The number of cytokinesis nodes in mitotic fission yeast scales with cell size

Two challenges complicate counting cytokinesis nodes in fission yeast. First, cytokinesis nodes form over 10 min, and after the SPB divides, they condense into a densely packed contractile ring over 10–12 min. Second, the protein composition of cytokinesis nodes changes dramatically during these 22 min (Bhatia et al., 2014; Lee and Wu, 2012; Pan et al., 2014; Rincon et al., 2014). The numbers per node of formin Cdc12, F-BAR Cdc15, anillin Mid1, myosin-II Myo2, and IQGAP Rng2 change rapidly as cytokinesis nodes form from interphase nodes (10 min prior to SPB separation), condense into contractile rings (10–12 min after SPBs separate) and then mature (over the next 15 min) (Wu and Pollard, 2005).

We solved the first issue by studying live cells with SPBs marked with Sad1-RFP. We selected cells with two narrowly separated SPBs and well-dispersed nodes in a broad band around the equator.

We dealt with the second issue by marking nodes with the scaffold protein Blt1 tagged with mEGFP. Blt1 is the only protein known to persist in nodes throughout the cell cycle, so it is found in interphase nodes, cytokinesis nodes, and contractile rings (Akamatsu et al., 2014; Goss et al., 2014; Moseley et al., 2009). Therefore, Blt1 is the only reasonable marker to measure the number of cytokinesis nodes. The fluorescent proteins were integrated into the haploid genome to ensure that all copies of each protein were tagged and expressed at normal levels.

We used a Zeiss Airyscan fluorescence microscope to acquire 40 Z-slices covering the entire volume of cells to measure the total and local fluorescence of Blt1-mEGFP in wild-type fission yeast cells in 2–3 s. The frame size and pixel dwell time were adjusted so that motions of cytokinesis nodes (20 nm/s; Vavylonis et al., 2008) did not blur the images during image acquisition. Cytokinesis nodes appeared as a broad band of fluorescence spots in the cortex around the middle of mitotic cells with two SPBs. The fluorescence intensities of these spots ranged from very bright to dim (Figure 1A, upper panel).

Figure 1

Download asset Open asset

To compare the high resolution and sensitivity of Airyscan to a conventional laser scanning confocal microscope, we used the same Airyscan detector in the confocal mode with a pinhole of 0.6 AU, 8× line averaging + linear constrained iterative deconvolution, and 2× laser intensity to compensate for the lower signal-to-noise ratio as assessed previously (Kolossov et al., 2018). Large nodes stood out in the confocal images, but the higher background noise made it difficult to detect the dim cytokinesis nodes in broad bands. Contractile rings were brighter and narrower in Airyscan than confocal images (Figure 1A, C, lower panels). Fast Fourier transform analysis of images of cells with contractile rings showed that Airyscan retrieved higher frequencies and more extensively distributed information. The horizontal line in the frequency domain of the Airyscan image corresponds to the contractile ring (Figure 1B), which was weak in the frequency domain of the confocal image (Figure 1D). In addition to providing data for 3D reconstructions, Airyscan imaging can resolve small, dim cytokinesis nodes, which we interpret as single nodes, while the brighter nodes consist of two or more single nodes (Akamatsu et al., 2017; Laplante et al., 2016) located too closely to be resolved.

Time-lapse imaging showed that cytokinesis nodes of all sizes, including single nodes, condensed into bigger and brighter nodes as they coalesced into a contractile ring during 10–12 min after separation of the SPBs as described for the brighter fraction of the population of nodes by confocal microscopy (Vavylonis et al., 2008). Neither the Airyscan nor confocal microscopy resolved cytokinesis nodes known to be present in contractile rings (Figure 1A, C, lower panels) by single-molecule localization microscopy (Laplante et al., 2016).

The methods section explains how we used the three-dimensional distribution of fluorescence of spots around the equator to identify nodes (Figure 2A) and measure their fluorescence. To qualify as a single node, the fluorescence had to be confined in the X–Y plane within a circle of 12-pixels (0.5 µm) in diameter and in the Z direction in more than 3 and less than 7 Z-slices of 170 nm (1.19 µm). The total fluorescence within the 12-pixel diameter circle in 7 Z-slice images was sum projected and measured as a node fluorescence (Figure 2B). From the same sum projected image we calculated the average fluorescence from 8 equivalent volumes of cytoplasm on both sides of the node broad band (Figure 2B). The fluorescence of each candidate node was its total fluorescence minus the average fluorescence of an equivalent volume (~0.9 µm³) of cytoplasm. We used 3D reconstructions of stacks of Airyscan images to measure the total fluorescence of all the cytokinesis nodes around the equator of each cell to compare with the average fluorescence of single nodes.

