Fig. 1.

BSCaM_IQ, a sensor and a sink for calmodulin, rescues toxicity induced by α-syn by decreasing the total levels of free Ca²⁺–CaM. (A) Strains of control (no α-syn), NoTox (low copy number of α-syn), IntTox (intermediate copy number of α-syn), and HiTox (high copy number of α-syn) were transformed with aequorin, a genetically encoded Ca²⁺ indicator. Cytosolic Ca²⁺ was measured by aequorin luminescence over time after α-syn induction. Cytosolic Ca²⁺ levels are expressed as fold induction relative to control. (B) Cmd1p_free levels assayed by FRET 0, 4, and 8 h after α-syn induction in the presence of BSCaM_IQ in control (blue), HiTox strain (red), and HiTox strain transfected with cmd1 (red dashed line). Cmd1p_free = K_d [(R_max − FRET/CFP)/FRET/CFP − R_min] (see Supporting Information). (C) Yeast strains were spotted onto plates containing uninducing media [synthetic defined (SD) −Ura; GPD-BSCaM_IQ selective; Lower] and replica platted in threefold serial dilutions onto α-syn–inducing plates containing selective media and (SGal −Ura) (Upper). YFP is used as control plasmid. (D) Yeast strains were spotted onto plates containing uninducing media (SD −Ura, Leu; BSCaM_IQ and cmd1 selective; Lower) and replica platted in threefold serial dilutions onto α-syn–inducing plates containing selective media and SGal −Ura, Leu (Upper). YFP and empty vector (vec) were used as control plasmids, cmd1 = yeast calmodulin and cmd1_X = D94A, E105V; unable to bind calcium at the third EF hand. (E) BSCaM_IQ (green), neuronal (red MAP2 positive), and nuclear (blue, Hoechst) stainings from representative pictures of rat primary neuronal cultures coinfected with either control lentivirus LacZ, LacZ and α-SynA53T, LacZ and α-SynA53T and BSCaM_IQ, or LacZ and BSCaM_IQ. (F) Percentages of neurons (MAP2 positive) relative to control (LacZ infected) in the conditions described in B. *P < 0.05, one-way ANOVA, Dunnett’s multiple comparison test.