Extended Data Fig. 7: Reconstitution of DRP1 KO cells with GTPase-active WT DRP1 rescues IDR-induced mitophagy. | Nature Cell Biology

Extended Data Fig. 7: Reconstitution of DRP1 KO cells with GTPase-active WT DRP1 rescues IDR-induced mitophagy.

From: The intrinsically disordered regions of organellophagy receptors are interchangeable and control organelle fragmentation, ER-phagy and mitophagy flux

Extended Data Fig. 7

(a) WT MEF were exposed to BafA1 to preserve mitochondria (labeled with TOMM20, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan). Mitochondria were delivered into the lumen of the endolysosomes in the absence of ectopically expressed DRP1. Scale bar: 10 µm, inset magnification: 4x. (b) DRP1 KO MEF cells transfected with Mito-GSEC62IDR show reduced delivery of mitochondria into the lumen of LAMP1-positive endolysosomes, demonstrating reduced mitophagy. (c) Co-transfection of mCherry-DRP1 construct rescues mitophagy induced by Mito-GSEC62IDR as shown by the increased prevalence of TOMM20-positive mitochondria inside LAMP1-positive endolysosomes. (d) GTPase-inactive mutant DRP1 K38A fails to rescue mitophagy inhibition in DRP1 KO MEF cells. (e) Same as (c) for Mito-GSEC62IDRLIR, showing no induction of mitophagy. (f-h) Same as (c-e) for Mito-GFAM134BIDR chimeras. (i-k) Same as (c-e) for Mito-GTEX264IDR chimeras. (l) LysoQuant quantification of the percentage of cellular endolysosomes that are degrading TOMM20-positive mitochondria fragments in panels (a-k) (N = 3 biological replicates for panels c-e, N = 2 for a-b and f-k, n = 9, 11, 40, 37, 28, 9, 5, 11, 12, 15, and 10 cells for panels a-k, respectively). Ordinary one-way ANOVA with Turkey’s multiple comparisons test. F = 22.48. Mean bar is shown. Adjusted P value: **** p < 0.0001, ***p = 0.0003, **p = 0082.

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