Figure 2

Download asset Open asset

Figure 2—source data 1

Related to Figure 2D, E.

https://cdn.elifesciences.org/articles/76249/elife-76249-fig2-data1-v1.xlsx

Download elife-76249-fig2-data1-v1.xlsx

We measured the fluorescence of 235 nodes in 21 mitotic, wild-type cells satisfying the aforementioned ROI conditions and plotted a histogram to determine the distribution of intensities and the average fluorescence intensity (Figure 2D). Given the small number of molecules in each node (Akamatsu et al., 2017; Laplante et al., 2016), the exchange of molecules between nodes and cytoplasm (Laporte et al., 2011 ) and the fact that less than 70% of mEGFP is mature and emitting photons (Balleza et al., 2018), a range of intensities is expected, which does not detract from the usefulness of an average node intensity for calculating the total number of nodes from the total fluorescence intensity of the broad band of nodes. An initial Gaussian fit peaked at 2486 AU with a few higher intensity nodes. We assumed that the sample included a few closely spaced pairs of nodes, so we eliminated nodes with fluorescence values more than twice the peak of the Gaussian curve (2% of the total), before calculating the mean fluorescence of the single nodes. For the sample of 231 single nodes, the Shapiro–Wilk W test calculated W = 0.9842, which does not reject the null hypothesis that the data are from a normal distribution (King and Eckersley, 2019; Royston, 1992). A descriptive statistical test also gave a Z score of 1.7, which also does not reject the null hypothesis that the data are from a normal distribution (Ghasemi and Zahediasl, 2012).

We estimated that wild-type fission yeast cells have on average ~190 (standard deviation [SD] = 40) single cytokinesis nodes from the ratio of total Blt1-mEGFP fluorescence intensity in the broad band (corrected for cytoplasmic background) of a given cell to the average fluorescence intensity of Blt1-mEGFP in a single node, 2300 ± 900 AU (Figure 2D).

To determine if all the nodes in the broad band combine to form a contractile ring in wild-type cells, we measured the total Blt1-mEGFP fluorescence of contractile rings (after background subtraction) and of the broad band of nodes (after background subtraction) (Figure 2E). The average total fluorescence intensities are 440,000 ± 95,000 AU in broad bands and 372,000 ± 111,000 AU in contractile rings. An unpaired t-test showed that these numbers are significantly different with p = 0.035. Thus, about 85% of the Blt1 in nodes is incorporated into contractile rings.

We used five different mutant strains to determine the numbers of cytokinesis nodes in cells with a range of sizes. These mitotic cells had volumes from 50 to 450 µm³ (Figure 3).

Figure 3 with 2 supplements see all

Download asset Open asset

Figure 3—source data 1

Related to Figure 3G-K.

https://cdn.elifesciences.org/articles/76249/elife-76249-fig3-data1-v1.xlsx

Download elife-76249-fig3-data1-v1.xlsx

The Wee1 kinase catalyzes the phosphorylation of Cdc2/Cdk1 to prevent cells from entering mitosis (Russell and Nurse, 1987). At the restrictive temperature of 36°C, wee1-50 cells enter mitosis prematurely, so they are smaller than normal (Nurse, 1975). After 4 hr at the restrictive temperature, the average cell length of wee1-50 mutant cells (8.0 ± 0.9 µm) was about half the average length of wild-type cells (14.2 ± 0.8 µm) (Figure 3C). The diameter of wee1-50 mutant cells (3.8 ± 0.1 µm) was the same as wild-type cells (3.7 ± 0.1 µm). When released to the permissive temperature, most cells started to form a contractile ring in 10–15 min. The broad band of nodes looked more dispersed in these cells than in other strains.

The Cdc25 phosphatase removes the inhibitory phosphates added by Wee1 from Cdc2/Cdk1 to allow cells to enter mitosis (Nurse, 1990; Russell and Nurse, 1986; Russell and Nurse, 1987). At the restrictive temperature of 36°C, cdc25-22 mutant cells arrest at the G₂/M transition but continue growing at both ends. We varied the time at 36°C to obtain a range of lengths. After 4–6 hr at 36°C cells were 26.6 ± 5.7 µm long, twice the average length of wild-type cells but did not differ significantly in diameter (3.9 ± 0.1 µm) (Figure 3A) and had broader equatorial bands with more cytokinesis nodes (Vavylonis et al., 2008). The cdc25-22 mutant cells produced a wide range of cell sizes, so we grouped them by size. When released to the permissive temperature, most of the cells synchronously entered mitosis within 1 hr and formed a broad band of cytokinesis nodes.

The RhoGAP Rga4 localizes to the division site of cells and inactivates the GTPase Cdc42 (Das et al., 2007). Cells with deletion mutations of rga4 are shorter (11.6 ± 0.7 µm) but have larger diameters (4.4 ± 0.2 µm) than wild-type cells (Das et al., 2007; Tatebe et al., 2008; Figure 3D). Treating rga4Δ cells with 15 mM hydroxyurea for 4–6 hr inhibited DNA synthesis by inactivating ribonucleoside reductase (Krakoff et al., 1968) and arrested the cells in the S phase during which they grew longer (Mitchison and Creanor, 1971; Figure 3F). On the other hand, RhoGAP Rga2 localizes to growing cell tips and inactivates the Rho2 GTPase (Villar-Tajadura et al., 2008). Deletion of rga2 does not change the cell length but decreases the cell diameter (3.4 ± 0.2 µm) (Villar-Tajadura et al., 2008; Figure 3E).

