Fig. 2: Proof-of-concept characterization of the Ly–Mt reporter. | Nature Communications

Fig. 2: Proof-of-concept characterization of the Ly–Mt reporter.

From: A SPLICS reporter reveals \({{{{{\boldsymbol{\alpha }}}}}}\)-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation

Fig. 2

a Cartoon of lysosome-mitochondria interface modulation by of Rab7A/TBC1D15 interaction. Rab7aT22N is a constitutively GDP-binding Rab7A mutant; TBC1D397A is constitutively inactive GTPase activating mutant and Rab7aQ67L is a constitutively GTP-binding Rab7A (Q67L). b Representative Z-projection merge images of HeLa cells transfected with SPLICSS/L- P2ALY-MT alone or plus Rab7A WT or Rab7A Q67L or Rab7A T22N or TBC1D15 WT or TBC1D15 D397A. In merge panels, SPLICSS/L- P2ALY-MT (488 nm), mCherry -Rab7A WT or -Rab7A Q67L or -Rab7A T22N (568 nm) and c-myc -TBC1D15 WT or -TBC1D15 D397A (594 nm) (red signal) are shown, mitochondria or lysosomes were detected by anti-Tom20 or anti-LAMP1 (405 nm) (blue and cyan fluorescence in pseudocolor, respectively). c Representative Z-projection images of HeLa cells transfected with SPLICSS/L- P2ALY-MT plus scramble or siRNA Rab7A #1 or siRNA Rab7A #2 or siRNA TBC1D15 #1 or siRNA TBC1D15 #2. Mitochondria morphology was detected by MitoTracker ® Red CMXRos upon excitation at 568 nm. d, e Quantification of short (Mean ± SEM: SPLICSS- P2ALY-MT + Mock 76.8 ± 3.5 n = 60, SPLICSS- P2ALY-MT + Rab7A 95.9 ± 9.6 n = 43, SPLICSS- P2ALY-MT + Rab7A (Q67L) 123.8 ± 9.8 n = 39, SPLICSS- P2ALY-MT + Rab7A (T22N) 71.9 ± 7.7 n = 37, SPLICSS- P2ALY-MT + TBC1D15 63.2 ± 5.5 n = 33, SPLICSS- P2ALY-MT + TBC1D15 (D397A) 103.2 ± 7.1 n = 38) and long (Mean ± SEM: SPLICSL- P2ALY-MT + Mock 97.4 ± 3.9 n = 52; SPLICSL- P2ALY-MT + Rab7A WT 105.8 ± 6.5 n = 30, SPLICSL- P2ALY-MT + Rab7A (Q67L) 106.4 ± 6.8 n = 31, SPLICSL- P2ALY-MT + Rab7A (T22N) 75 ± 5.8 n = 34, SPLICSL- P2ALY-MT + TBC1D15 WT 81.9 ± 4.6 n = 35, SPLICSL-P2ALY-MT + TBC1D15 (D397A) 108.5 ± 7.2 n = 37) LY-MT contacts in HeLa cells overexpressing WT and mutants Rab7 and TBC1D15. f, g Quantification of short (mean ± SEM: SPLICSS- P2ALY-MT + SCR 79.5 ± 5.8 n = 40; SPLICSS- P2ALY-MT + siRNA Rab7A #1 83.8 ± 8.1 n = 33; SPLICSS- P2ALY-MT + siRNA Rab7A #2 79.4 ± 6.2 n = 32; SPLICSS- P2ALY-MT + siRNA TBC1D15 #1 110.2 ± 7.1 n = 34; SPLICSS- P2ALY-MT + siRNA TBC1D15 #2 97.9 ± 6.3 n = 40) and long (mean ± SEM: SPLICSL- P2ALY-MT + SCR 107.1 ± 5.8 n = 47; SPLICSL- P2ALY-MT + siRNA Rab7A #1 80 ± 5.2 n = 39 *p ≤ 0.05; SPLICSL- P2ALY-MT + siRNA Rab7A #2 91 ± 7.7 n = 30, SPLICSL- P2ALY-MT + siRNA TBC1D15 #1 95.3 ± 8 n = 37; SPLICSL- P2ALY-MT + siRNA TBC1D15 #2 101.4 ± 9.1 n = 34) LY-MT contacts in HeLa cells upon downregulation of Rab7 and TBC1D15. The SPLICS dots were quantified from the 3D rendering of a complete Z-stack. Scale bar 10 μm. n= cells over 3 independent transfections. (*p ≤ 0.05, **p ≤ 0.01 one-way ANOVA). Source data are provided as a Source Data file.

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