Fig. 1: ISGylation of DRP1 by HERC5 and effect on mitochondrial dynamics.

A A549 cell lysates were immunoprecipitated (IP) with anti-ISG15 antibody. Western blot (IB) analysis of extract with anti-DRP1 antibody shows co-immunoprecipitation (IP) of ISG15-modified DRP1. Note a shift in band size detected between input and immunoprecipitated samples. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. endogenous DRP1, ISGylated endogenous DRP1. B Cell lysates co-immunoprecipitated to verify interaction between HERC5 with DRP1. The proportion of lysate loaded as input and used for immunoprecipitation is denoted in brackets by ‘X’. endogenous DRP1, ISGylated endogenous DRP1. C Mock or HERC5 siRNAs-treated cell lysates were immunoprecipitated (IP) with anti-ISG15 antibody (left panel). Note Western blot (IB) analysis with anti-DRP1 antibody shows decrease in ISGylated-DRP1 in HERC5-depleted samples. Reverse co-IP on the right corroborates the same. HERC5 and VINCULIN confirm knockdown and loading efficiencies. D A549 cells treated with 10 ng/ml of human IFNα1 (hIFNα1) for 48 h were lyzed and checked for ISGylation of DRP1 by co-IP between ISG15 and DRP1. Note that treatment with hIFNα1 induces enhanced ISGylation of DRP1. Similarly hIFNα1 treatment results in higher ISG15 expression, VINCULIN served as loading control. The proportion of lysates and immunoprecipitates denoted by ‘X’ in brackets. endogenous DRP1, ISGylated endogenous DRP1. E Cells treated with the hIFNα1 as described in panel D and treated with MitoTracker Red FM were imaged under live-cell conditions. Scale bar, 20μm. Box plots showing quantification of mitochondrial length for the experiment. ~150 cells from 3 independent experiments were analysed. The central line and the plus (+) symbol in each box show the median and mean value, respectively. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. F A549 cells were transfected with indicated mCherry-tagged constructs and co-immunoprecipitated against ISG15 and DRP1. Note that K532R mutation in DRP1 compromises its ISGylation; ISGylated DRP1 band being more prominent in the dark exposure. ISG15 and VINCULIN served as loading control. ISGylated endogenous DRP1, ◄ ISG15-modified mCherry-tagged DRP1, endogenous DRP1, ← mCherry-tagged DRP1. G Cells were transfected with indicated mCherry-tagged constructs and treated with MitoTracker Green FM were imaged under live-cell conditions. Scale bar, 20μm. Box plots showing quantification of mitochondrial length for the experiment. ~200 cells from 3 independent experiments were analysed. The central line and the plus (+) symbol in each box show the median and mean value, respectively. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. H Experimental hypothesis regarding differential effects of post-translational modifications (ISGylation and ubiquitylation) of DRP1 on mitochondrial dynamics.