After correcting for cytoplasmic background fluorescence, we measured the total Blt1 fluorescence in the broad band around the equator of each cell (Figure 2). Assuming that this Blt1 is located in single nodes, we estimated the numbers of single cytokinesis nodes in the broad bands by dividing the total Blt1 fluorescence around the equator by the average intensity of the single nodes in that strain. The distributions of Blt1 fluorescence intensities of single cytokinesis nodes were similar (around 2300 AU) in wild-type cells (Figure 2C) and all the experimental strains (Figure 3G).

The numbers of cytokinesis nodes in the broad band of our sample of 125 cells were strongly correlated with cell volume (Figure 3H) with a slope of about 0.7 nodes per µm³ of volume. This line does not extrapolate to the origin. Plots of the same data on node numbers versus cell length (Figure 3I) or surface area (Figure 3—figure supplement 1A) showed the same linear relationship seen for cell volume. Node numbers did not correlate with cell diameter (Figure 3—figure supplement 1B).

Comparing the numbers of cytokinesis nodes in the broad band of wild-type cells, rga4Δ cells, and rga2Δ cells provided data on the effect of cell diameter on the number of cytokinesis nodes. The number of cytokinesis nodes in long, narrow rga2Δ cells (193 ± 39) was indistinguishable from wild-type cells (191 ± 41) and aligned with the data from the other strains in plots of node number versus length and volume. On the other hand, the short, wide rga4Δ cells had fewer cytokinesis nodes (115 ± 40) than the wild-type and rga2Δ mutant cells with similar volumes (Figure 3H). The rga4Δ cells grew longer in 15 mM hydroxyurea for 4–6 hr and had the same number of nodes per volume as the other strains (Figure 3H).

The cells might use two strategies to accommodate a total number of nodes in a broad band determined by their size. Either the surface area (width) of the broad band or the density of nodes might increase with the total node number. We observed both. The very large cdc25-22 mutant cells simply expanded the width of the broad band in proportion to the number of nodes, while the density of nodes and width of broad band both increased with their numbers in the other strains of freely cycling cells (Figure 3J, K).

We used five different strains to determine if the total Blt1-mEGFP fluorescence in contractile rings scales with cell size. The total Blt1-mEGFP fluorescence in contractile rings was generally higher in large cells (Figure 3—figure supplement 2A), but did not scale cleanly with size because the five tested strains incorporated different fractions of the total Blt1-mEGFP in the cytokinesis nodes into their contractile rings: 85% in wild-type cells; 40–70% in cdc25-22, wee1-50, and rga2Δ mutant cells; and nearly 90% in rga4Δ mutant cells. In cdc25-22 mutant cells, the total Blt1-mEGFP fluorescence in cytokinesis nodes increased with cell volume (Figure 3H) but did not scale with cell size in their contractile rings (Figure 3—figure supplement 2B). Therefore, the largest cdc25-22 cells had as little as ~50% of the cytokinesis node Blt1-mEGFP fluorescence in their contractile rings. In contrast, cdc25-22 mutant cells did recruit mEGFP-Myo2 to their contractile rings in proportion to their volumes (Figure 3—figure supplement 2B).

We performed two types of experiments to investigate the relationship between the membrane-bound Pom1 kinase and the number of cytokinesis nodes. Concentrating Pom1 at the poles depends on microtubules that transport the polarity proteins Tea1 and Wsh3/Tea4 to the poles (Bähler and Pringle, 1998; Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009; Tatebe et al., 2005). First, we measured the distributions of Pom1 in cells with a wide range of sizes by time-lapse, spinning disk confocal microscopy (Figure 4). The five strains were wild-type, rga4Δ, rga2Δ, cdc25-22, and wee1-50. The cells expressed Pom1-GFP along with Sad1-RFP to identify mitotic cells with two SPBs and Blt1-mCherry to track nodes. Second, we varied the concentration of Pom1 and measured the number of nodes (Figure 5).

Figure 4

Download asset Open asset

Figure 4—source data 1

Related to Figure 4C, D.

https://cdn.elifesciences.org/articles/76249/elife-76249-fig4-data1-v1.xlsx

Download elife-76249-fig4-data1-v1.xlsx

Figure 5

Download asset Open asset

Figure 5—source data 1

Related to Figure 5A–H.

https://cdn.elifesciences.org/articles/76249/elife-76249-fig5-data1-v1.xlsx

Download elife-76249-fig5-data1-v1.xlsx

Figure 4A shows time series of fluorescence micrographs of individual cells from three strains paired with scans of the fluorescent intensity of Pom1-GFP along the lengths of these cells, while Figure 4B shows the line profiles of fluorescence of Blt1-mCherry plus Sad1-RFP (magenta) and Pom1-GFP (green) along the lengths of seven to nine mitotic cells of the five different strains. To our knowledge, this is the first time that the distribution of Pom1 was documented in cells with such a wide range of sizes (lengths of 8–40 µm).

Wild-type cells imaged at 22°C concentrated Pom1 at the cell tips throughout interphase and then at the division site in anaphase (Figure 4A, second panel from top) as reported previously (Bähler and Pringle, 1998) Time-lapse imaging showed that Pom1 followed the constricting contractile ring and disappeared from the division site after the cells separated. Consequently, after cell division, the concentration of Pom1 was higher at the old cell ends than at the new ends. As the growth of the new ends took off, Pom1 concentrated at the new ends with gradients toward the middles of the cells.

