G1 CYCLINS FUNCTION THROUGH CELL SIZE TO MODULATE MEIOSIS IN SACCHAROMYCES CEREVISIAE

Audra R. Day*, Jody Markwardt, Kedar Purnapatre, Saul Honigberg, and Brandt L.Schneider

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street MS6540, Lubbock, TX 79430 *Audra.Day@ttuhsc.edu

INTRODUCTION. Mitotic cell cycle progression depends upon G1-phase cyclin dependent kinase (Cln-Cdc28) activity and the attainment of a minimum cell size.  In contrast to mitosis, Cln-Cdc28 inhibits entry into meiosis.  However, because Cln-Cdc28 influences both cell size and entry into meiosis, it has remained difficult to establish how cell size, in the absence of Cln-Cdc28 activity, affects entry into meiosis.  It has been previously suggested that cell size may affect entry into meiosis [1].  However, in these studies, cell size was altered by changing cell growth rates [1].  Therefore, we sought to determine if cell size influences in meiosis initiation in synchronous cultures that have not been modified by changing cell growth rate. Additionally we investigated if the loss of G1-phase cyclins, the presence of a temperature sensitive CDC28 mutation, or ectopic expression of IME1 would influence meiotic initiation in cells of various sizes. 

METHODS. Centrifugal elutriation was carried out using standard methods to isolate small (<90fL) and large (>130fL) cell fractions.  This technique was performed on wild type yeast, a conditional CLN strain (Dcln1, Dcln2, Dcln3:Gal-CLN3), a cdc28-4ts strain, and a strain harboring the tetO-IME1 expression plasmid. The wild type strain was elutriated and fractions of cells ranging from 64fL to 210fL were collected and placed in sporulation conditions. The conditional CLN strain was proliferated mitotically in the presence of galactose, elutriated, and placed in sporulation medium lacking galactose to eliminate CLN expression. The cdc28-4ts strain was grown, elutriated, and sporulated at permissive temperature. The strain harboring the tetO-IME1 expression plasmid was studied in the presence (IME1 off) and absence (IME1 on) of tetracycline. After being placed in sporulation conditions, samples were taken at specific time points and utilized for flow cytomentry, microscopic study, and Northern analysis. 

RESULTS. We found that in wild type yeast cell growth is required to achieve a minimum cell size requirement (>90 fL) for the initiation of meiosis.  However, cells above this threshold commit to meiosis in the absence of cell growth.  We found that the absence of CLN expression or partial loss of Cdc28 activity allowed small cells to form spores nearly as efficiently as wild type large cells. Interestingly, induced expression of IME1 promoted early entry into pre-meiotic S-phase and spore formation in both large and small cells. As an indicator of Ime1 activity and to determine the effect of cell size on SPS1 expression, we examined SPS1 mRNA in large and small cells. In wild type yeast there was a dramatically lower expression of SPS1 in small cells as compared to large cells. Interestingly, small cells containing a deletion of CLN3 or a temperature sensitive CDC28 mutation were able to express SPS1. 

DISCUSSION. The results described above indicate that the timing of meiotic initiation is directly proportional to cell size. In addition, data from conditional CLN and temperature sensitive cdc28-4 strains suggest that the effect of cell size on meiotic initiation is attributable in large part to high Cln:Cdc28 activity in small cells.  However, cell size also affects meiosis through mechanisms that function independent of Cln-Cdc28. These data imply Cln:Cdc28 activity functions to prevent small cells from entering meiosis until they have reached a minimum cell size. Finally, we demonstrated that IME1 activity is defective in small cells, and ectopic expression of IME1 allows small cells to initiate meiosis sooner and more efficiently. Together these data suggest that the effect of cell size on meiosis is mediated through IME1.  

ACKNOWLEDGEMENTS: This work was supported by National Institutes of Health Grant T32HD07271-19 Regulation of Reproductive Processes. 

REFERENCE.

1. Calvert, G.R. and I.W. Dawes, (1984) Nature. 312, 61-3.