cell cycles are interesting both for the ways they are similar from one cell type to the next and for the ways they are different. To bring the subject in relief, we consider the cell cycles in a variety of different organisms including a model prokaryote, for mammalian cells in tissue culture and during embryonic development in the fruit fly. Specifically, we ask what are the individual steps that are undertaken for one cell to divide into two and how long do these steps take?
Figure 1: The min cell cycle of Caulobacter is shown, highlighting some of the key morphological and metabolic events that take place during cell division.
M phase is not indicated because in Caulobacter there is no true mitotic apparatus that gets assembled as in eukaryotes. Much of chromosome segregation in Caulobacter and other bacteria occurs concomitantly with DNA replication.
The final steps of chromosome segregation and especially decatenation of the two circular chromosomes occurs during G2 phase. Adapted from M. Laub et al. Arguably the best-characterized prokaryotic cell cycle is that of the model organism Caulobacter crescentus.
One of the appealing features of this bacterium is that it has an asymmetric cell division that enables researchers to bind one of the two progeny to a microscope cover slip while the other daughter drifts away enabling further study without obstructions.
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