The behavior of Pom1 in the very short wee1-50 cells grown at 36°C for 4 hr and imaged at 22°C was mostly similar to wild-type cells. However, when cells formed a contractile ring, the Pom1 decreased at the cell tips as it distributed more homogeneously along the length of the cells. Then it concentrated at the division site and followed the constricting contractile ring as seen in wild-type cells (Figure 4A, upper panel).

The behavior of Pom1 was similar in wild-type, rga2Δ, and rga4∆ cells grown at 25°C. During interphase, the Pom1-GFP intensity was highest at the tips and low in the middle of the cells. This was exaggerated in the rga4∆ cells (Figure 4B, second panel from top). Pom1-GFP accumulated at the division site and followed the cleavage furrow as the cells divided.

During interphase at the nonrestrictive temperature Pom1 concentrated at the cell tips of cdc25-22 mutant cells as in other strains (Figure 4A, third panel). However, at the G₂/M transition, the Pom1 concentration decreased at both cell tips, and the fluorescence redistributed homogeneously in the cytoplasm. Similarly, when cdc25-22 cells were grown at a restrictive temperature of 36°C for 4 hr and released at the imaging temperature of 22°C to synchronize them at the G₂/M boundary, Pom1 was initially distributed throughout the cytoplasm (Figure 4A, fourth panel). Then Pom1 accumulated at both cell tips peaking during anaphase when the contractile ring formed and matured. Similar to wild-type cells, Pom1 concentrated at the cleavage site and appeared to follow the constricting contractile ring (CS) until it redistributed from the division site to the new ends of daughter cells as they took off and grew (Figure 4A, third and fourth panels).

The local intensities of Pom1-mEGFP along the plasma membrane in the broad band did not vary significantly in the five strains with a fivefold range of sizes (Figure 4C) and 100–400 nodes (Figure 3H). The local Pom1 concentration did not vary like the Blt1-mCherry intensity along the plasma membrane in the middle focal plane of these cells (Figure 4D). These measurements extend a previous report over a narrower range of cell lengths (Pan et al., 2014). We conclude that during the G₂/M phase transition of the cell cycle the local Pom1 concentration in the broad band does not control the variation in node number with cell size.

We used the nmt81 (no message in thiamine) thiamine-repressible promoter (Maundrell, 1993) to control the expression of Pom1 and measured the effects on the cells and their cytokinesis nodes (Figure 5). Pom1 expression was lowest under repressing conditions (EMM5S with thiamine) and three times higher than wild-type cells grown in EMM5S without thiamine for 40 hr (Figure 5A). The intensity of mEGFP-Pom1 in the broad band along the plasma membrane (Figure 5B) and the length of cells undergoing the G₂/M transition (Figure 5C) increased with the average concentration of Pom1 in the cells. The Blt1-mCherry intensity per pixel in the broad band also increased with the Pom1 concentration (Figure 5D), but the single node fluorescence measured with Blt1-mCherry did not change (Figure 5E). The width of the broad band of nodes did not change with the total Pom1 (Figure 5F). Therefore, the density of nodes in the broad band increased modestly with the cellular Pom1 concentration as the broad band occupied a smaller fraction of the cell length (Figure 5G). The ratio of the broad band width to the cell length decreased with the total cellular Pom1, indicating that Pom1 controls the width of the broad band (Figure 5H) as expected from previous work (Allard et al., 2019).

The kinase Cdr2 is the scaffold protein for interphase type 1 nodes, which cluster around the middle of the cell and later merge with type 2 nodes to form cytokinesis nodes. To determine whether the numbers of cytokinesis nodes depend on the concentration of Cdr2, we replaced the Cdr2 promoter in the S. pombe genome with the nmt81 thiamine-repressible promoter (Maundrell, 1993). To quantify Cdr2 expression in these cells the N-terminus of Cdr2 was tagged with mEGFP. This strain also had a C-terminal RFP tag on Sad1 to mark SPBs and a C-terminal mCherry tag on Blt1 to locate and count cytokinesis nodes. Thiamine suppressed the expression of mEGFP-Cdr2 from the nmt81 promoter while growth without thiamine produced mEGFP-Cdr2 concentrations higher than in wild-type cells (Figure 6A).

Figure 6

Download asset Open asset

Figure 6—source data 1

Related to Figure 6A–F.

https://cdn.elifesciences.org/articles/76249/elife-76249-fig6-data1-v1.xlsx

Download elife-76249-fig6-data1-v1.xlsx

The length of the cells as they entered mitosis decreased modestly with the concentration of mEGFP-Cdr2 (Figure 6B), but the width of the broad band of nodes did not change (Figure 6C). The average single node intensity measured with Blt1-mCherry in cells grown for 12, 25, or 40 hr in EMM5S media and 24 hr in EMM5S + thiamine did not differ from wild-type cells (Figure 6D). Both the intensity of Blt1-mCherry per pixel in the broad band and the number of cytokinesis nodes increased with the expression of mEGFP-Cdr2 (Figure 6E, F). We conclude that the cellular concentration of Cdr2 has a modest effect on the number and density of cytokinesis nodes and the size of cells entering mitosis but not the width of the broad band of nodes.

