CiteAb

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

In Nat Commun on 10 March 2025 by Tröger, J., Dahlhaus, R., et al.

Fig.2.E

Rhotekin2 associates with the outer mitochondrial membrane.a Sum intensity projections of ApoTome immunofluorescence images of NIH3T3 cells showing the localizations of endogenous rhotekin2 (RTKN2; red in merge) and cytochrome c (Cytc; mitochondrial marker; green in merge). The boxed area is show... more

Rhotekin2 associates with the outer mitochondrial membrane.a Sum intensity projections of ApoTome immunofluorescence images of NIH3T3 cells showing the localizations of endogenous rhotekin2 (RTKN2; red in merge) and cytochrome c (Cytc; mitochondrial marker; green in merge). The boxed area is shown at higher magnification as an inset in the respective image. The yellow line in the merge inset represents the line for showing an intensity profile histogram of fluorescence intensities of both channels, clearly demonstrating rhotekin2’s spatial overlap with cytochrome c (yellow in merges). Insets, enlargement of boxed area. Bar, 20 µm. b Pearson’s coefficient (calculated using the ImageJ plug-in JaCoP) of the overlap of the anti-RTKN2 and anti-cytochrome c signals (a). c Maximum intensity projections (MIPs) of anti-rhotekin2 (red in merge), anti-cytochrome c (Cytc; green in merge) and anti-MAP2 (neuronal marker for dendritic compartment; blue in merge) immunostained DIV10 hippocampal neurons showing that the localization of endogenous rhotekin2 overlaps with that of mitochondria in the cell soma and in dendrites. Insets, enlargement of boxed area. Bars, 10 µm. d Immunoblotting analyses of a fractionation experiment of NIH3T3 cells confirming that rhotekin2 is present and enriched at mitochondria (marker, inner mitochondrial membrane protein cytochrome c oxidase (COX IV) also showing enrichment in mitochondrial fractions). S, supernatant. P, pellet. Immunoblottings for further components reflecting other cellular compartments (insulin receptor tyrosine kinase (IRTK), plasma membrane; GM130, Golgi apparatus; GAPDH, cytosol) confirm the specific RTKN2 enrichment in the mitochondrial fractions. e Immunoblotting analyses of biochemical fractionations of brain homogenate obtained from adult rats towards mitochondria and of a Na2CO3 extraction of purified mitochondria. Markers of enriched mitochondria (COX IV) and markers as above in d representing other cellular compartments and additionally laminB1 representing the nucleus. f MIPs of high-resolution structured illumination microscopy (SIM) images showing two examples of dendritic mitochondria of hippocampal neurons (DIV7). Note that the anti-RTKN2 (red) immunolabeling extends beyond the localization of the immunolabeling of the internal mitochondrial protein cytochrome c (Cytc). Dashed line in right example delineates the course of the dendrite (as tracked by CherryF coexpression). Bars, 100 nm. g, h MIPs of non-permeabilized (w/o Triton X-100), isolated mouse brain mitochondria immunostained with antibodies against rhotekin2 and cytochrome c (g), as well as of Triton X-100-permeabilized mitochondria (h). Note that rhotekin2 was detected in both preparations (i.e. is associated with the cytosolic leaflet of the outer mitochondrial membrane), whereas cytochrome c immunodetection required membrane permeabilization (internal protein). Insets, enlargement of boxed area. Bars, 10 µm. i, j Immunoblotting analyses of Na2CO3-extracted and untreated mitochondria that were incubated with GST-RTKN2’ (i) and GST (j), respectively. Note that rhotekin2’ (pointed out by blue arrowhead) is able to bind to Na2CO3-extracted mitochondria. k, l Anti-GST immunoblottings of density gradient fractionations showing a membrane-binding ability of rhotekin2’. Liposomes were incubated with either GST-RTKN2’ (k) or GST (l). Note that GST-RTKN2’ was detected in the liposome-containing fraction F2 (blue arrowhead), whereas GST-RTKN2’ stayed at the bottom of the gradient (F4-6) in the absence of liposomes (k). Grey arrowheads mark the F2 density gradient fractions of control conditions devoid of accumulated, floating protein. All experiments shown as representative images (a, c–l) have been done at least twice with similar results. less

Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license

Product Citations: 480

41 images found

Fig.2.E

Fig.2.C

Fig.3.D

Fig.2.A

Fig.6.A

Fig.6.B

Fig.3.C

Fig.4.E

Fig.3.A

Fig.6.B

Fig.4.D

Fig.1.F

Fig.1.D

Fig.1.F

Fig.2.A

Fig.3.A

Fig.3.F

Fig.4.A

Fig.3.D

Fig.3.E

Fig.4.A

Fig.6.A

Fig.5.B

Fig.7.C

Fig.1.B