EGL-19- and RYR-mediated Ca²⁺ influx are components of the proteostasis rescue mechanism.

Abstract

<p>Ca²⁺ relevance for <i>gei-11</i> effect on (A) Q35 aggregation (B) and <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) upregulation, tested by employing a hypomorphic mutant <i>egl-19(n582)</i>, a weak hypermorph <i>egl-19(n582ad952)</i>, a hypermorph <i>egl-19(ad695), egl-19</i> RNAi (control RNAi in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003711#pgen.1003711.s008" target="_blank">Table S1</a>) or the specific EGL-19 antagonist Nemadipine A (0.75 µM, DMSO). Student t-test *<i>p</i><0.05, ***<i>p</i><0.001, ns/not significant; data relative to vector control or control in DMSO (±SD). (C) The RYR agonists ryanodine (50 nM, EtOH) and 4-CmC (10 µM, water) suppressed Q35 aggregation in a similar way to <i>gei-11</i> RNAi, but were less efficient in Q35<i>;egl-19(n582)</i> hypomorphic mutant animals. Treatment with the RYR antagonist DS (DMSO) together with <i>gei-11</i> RNAi prevented suppression of Q35 aggregation. Student t-test ***<i>p</i><0.001, ns/not significant; data relative to vector control in respective compound % solvent (±SD). (D) Real-time qPCR analysis of <i>hsp-70</i> (<i>C12C8.1, F44E5.4</i>) levels: RYR agonists Ryr (50 nM) and 4-CmC (10 µM) up-regulated <i>hsp-70</i> in wt animals but not in mutant <i>egl-19(n582</i>) animals. Chaperone induction by <i>gei-11</i> RNAi was prevented in the RYR mutant (<i>unc-68(kh30)</i>) and by co-treatment with DS (±SD). <i>gei-11</i> levels were 0.27±0.150 upon RNAi, relative to vector sample. (E) Model for <i>gei-11</i> modulation of proteostasis in BWM. [a] Knockdown of <i>gei-11</i> by RNAi leads to an increase in L-AChR expression at the NMJ (dashed line: proposed genetic interaction). [b] This causes a shift in the cholinergic/GABAergic signaling at the NMJ towards higher (thick arrow) excitatory signaling into the muscle. ACh binding to AChRs activates the VGCC EGL-19. [c] Depolarization, conformational changes and Ca²⁺ influx through EGL-19 triggers the opening of RYR at the SR and further release of Ca²⁺ into the cytosol [d]. Ca²⁺ activates signaling cascades to promote muscle contraction [e], HSF-1 activation [f ] and expression of cytosolic chaperones that rescue protein folding in the cytosol [g]. Dashed lines represent proposed and simplified sequence of events.</p