Figure 2: Membrane targeting enhances the efficiency of miniSOG mediated cholinergic neuron ablation.
From: Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG
(a) Photoablation of cholinergic motor neurons by PH-miniSOG results in uncoordinated movement immediately after 4 min blue light illumination. Scale, 250 μm. (b) Quantitation of Unc animals immediately after blue light illumination, for the indicated times. Membrane targeted miniSOG transgenic animals required shorter illumination times to display comparable Unc phenotypes. Numbers are the animals that were analyzed in three independent experiments. (c) Morphology of cholinergic motor neurons 4 h and 24 h after 4 min blue light illumination of PH-miniSOG. Motor commissures have disappeared and the remaining dorsal and ventral cord processes have degenerated into puncta. The unc-17β and acr-2 promoters are both expressed in the DA, DB, VA and VB neurons. White arrows indicate ventral and dorsal nerve cords. White arrowheads indicate motor neurons, red arrowheads indicate accumulation of mCherry in the epidermis, and yellow arrow indicates accumulation of mCherry in coelomocytes. Scale, 20 μm. (d) Quantitation of locomotion velocity immediately after blue light illumination (2 min, 2 Hz). PH-miniSOG transgenic animal displayed significantly reduced speed compared to mito-miniSOG. Worm locomotion was analyzed by multi worm tracker; in this and subsequent panels n in bars denotes numbers of animals analyzed in three independent experiments. mean ± SEM. ***P < 0.001, t-test. (e) Representative morphology of cholinergic motor neurons before and 6 h after 3 min continuous blue light illumination in WT and sod-1(tm776) mutant animals expressing Punc-17β-PH-miniSOG. Blue light illumination was performed on L3 larvae, and animals imaged 6 h later (early L4 stage). White arrows indicate damaged cell bodies in the ventral nerve cord. The damaged cells eventually died. Yellow arrow indicates accumulation of mCherry in coelomocytes. Scale, 10 μm. (f) Quantitation of damaged VNC cholinergic motor neurons 6 h after 1 min and 3 min continuous blue light illumination (see representative images in Panel c) in WT and sod-1(tm776) mutant animals. n, number of cells. ns, not significant, Student’s t-test.