Using Airyscan imaging we measured the number of cytokinesis nodes in S. pombe and discovered that the number during early mitosis is proportional to cell size (Figure 3). Previous studies underestimated the number of cytokinesis nodes due to limitations of imaging techniques and/or methods used to choose nodes (Akamatsu et al., 2014; Akamatsu et al., 2017; Allard et al., 2018; Deng and Moseley, 2013; Laplante et al., 2016; Lenz et al., 2011; Pan et al., 2014; Vavylonis et al., 2008; Willet et al., 2019). Conventional confocal microscopy cannot resolve closely spaced nodes, so confocal images give the impression that cytokinesis nodes vary considerably in size and fluorescence intensity. Several studies considered only the largest nodes when estimating their numbers. On the other hand, super-resolution single-molecule localization microscopy showed that nodes are uniform in size and composition (Laplante et al., 2016). However, the method cannot make a 3D reconstruction of live cells to count the total number of nodes, so the numbers were estimated by extrapolation.

Airyscan imaging overcomes these limitations by allowing 3D reconstructions of live cells at a higher resolution than conventional confocal microscopes. We imaged 40 Z-slices covering the entire thickness of the cells and used the method of Akamatsu et al., 2017 to count cytokinesis nodes. The first step was to measure the average fluorescence intensity of a sample of single nodes marked with Blt1-mEGFP (Figure 2D). The Airyscan microscope resolved single nodes better than the confocal microscope (Akamatsu et al., 2017; Figure 1), so we observed a large, main peak in the distribution of fluorescence intensities and only a few spots in our sample with higher fluorescence intensities, likely arising from overlapping single nodes. These single nodes move closer to each other to form binary and multiples of single nodes and eventually coalesce into a contractile ring. The second step was to measure the total Blt1-mEGFP fluorescence in the broad band of nodes in the cortex around the equator of the cell, correcting for background cytoplasmic fluorescence. Dividing the total Blt1-mEGFP fluorescence in the broad band by the average fluorescence of a single node gave ~190 single nodes in wild-type fission yeast cells.

Similar measurements on contractile rings in wild-type cells (Figure 2E) showed about 85% of total Blt1-mEGFP fluorescence from the broad band of nodes is incorporated into contractile rings. This may be related to the observation that 10 min before SPB divide, about 75% of late interphase nodes contained markers of both type 1 and 2 nodes (Akamatsu et al., 2014) at the time they begin to accumulate Myo2, IQGAP Rng2, F-BAR Cdc15, and formin Cdc12 (Wu et al., 2003). During 10–15 min after SPBs separate, cytokinesis nodes containing these proteins and Blt1 condense into a contractile ring (Vavylonis et al., 2008; Wu et al., 2006). The type 1 node scaffold protein Cdr2 leaves the contractile ring as it forms (Akamatsu et al., 2014). Anillin Mid1 is released from the fully formed contractile rings as unconventional myosin-II Myp2 joins the ring (Wu et al., 2003). It is possible that the 25% of cytokinesis nodes with Blt1 but lacking Cdr2 are left behind when the contractile ring forms, because they failed to grow actin filaments or were deficient in Myo2, which are required for contractile ring formation.

Our approach may be useful to extend studies on how the density of nodes marked with Cdr2 senses the cell surface area, activates cyclin-dependent kinase Cdk1 through inhibitory phosphorylation of wee1, and promotes the transition into mitosis (Facchetti et al., 2019b; Pan et al., 2014; Russell and Nurse, 1987; Simanis and Nurse, 1986). Rather than measuring the total intensity of Cdr2 around the equator (Facchetti et al., 2019b; Pan et al., 2014; Russell and Nurse, 1987; Simanis and Nurse, 1986) or counting only bright nodes (Pan et al., 2014), we measured the number of single nodes marked with Blt1 and found that their density on the membrane in the broad band varied with cell sizes except in cdc25-22 mutant cells (Figure 3K). Using our method to count single nodes marked with Cdr2 or Blt1 may provide further insights into the relation of node numbers to the timing of cell division.

Starting from the observation that the number of interphase nodes in wild-type cells increases with the twofold increase in cell size during the cell cycle (Akamatsu et al., 2017; Allard et al., 2018; Deng and Moseley, 2013; Pan et al., 2014), we measured the number of cytokinesis nodes in wild type and mutant cells that varied fivefold in size as they entered mitosis (Figure 3). To avoid the time-dependent compositional changes in cytokinesis nodes during their ~22 min life span and to be able to compare the number of cytokinesis nodes as well as the cell size among the wild-type and four mutant cells, we restricted our measurements to a specific cell cycle time point. The average fluorescence intensity of the single nodes was the same in wild-type and these mutant cells (Figure 3G). The total Blt1-mEGFP fluorescence in a broad band of nodes and therefore the total number of nodes increased with cell size measured as volume (Figure 3H), length (Figure 3I), or surface area (Figure 3—figure supplement 1A).

