A conserved G₁ regulatory circuit promotes asynchronous behavior of nuclei sharing a common cytoplasm

Figures & data

Figure 1 G₁ is variable between nuclei in the same cell. (A) Cells with GFP-localized to nuclei and SPBs visualized with mCherry were filmed using 4D fluorescence microscopy (AG290, Whi5-GFP, Tub4-mCherry). Histogram represents the distribution of times observed from birth to SPB duplication for 40 nuclei. Grey bars represent nuclei that were born but never duplicated their SPBs during the observation period and therefore ultimate G₁ times are unknown but are greater than the movie length (65–100 min). The average G₁ duration includes the minimal G₁ lengths of these nuclei that did not transition during observation. (B) Example pedigree showing variable G₁ lengths between sister nuclei. (C) Overlapping barplot of G₁ times for 11 pairs of sister nuclei. The G₁ time for the slow sister is in black and the G₁ time for the fast sister is overlapping in grey. (D) Schematic summarizing how pairs of neighboring nuclei are considered for scoring in Table 1.

Figure 2 Whi5p is nuclear in all the stages of the cell cycle. (A) A young mycelium expressing Whi5-GFP and Tub4-mCherry shows that all nuclei have Whi5-GFP (AG290). Bar, 5 µm. (B) Individual nuclei in different stages of the nuclear division cycle containing Whi5-GFP. Bar, 5 µm. (C) Images from a time series showing Whi5-GFP appears equally segregated in mitosis between sister nuclei. The nucleus undergoing mitosis is indicated by an asterisk and the resulting sister nuclei are indicated by arrowheads. Bar, 5 µm. (D) Histogram depicting the percent difference in Whi5-GFP mean fluorescence intensity between sister nuclei born from one mitosis. This difference in intensity is calculated by dividing the mean fluorescence intensity of each sister nucleus by the total intensity of the two sister nuclei and taking the absolute value of the difference between the values (n = 19 sister pairs).

Figure 3 Model for asynchrony arising in G₁. Neighboring nuclei divide asynchronously and newborn sister nuclei spend different amounts of time in G₁. In mutants with altered regulation of the G₁/S transition, nuclei become more synchronous.

Table 1 Observed frequency distributions of nuclear cycle stages and neighbor relations

	SPB category (%)			Total nuclei	Synchronous pairs (%)			Asynchronous pairs (%)			Total pairs
Genotype	1SPB	2SPB	M	n	1SPB-1SPB	2SPB-2SPB	M-M	1SPB-2SPB	2SPB-M	1SPB-M	n
WT	61	26	13	483	35	6	3	34	8	14	439
pCLN1/2	70	20	10	590	60	7	1	24	2	6	485
pHIS3pr-CLN1/2	66	22	12	512	47	9	3	23	3	15	462
swi4Δ	54	23	23	765	48	15	14	10	6	7	610
mbp1Δ	56	25	19	620	47	13	8	14	10	8	495
whi5Δ	14	45	41	429	13	39	33	3	3	9	352

n is the number of nuclei or nuclear pairs and all other values shown above are percentages rounded to the nearest whole number.

Table 2 Synchrony index statistics

Genotype	% Observed synchrony	% Chance synchrony	Synchrony index (SI)	p-value mutant vs. WT t-test (1-tailed)
WT	43.6	45.3	0.96	-
pCLN1/2	67.6	54.5	1.24	<0.01
pHIS3pr-CLN1/2	59.3	50.2	1.18	<0.02
swi4Δ	76.4	39.7	1.93	<0.001
mbp1Δ	67.9	41.6	1.63	<0.001
whi5Δ	84.9	39.0	2.18	<0.001

Percent observed and chance synchrony are rounded to the nearest 10^th of a percent. SI is calculated as observed synchrony count divided by chance synchrony count. p values [mutant vs. WT t-test (1-tailed)] refer to significance of a one-tailed, unequal variance, t-test comparing the SI values between each mutant and WT.