Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration (2022)
Figure 2

Figure 2. DLK vesicle recruitment induces kinase activation and signaling upon stress.

A. Representative stills from time-lapse recordings of DIV6 DRG neurons overexpressing DLK-GFP. Arrowheads point to individual moving DLK-GFP positive structures.

B. Kymographs made from the complete time-lapse recording shown in A. The left shows the original kymograph, and the right shows an illustration of the manually traced structure displacements for better visualization.

C. Representative images of DIV3 DRG neurons nucleofected with a non-targeting siRNA (NT siRNA) or a siRNA against DLK (DLK siRNA), and stained for endogenous DLK and β3-tubulin (TUBB3). On the right, quantification of the DLK mean intensity in neurons expressing control NT or DLK siRNAs (n = 25 neurons/condition; Unpaired t test).

D-F. Representative kymographs (D), quantification of GFP-positive particle number (E) and percentage of anterograde, retrograde, reverse or non-moving GFP-positive particles (F) from DLK-GFP time-lapse recordings in DIV6 DRG neurons, in control situation (+ NGF) or after 3h of NGF deprivation (-NGF) (n = 45-46 neurons/ condition; Mann-Whitney U test in E and Chi-square test in F).

G. Representative images of DIV3 cultured embryonic DRG neurons maintained in the presence (+NGF) or absence (-NGF) of NGF for 3h and stained for endogenous DLK. On the right, quantification of the number of DLK- positive puncta in control situation or after 3h of NGF deprivation (n = 18-19 neurons/condition; Mann-Whitney U test).

H. Representative kymograph and stills from a time lapse recording of 6DIV neurons overexpressing a GFP-tagged N-terminal region of DLK (NtermDLK-GFP). Arrowheads point to individual moving NtermDLK-GFP positive structures.

I-J. Representative Western blots of p-cJun and GAPDH (I), and quantification of relative c-Jun phosphorylation levels (J) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK-GFP, and subjected to NGF withdrawal for 3h (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction).

K. Diagram of different DLK constructs fused with the transmembrane domain of VAPB (DLK-ER), the C-terminal CAAX motif of Ras proteins (DLK-CAAX) or the membrane targeting sequence of the ActA protein of Listeria monocytogenes (DLK-MTS), and their expected localization.

L. Representative images of HeLa cells expressing the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS), and stained with markers of the endoplasmic reticulum (KDEL), plasma membrane (CellMask) and mitochondria (cytochrome C).

M-N. Representative Western blots of DLK, p-cJun and GAPDH (M) and quantification of relative c-Jun phosphorylation levels (N) from HeLa cells transfected with HA alone (HA) and the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS) (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT or DLK-CS followed by a Holm-Sidak correction).

Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (B) and (C), 5 μm in (A), (D), (G), (H) and (L), and 2 μm in zooms in (C). See also Figure Appendix Figure S2.