We investigated the roles of Pom1, the kinase that restricts type 1 nodes from the poles of cells, and Cdr2, the kinase and scaffold protein for type 1 nodes, in setting the number of cytokinesis nodes. Pom1 forms a gradient from the cell tips to their middles and phosphorylates Cdr2 at multiple sites. Phosphorylation of one site reduces Cdr2 oligomerization and membrane binding at the poles, while phosphorylation of another site inhibits Cdr2 from phosphorylating the Wee1 kinase (Bhatia et al., 2014; Deng and Moseley, 2013; Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009; Rincon et al., 2014). However, both cdr2∆ and pom1∆ cells form nodes and complete cytokinesis, although the cleavage site is off-center in pom1∆ cells because they only exclude nodes from one pole by an unknown mechanism (Martin and Berthelot-Grosjean, 2009; Moseley et al., 2009).

Pan et al., 2014 found a constant concentration of Pom1 along the equatorial plasma membrane of wild-type cells as they grew twofold, and we extended the range of sizes to fivefold. Thus, the scaling of interphase or cytokinesis node numbers with cell size is not a consequence of differences in the local concentration of Pom1 in the broad band. Furthermore, Pan et al., 2014 also reported that total Cdr2 and Cdr2 in interphase nodes increased with cell size during the cell cycle in cells with or without Pom1. This is very strong evidence that Pom1 does not control type 1 node formation in the cell middle directly, rather as we explain below, Pom1 controls the cytokinesis node number indirectly via its influence on cell size.

Although neither Cdr2 nor Pom1 controls the scaling of interphase node number with cell size, both can influence the number and density of cytokinesis nodes in the broad bands of mitotic cells. Artificially varying the total cellular concentration of Pom1 revealed the following dependencies as cells entered mitosis: the lengths of the cells (Figure 5C), the concentration of Pom1 along the plasma membrane in the broad band (Figure 5B), and the numbers of cytokinesis nodes (Figure 5G) all increased with the cellular concentration of Pom1, while both the fluorescence of single nodes (Figure 5E) and the width of the broad band (Figure 5F) were constant. Therefore, high Pom1 increased the density of cytokinesis nodes (Figure 5F) as the broad band occupied a smaller fraction of the cell (Figure 5H). The numbers of cytokinesis nodes in cells expressing high Pom1 concentrations exceeded the numbers in cells of similar lengths and normal Pom1 concentrations (Figure 3I).

On the other hand, cells expressing low concentrations of Pom1 were shorter when they divided (Figure 5C) with fewer cytokinesis nodes (Figure 5G) than cells with high Pom1 concentrations. The node numbers were higher than in cells of a given length with normal Pom1 levels (Figure 3I).

Artificially increasing the total cell concentration of Cdr2 (Figure 6) reduced cell length at the time of division (Figure 6B) but did not change the width of the broad band of cytokinesis nodes (Figure 6C), so the broad band occupied a larger fraction of the cell surface. High Pom1 concentrations had the opposite effects on cell size (Figure 5C) and broad band width (Figure 5F). On the other hand, the number of cytokinesis nodes increased with the concentration of Cdr2 (Figure 6E). The short cells expressing high Cdr2 concentrations had almost double the number of cytokinesis nodes in their slightly wider broad bands than cells of similar lengths with wild-type levels of Cdr2 (Figure 3I). Longer cells expressing low concentrations of Cdr2 had numbers of cytokinesis nodes similar to cells of the same lengths in Figure 3I. The plot of cytokinesis node number versus Cdr2 concentration extrapolates to more than 190 nodes when the Cdr2 concentration is zero (Figure 6E), as expected for cells longer than wild-type cells at division (Figure 6B). Therefore, Cdr2 can drive cytokinesis node numbers above the basal level of about 190.

Most of the proteins comprising cytokinesis nodes during mitosis (Blt1, anillin Mid1, F-BAR Cdc15, IQGAP Rng2, and Myo2) are initially incorporated into contractile rings (Goss et al., 2014; Wu and Pollard, 2005). Thereafter, Myo2 is retained and concentrated along with newly arriving Myp2, while first Mid1 and then subsequently actin are lost in proportion to the declining circumference of the constricting contractile ring (Courtemanche et al., 2016; Malla et al., 2021). This gave the strong impression that the core of the cytokinesis nodes, with the exception of Mid1, remained intact in the contractile ring. However, we report here that the fraction of Blt1 in cytokinesis nodes incorporated into contractile rings varies: nearly 90% in rga4Δ mutant cells, 85% in wild-type cells, and 40–70% in cdc25-22, wee1-50, and rga2Δ mutant cells. In the case of the longest cdc25-22 mutant cells, a much larger fraction of the Myo2 from the cytokinesis nodes was incorporated into the rings than Blt1 (Figure 3—figure supplement 2). Arresting and then releasing the cell cycle of the cdc25-22 mutant cells at the G₂/M transition may contribute to this imbalance, which is greater than in any of the strains allowed to cycle normally. In any case, this new observation indicates that nodes observed in rings by single-molecule localization microscopy (Laplante et al., 2016) are likely to have different compositions than cytokinesis nodes. Documenting and characterizing this maturation of nodes transitioning into contractile rings is worthy of further exploration.

