Fig. 3: Rhotekin2 shapes mitochondria in the dendritic arbor of hippocampal neurons and interacts with syndapin I in the brain. | Nature Communications

Fig. 3: Rhotekin2 shapes mitochondria in the dendritic arbor of hippocampal neurons and interacts with syndapin I in the brain.

From: Mitochondria are positioned at dendritic branch induction sites, a process requiring rhotekin2 and syndapin I

Fig. 3

a–d Sum intensity projections of anti-MAP2 immunostained primary hippocampal neurons (DIV6) that were transfected with scr. RNAi and IRES-mitoGFP (a) or RTKN2 RNAi and IRES-mitoGFP (b), RTKN2 RNAi and RTKN2*-IRES-mitoGFP (c) or RTKN2 RNAi and RTKN2*ΔKRAP-IRES-mitoGFP (d) at DIV4 (RTKN2*, wild-type, RNAi-insensitive rhotekin2; RTKN2*ΔKRAP, RNAi-insensitive rhotekin2 mutant lacking the syndapin binding site). Boxed areas are shown as magnifications below. Bars, 10 µm. e Determinations of the length of dendritic mitochondria employing mitoGFP and the skeleton software tool show a syndapin binding site-dependent function of rhotekin2 in mitochondria length control. f Length distributions for dendritic mitochondria of mitoGFP-transfected control neurons (scr. RNAi) and of rhotekin2-deficient neurons (RTKN2 RNAi). g Immunoblotting analyses of coprecipitation experiments showing that rhotekin2 interacts with endogenous syndapin I from mouse brain lysates. h Immunoblotting analyses of endogenous coimmunoprecipitation experiments. Guinea pig anti-rhotekin2 but not unrelated guinea pig antibodies (IgG) immunoprecipitated endogenous rhotekin2 and coimmunoprecipitated syndapin I from brain lysates. White lines/lanes indicate lanes of the blot omitted (g, h). i Scheme of a neuron highlighting the positions of the proximal and peripheral dendritic segments analyzed. (j, k) Analysis of mitochondrial distribution within peripheral (j) and proximal (k) dendritic arbor segments of 20 µm in length each. l, m Quantitative analyses of mitochondrial length in peripheral (l) and in proximal (m) dendritic segments of neurons demonstrating that the rhotekin2 loss-of-function effects, the successful rescue by reexpressing RNAi-insensitive rhotekin2 (RTKN2*) and the failed rescue attempt with RTKN2*∆KRAP were observed irrespective of the position of the mitochondria in the dendrites. All experiments shown as representative images (g, h) have been done at least twice with similar results. Data, mean ± SEM visualized as bar (e, l, m) and bar/dot plots (j, k), respectively. e, f Scr. RNAi, n = 909; RTKN2 RNAi, n = 1035; RTKN2 RNAi+RTKN2*, n = 926 and RTKN2 RNAi+RTKN2*ΔKRAP, n = 933 dendritic mitochondria in 63, 56, 64 and 57 neurons, respectively, from 5 independent coverslips and 2 neuronal preparations; j Peripheral, scr. RNAi, n = 60; RTKN2 RNAi, n = 68 dendritic segments. k Proximal, scr. RNAi, n = 63; RTKN2 RNAi, n = 76 dendritic segments. l Scr. RNAi, n = 418; RTKN2 RNAi, n = 536; RTKN2 RNAi+RTKN2*, n = 491 and RTKN2 RNAi+RTKN2*ΔKRAP, n = 527 mitochondria. m Scr. RNAi, n = 491; RTKN2 RNAi, n = 499; RTKN2 RNAi+RTKN2*, n = 435 and RTKN2 RNAi+RTKN2*ΔKRAP, n = 406 mitochondria from 63, 56, 64 and 57 neurons, respectively, in 5 independent coverslips and 2 neuronal preparations. Statistical significances, Kruskal-Wallis/Dunn´s post-test (e, l, m) and Mann-Whitney (j, k), respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. For exact P values ≥ 0.0001 see figure.

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