We used structured-illumination super-resolution microscopy (3D-SIM) to assess the relationship between mitochondria, Cenp-F and microtubules. This, together with the fact that Cenp-F is a microtubule-binding protein, suggested that Cenp-F mediated mitochondria–cytoskeleton interaction. Interestingly, during S/G2, Cenp-F was enriched at the distal tip of mitochondria projecting away from the cell centre ( Fig. Scale bars, 5 μm (top panel), 500 nm (bottom). Structured-illumination microscopy of U2OS cells stained with mitotracker-red (red), Cenp-F (green) and tubulin-α (white). ( e) Cenp-F colocalizes with mitochondria and microtubules. The right panels are higher magnifications of the boxed area. ( d) Cenp-F localizes to mitochondrial tips. The experiment was repeated at least three times. Number of selected regions: G1: 14 S/G2: 39, M: 48, cytokinesis: 17. For quantifications, 2–4 regions were selected per cell. M represents mitosis, from prometaphase to anaphase, * P value <10 −6 from a Mann–Whitney–Wilcoxon U-test. ( c) Quantification of Cenp-F–mitochondria colocalization in different cell cycle phases. Interphase cells were classified as G1 and S/G2 based on Cenp-F levels. Where indicated, cells were labelled with cell-cycle markers: α-Phosphorylated histone H3 (phospho-H3, blue) α-Aurora-B (Aur-B, blue). Immunofluorescence using an α-Cenp-F antibody (green in lower panel) of KERMIT cells expressing a mitochondrial marker (mtBFP, red in lower panel). ( a) Cenp-F staining is heterogeneous in a field of unsynchronized cells. In all panels, mitochondria are shown in red and Cenp-F in green. 2C), and must therefore indicate a regulated process. 1c) was not merely due to differences in Cenp-F levels ( Supplementary Fig. The profound difference in recruitment between early and late mitosis ( Fig. Strikingly, at the end of mitosis, Cenp-F was strongly recruited to mitochondria ( Fig. In addition, in S/G2, a fraction of Cenp-F was found on mitochondria ( Fig. Cenp-F showed expected localization patterns Cenp-F was undetectable in G1 and was found in the nucleus, the nuclear envelope, the kinetochores and the midbody in G2, early prophase, (pro)metaphase and cytokinesis, respectively ( Fig. We imaged Cenp-F together with cell cycle markers, including cyclin A that accumulates in G2 and is degraded in mitosis, phosphorylated histone H3 (Phospho-H3) that is a marker of mitotic chromatin and Aurora-B (Aur-B) that localizes to the midbody during cytokinesis 14. 1a), likely due to the fact that cells were at different cell cycle phases. Cenp-F displayed highly variable immunofluorescence staining from cell to cell ( Fig. To assess whether Cenp-F localized to the mitochondria, we generated a U2OS-derived stable cell line co-expressing a mitochondrial and an ER marker (mtBFP and sec61α-GFP, respectively), which we called KERMIT (for Kinesis of ER and MITochondria). Cenp-F localizes at mitochondria–microtubule interfaces Cenp-F interacts with several cytoskeletal components, such as Dynein motor complexes and microtubules themselves 9, 10, 11, 12, 13. Cenp-F functions at the kinetochore and participates in nuclear envelope disassembly. It is a large (367 kDa) coiled-coil protein that accumulates during the G2 cell cycle phase, culminating during mitosis 8. 1D) that showed non-labelled peptides enrichment: Miro1, Miro2 and centromeric protein F (Cenp-F).Ĭenp-F came as a surprise. Using this approach, we detected only three proteins ( Supplementary Fig. In such conditions, illegitimate proteins that bind Miro1 or the immunoprecipitation matrix consequent to cell lysis should derive from both the heavy-labelled and the non-labelled proteins, while bona fide interactors should be enriched for non-labelled proteins. We mixed cells expressing Flag-tagged Miro1 with 15N- 13C-Arg/ 15N- 13C-Lys-labelled non-transgenic cells, prior to lysis and immunoprecipitation ( Supplementary Fig. We therefore devised a stable-isotope amino-acid labelling in culture (SILAC) strategy 7. This analysis also identified a large number of unrelated proteins, indicating possible unspecific interactions. This analysis identified Miro2 in FLAG-Miro1 pull-downs and vice versa, indicating that Miro1 and Miro2 are part of the same complex and underscoring the validity of our approach ( Supplementary Data Set 1). To identify Miro interactors, we established stable HEK293 cell lines that expressed doxycycline-inducible 3 × Flag-tagged versions of the two Miro paralogues encoded in the human genome-Miro1 and Miro2-and used them for immunoprecipitation ( Supplementary Fig. Miro GTPases interact with the centromeric protein F
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