Table 1 lists the strains used in this work. We used standard methods for S. pombe growth and genetics (Moreno et al., 1991). We made most observations on a strain with SPBs marked by Sad1-RFP, and nodes marked by Blt1-mEGFP (Goss et al., 2014). The sequences encoding the fluorescent proteins were incorporated into the haploid genome, so all copies of these proteins were tagged. The yeast strains rga2Δ (FC3158) and rga4Δ (FC3157) were a kind gift from the laboratory of Prof. Fred Chang (University of California, San Francisco). All the other strains were obtained from laboratory stocks or by crossing laboratory stock strains. Strains were confirmed by growth on selective media and microscopy. Except for Figures 5 and 6, cells were grown in YE5S medium for 24 hr at 25°C and then overnight in synthetic EMM5S medium at 25°C before imaging.

To control the expression of Cdr2 and Pom1 (Figures 5 and 6), we inserted by homologous recombination the weak nmt81 promoter along with mEGFP upstream of the open reading frames for Cdr2 or Pom1 in their chromosomal loci. Cells were grown in the EMM medium for 25 hr at 25°C to induce moderate expression or 40 hr at 25°C to overexpress mEGFP-Cdr2 or mEGFP-Pom1. Cells were grown in 5 µg/ml thiamine for >24 hr at 25°C to repress the expression of these two fusion proteins. The OD₅₉₅ of the cultures was kept below 0.6 over the entire period.

Imaging was performed on LSM 880 laser scanning confocal microscope (ZEISS) equipped with 100x Plan Apochromatic 1.46 NA oil objective, an Airyscan super-resolution module, GaAsP detectors, and Zen Black acquisition software (ZEISS). We acquired 40 Z-slices with a step size of 170 nm covering the entire volume of a cell. Pixel size was 41 nm by 41 nm. Images were collected with a pixel dwell time of 1.02 μs and an image size of 520 × 520 pixels corresponding to 22 × 22 μm. Cells were imaged for mEGFP with a 488 nm laser and BP 420–480 excitation filter and BP 495–620 emission filter; and for RFP with a 561 nm laser and with a BP 495–550 excitation filter and LP 570 emission filter. The pixel dwell time, laser intensity, and detector gain were kept low to avoid saturation and photobleaching during image acquisition. To increase signal-to-noise ratio and resolution, acquired images were processed by 3D Airyscan filter strength 7.0 with Zen Black software.

We identified cytokinesis nodes as spots of Blt1-mEGFP fluorescence around the equator of cells with two SPBs marked by Sad1-RFP, indicating that the cells were in mitosis. Since these cytokinesis nodes formed contractile ring within 10 min of their formation, we chose cells with broad band of nodes and neglected others which show partially formed contractile rings. We identified and measured the fluorescence intensity of single cytokinesis nodes using the following procedure to process the fluorescent images. First, we used ImageJ software to make a maximum intensity projection from 40 Z-slices of Airyscan images containing all of the fluorescence spots in the broad band of a cell (Figure 2A). These spots varied considerably in size and brightness depending on the number of ‘single nodes’ clustered in a diffraction-limited spot (Akamatsu et al., 2017; Laplante et al., 2016). We searched for small spots and marked their X–Y positions. Then we inspected the fluorescence along the Z-axis at these X–Y positions in 40 Z-slices. To qualify as a single node, a spot had to satisfy two conditions: (1) the fluorescence was within a circle 12-pixels (~0.5 µm) in diameter, and (2) the fluorescence was present in more than 3 and up to 7 consecutive Z-slices, with a peak in the middle slice and diminishing toward either or both sides. This middle slice was designated as the Z-axis position of the node. The X–Y–Z positions of each node were stored in the ROI manager of ImageJ software and exported to MATLAB using the add-on @ReadImageJROI (Muir, 2021). We developed a MATLAB code for further analysis (Source code 1). We sum projected the 7 Z-slices containing the putative single node and measured the total fluorescence intensity within a circle 12-pixels in diameter at the X–Y position of the node (Figure 2B, black dotted circle). From the same sum-projected image of 7 Z-slices, we measured the average cytoplasmic fluorescence from 8 circles 12-pixels in diameter from regions of cytoplasm flanking the node broad band (Figure 2B, blue dotted circles). We subtracted this average cytoplasmic fluorescence from the fluorescence of the node to remove the background. For each node, we used SUM projected image of 7 Z-slices containing all the fluorescence of the same node to calculate its total and average cytoplasmic background as explained above. A histogram of the sample of fluorescence intensities of single nodes after subtracting their respective average cytoplasmic background was fit with Gaussian distribution using Solver add-in Microsoft Excel, which minimizes the sum of the chi-squared values, showed a dominant peak. The analysis method is similar to Figure 2 of Akamatsu et al., 2017, but the higher resolution of Airyscan microscopy separated some closely space nodes that would be counted as one by confocal microscopy. To calculate the mean value of fluorescence of single nodes, we considered only the nodes with fluorescence values less than or equal to twice the peak of the Gaussian curve fitted to the distribution and average it. In Figures 2 and 3G, the Gaussian fits show the distribution of fluorescence of only single nodes on histograms.

