Study of organelle size control in yeast
Kazunori KUME
Graduate School of Integrated Sciences for Life, Hiroshima University
Research objective
Membrane-bound organelles are compartmentalized by membranes in eukaryotic cells
and are of an appropriate size. Eukaryotic cells are thought to have acquired multiple cellfunctions through the uptake of organelles; however, it is unclear how cells control organelle
size and how this affects cell function. Therefore, the purpose of this study was to elucidate
the mechanism of organelle size control and to investigate the effect of its perturbation on cellfunction using fission yeast as a model organism. To understand the mechanism of nuclear
size control, we screened a fission yeast deletion library for nuclear size mutants.1,2) The
analysis of these mutants revealed that, in addition to nuclear enlargement, they presented
defects in the structure of other organelles. Therefore, we focused on the nucleus and otherorganelles of the nuclear size mutants to elucidate the mechanism of organelle size control.
Furthermore, to clarify the effect of organelle size control on cell function, we performed
transcriptome analysis and cell proliferation assays using these organelle size mutants.
Methods
1. Observation of organelles in living cells and image analysis
Organelles were visualized using fluorescent probes that specifically stain certain
organelles in a yeast strain whose nucleus is labeled with a fluorescent protein. We used Cut11
as a nuclear membrane marker. ImageJ free software was used to analyze organelle structures.
For any organelle that could not be clearly stained with a fluorescent probe, we used yeast
strains in which GFP or mCherry was fused with a protein localized at that specific organelle.
2. Transcriptome analysis and cell proliferation assay
After growing cells until the exponential growth phase, RNA was extracted andsubjected to RNA sequencing (RNA-seq) for transcriptome analysis. Cell proliferation wasassessed by spot assay on an agar medium using several inhibitors, including a DNA
synthesis inhibitor, a DNA replication inhibitor, a protein synthesis inhibitor, and a lipid
synthesis inhibitor. Yeast cells were suspended in sterile water at a concentration of 5 × 106
cells/mL and spotted on synthetic medium containing yeast extract. Yeast cell growth wasevaluated at different temperatures (25–36 °C).

Results
1. Analysis of the mechanism of organelle size control
To investigate the size of the nucleus and that of other organelles in living cells, we searched
for an experimental condition allowing simultaneous visualization of the nucleus and otherorganelles, such as lipid droplets, vacuoles, mitochondria, and the endoplasmic reticulum.
We tested commercially available fluorescent probes and found that probes for lipid droplets(BODIPY, Thermo Fisher Scientific), vacuoles (FM4-64, Thermo Fisher Scientific), andmitochondria (MitoTracker, Thermo Fisher Scientific) were suitable for the experimentalconditions. As for the endoplasmic reticulum, the signal of the fluorescent probe ER-Tracker(Thermo Fisher Scientific) was weak; therefore, a strain in which a fluorescent protein wasfused to a protein localized in the endoplasmic reticulum was constructed. We examined
multiple proteins localized in the endoplasmic reticulum (i.e., Sec63, Elo2, and Rtn1) andfound that a strain in which the fatty acid elongase Elo2 was fused with a fluorescent protein
was suitable for the experimental conditions. Under these conditions, we observed nuclei andother organelles in nuclear size mutants,1,2) and found that the structures of vacuoles andlipid droplets in these mutants were different from those in the wild-type strain. In particular,
the vacuole size in the nuclear size mutants was reduced to almost half of that in the wildtype strain. Conversely, the size of lipid droplets in the nuclear size mutants was similar tothat in the wild-type strain, but their number was lower. These results suggest that the size
control mechanism of the nucleus might be associated with that of vacuoles and lipid
droplets. To examine this possibility, we examined the effect of the deletion of the gene
mediating the conversion of lipid droplets, which are lipid storage organelles, into structural
lipids on the nuclear size of the mutants. Nuclear enlargement was partially suppressed in thedouble mutant, indicating that the size control mechanism of the nucleus and that of lipid
droplets were associated. We are currently investigating the possible relationship between
the size control mechanism of the nucleus and that of vacuoles.
2. Effects of the perturbation of organelle size control on cell function
Next, we examined the growth of organelle size mutants, in which the sizes of the nucleus
and other organelles were altered, on agar medium containing various inhibitors. We foundthat the mutants showed severe growth defects in presence of a protein synthesis inhibitor.
Next, we performed transcriptome analysis on an organelle size mutant strain and a wild-typestrain. A total of 434 genes were upregulated and 281 genes were downregulated in the mutant
strain compared to the wild-type strain. Gene Ontology (GO) enrichment analysis revealed
that genes related to transcription and translation were enriched among the downregulated
genes. We will soon examine the relationship between the perturbation of organelle size
control and these phenotypes.

Conclusion
Our study revealed that mutants displaying enlarged nuclear size presented defects also
in the size of other organelles. This suggests the existence of a size control mechanism sharedamong multiple organelles. In addition, organelle size mutants showed growth defects inpresence of a protein synthesis inhibitor and fluctuations in the expression of genes involvedin transcription and translation, indicating a functional linkage between organelle size control
and these phenotypes.
References
1) Kume, K., Cantwell, H., Neumann, F.R., Jones, A.W., Snijders, A.P., and Nurse, P. (2017)
A systematic genomic screen implicates nucleocytoplasmic transport and membrane
growth in nuclear size control. PLOS Genet. 13(5): e1006767.
2) Kume, K., Cantwell, H., Burrell, A., and Nurse, P. (2019) Nuclear membrane protein Lem2
regulates nuclear size through membrane flow. Nat. Commun. 10(1): 1871.