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. 2018 Jun 21;11:205. doi: 10.3389/fnmol.2018.00205

Figure 1.

Figure 1

LRRK2RCK triggers death of aging yeast cells. (A) Scheme of truncated leucine-rich repeat kinase 2 (LRRK2) constructs used in this study. The enzymatic core of human LRRK2 (amino acids 1300–2163) containing the Ras-of-complex (ROC) GTPase, the C-terminal-of-ROC (COR) and the protein kinase domain, was expressed in yeast cells during chronological aging under the control of a GAL1 promoter. The wild type form of truncated LRRK2 (hereinafter referred as LRRK2RCK) and the point mutant R1398LRCK with higher GTPase activity were used. The green star indicates GTPase activity, the star in magenta represents kinase activity. (B) Growth of control cells expressing LacZ compared to cells expressing LRRK2RCK and the R1398LRCK variant. OD600 was measured in intervals of 2 h starting from the induction of galactose-driven expression. Means ± SEM; n = 4. (C) Flow cytometric quantification of loss of membrane integrity as indicated with propidium iodide (PI) staining of cells as described in (B). In addition, cells harboring the empty vector were analyzed to validate the suitability of LacZ expression as a control. Significances represent simple main effects between different expression types at each time point. Significances shown are valid for day 3–5. Means ± SEM; n = 4. (D) Clonogenic survival on day 3 of chronological aging determined by counting colony forming units (cfu) after plating 500 cells with indicated expression types on YEPD agar plates. Means ± SEM; n = 8. (E,F) AnnexinV/PI co-staining on day 3 of aging. Representative epifluorescence micrographs (E) and flow cytometric quantification (F) are shown. Scale bar represents 10 μm. Means ± SEM; n = 4. For AnnexinV/PI staining at earlier time points, please see Supplementary Figures S1D,E. (G) Immunoblot analysis of protein extracts from cells as described in (B). Blots were probed with antibodies directed against the V5-epitope to detect V5-tagged LacZ, LRRK2RCK and R1398LRCK, and against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. ***p < 0.001, **p < 0.01, *p < 0.05, n.s. not significant.