To obtain the total fluorescence of a broad band of nodes, we sum projected 40 Z-slices of Airyscan images and measured the fluorescence of an ROI drawn around the broad band of nodes (Figure 2C, red dashed line area). The mean pixel intensity from polygonal ROIs from either side of the broad band (Figure 2C, yellow dashed line) was averaged and multiplied by the number of pixels in the broad band ROI to calculate the background fluorescence in the volume of the broad band of nodes. This value was subtracted from the fluorescence in the broad band of nodes to remove the background. To estimate the number of nodes in a cell we divided the total fluorescence of the broad band of nodes after subtracting the background by the average total fluorescence of individual single nodes after subtracting the background.

In Figure 3K, we calculated the density of nodes in broad bands by taking into account the cell diameters, which were the same in wild-type, wee1-50, and cdc25-22 mutant cells but larger in rga4Δ cells and smaller in rga2Δ cells.

To compare the confocal and Airyscan imaging shown in Figure 1 we obtained confocal images using the same Airyscan detector. To make and improve the signal-to-noise ratio comparable to Airyscan microscopy we used the pinhole 0.6 AU, 8× averaging, linear constrained iterative deconvolution, and twice the 488 nm laser intensity implemented in Airyscan imaging. We acquired 20 Z-slices with step size 340 nm covering the entire fluorescence of a cell with pixel size 82 nm by 82 nm. Images were processed for linear constrained iterative deconvolution using the ImageJ plugin ‘Deconvolution’.

To determine the Pom1 localization in Figure 5, we used an Olympus IX-71 microscope with a 100x, numerical aperture (NA) 1.4 Plan Apo lens (Olympus), and a CSU-X1 (Andor Technology) confocal spinning-disk system equipped with an iXON-EMCCD camera (Andor Technology). We used Coherent OBIS 488 nm LS 20 mW and OBIS 561 nm LS 20 mW lasers for excitation. Images were acquired using Micromanager 1.4 (Edelstein et al., 2014).

We acquired 15 Z-slices covering the fluorescence of entire cells every 2–5 min with 0.5 μm spacing between slices; 22 μW 561 nm laser power (measured after the objective) and 50 ms exposures; 89 μW 488 laser power and 200 ms exposures. Images were sum projected and corrected for camera noise and uneven illumination. Photobleaching was compensated by choosing a small ROI in both channels that was present in another nondividing cell throughout the acquisition and using the Fiji plugin ‘Bleach correction’ for Exponential Fitting.

We studied cytokinesis nodes in cells during the interval between SPB separation and the formation of the contractile ring. The intensity per pixel of an ROI where no cells were present was subtracted from both channels to remove substrate background intensity. Then a line equal to cell width was drawn along the length of the cell, and a line profile was obtained using the ImageJ-Plot profile plugin, which generated a graph of intensity per pixel across the width of a cell vs the cell length. The intensity per pixel of wild-type cells not expressing a fluorescent protein was subtracted from each point to remove auto-fluorescence.

For each strain except in the cdc25-22 mutant strain, 10–12 line profiles were obtained as above and aligned at the centers of the cells. To measure the distributions of the fluorescence from Pom1-GFP or Blt1-mCherry plus Sad1-RFP at different phases of cell cycles of wild-type, wee1-50, and cdc25-22 mutant cells, we plotted the intensities per pixel across the width of cell vs the cell length across the cell cycle (Figure 4B). The temperature-sensitive cdc25-22 mutant cells were grown at the restrictive temperature of 36°C for 4 hr and released for imaging at 22°C. The diffuse fluorescence of Pom1-GFP was higher on one side of the nucleus than the other, so the line profiles were aligned with the higher concentration of Pom1 on the same side (Figure 4B, lower panel). We measured the width of the broad band of cytokinesis nodes as the zone where the Blt1-mEGFP signal was >20% of the peak value.

We outlined manually whole cells or the broad band area and used the ImageJ plugin ‘Measure’ to measure the Mean intensity in these areas from the intensity per pixel calculated as the total intensity in all the pixels in a region divided by the number of pixels, which is equivalent to the density (Figures 5 and 6).

In Figure 4C, the Pom1-mEGFP intensity along the equatorial plasma membrane was measured from confocal images of the middle focal plane (thickness ~0.5 µm) of the cells after subtracting the average background intensity from outside the cells in the same image. A rectangle 3 pixels wide (~0.4 µm) and as long as the broad band of nodes was manually drawn over the plasma membrane of cells in the image and used to calculate the Pom1-mEGFP intensity per pixel.

The mEGFP-Pom1 intensity in Figure 5B was measured similar to Figure 4C from the sum projected image of three consecutive middle focal planes Airyscan images (thickness ~0.5 µm) of the cells. The average pixel intensity outside cells was subtracted from the sum image. A rectangle 10 pixels wide (~0.4 µm) and as long as the broad band of nodes was manually drawn over the plasma membrane of cells in the image and used to calculate the Pom1-mEGFP intensity per pixel in the broad band as shown in Figure 4C, inset.

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM026132. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Prof. Fred Chang for the rga4∆ and rga2∆ mutant strains.

This work did not involve human subjects, animals or clinical trials.

© 2022, Sayyad and Pollard

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.