Journal: Nature cell biology

Article Title: FMRP regulates MFF translation to locally direct mitochondrial fission in neurons.

doi: 10.1038/s41556-024-01544-2

Figure Lengend Snippet: Fig. 1 | FMRP granules associate with the ends and midzone of mitochondria in neurons. a, Confocal image showing the association of EGFP–FMRP with mitochondria in a DIV8 rat hippocampal neuron. EGFP–FMRP brightness is increased in the insets. Arrowheads indicate individual FMRP granules associated with mitochondria. b, DNA-PAINT images of EGFP–FMRP and TOM20-marked mitochondria in dendrites of DIV8 rat hippocampal neurons. DNA-PAINT localizations (left). Segmentation for individual mitochondria and nearby FMRP granules from high-density DNA-PAINT localizations (right). c, Quantification of distance to nearest mitochondrion for EGFP–FMRP DNA-PAINT clusters. n = 3 biological replicates. Data points are shaped according to biological replicate. Line and bars show mean ± s.d. d,e, Kymograph and time series depicting the localization of an EGFP–FMRP granule to the ends and midzone of an axonal mitochondrion in a DIV7 mouse cortical neuron. f, Trajectory depicting EGFP– FMRP granule location along mitochondria over time for e, where mitochondrial

Article Snippet: The following plasmids were used: mito-DsRed2 (a gift from T. Schwarz, Harvard Medical School), EGFP–FMRP (gift from G. Bassell, Emory University), GFP-POLG2 (gift from W. Copeland, NIH), Halo–FMRP (subcloned from EGFP–FMRP into pFN21A-HaloTag-CMV vector from Promega), COX8A–BFP (Goldsmith et al.49), LAMP1–Halo (Gallagher and Holzbaur50), GFP–MFF (Addgene, 49153), GFP-RAB7 (Addgene, 12605), GFP–RAB7 T22N (Addgene, 12660), RFP–RAB5 (Addgene, 14437), EGFP– DRP1 (subcloned from pcDNA 3.1-Drp1 (Addgene, 34706) into pEGFP-C1 vector), pCRISPRia-vs2 (Addgene, 84832), PGK 4xMito-mEmerald (Addgene, 200430), pUbC-OsTIR1-myc-IRES-scFv-sfGFP (Addgene, 84563), AID–SunTag–MFF (subcloned from GFP–MFF, MFF cDNA clone (Transomic BC000797) and pUbC-FLAG-24xSuntagV4-oxEBFPAID-baUTR1-24xMS2V5-Wpre (Addgene, 84561) into pEGFP-N1 vector with EGFP removed), EGFP–TWINKLE (subcloned from TWINKLE– APEX2-V5 (Addgene, 129705) into pEGFP-N1 vector), EGFP–TFAM (subcloned from pCellFree_G03 TFAM into pEGFP-N1 vector) and Halo–Stop (Cason et al. 51).

Techniques:

Journal: Nature cell biology

Article Title: FMRP regulates MFF translation to locally direct mitochondrial fission in neurons.

doi: 10.1038/s41556-024-01544-2

Figure Lengend Snippet: Fig. 2 | FMRP associates with the mitochondrial midzone before fission. a,b, Kymograph and time series from an axon of a DIV7 mouse cortical neuron depicting the localization of an EGFP–FMRP granule (arrowhead) to the mitochondrial midzone, followed by mitochondrial fission. c, Trajectory depicting EGFP–FMRP granule localization along mitochondria before fission for b, where mitochondrial length is normalized from 0 to 1. d, Time series from a dendrite of a DIV13 rat hippocampal neuron showing an EGFP–FMRP granule that marks a mitochondrial fission site and remains at the newly formed mitochondrial end. e, Percent of mitochondrial fission events marked by EGFP–FMRP in axons and dendrites of rat and mouse neurons. n = 5 biological

Article Snippet: The following plasmids were used: mito-DsRed2 (a gift from T. Schwarz, Harvard Medical School), EGFP–FMRP (gift from G. Bassell, Emory University), GFP-POLG2 (gift from W. Copeland, NIH), Halo–FMRP (subcloned from EGFP–FMRP into pFN21A-HaloTag-CMV vector from Promega), COX8A–BFP (Goldsmith et al.49), LAMP1–Halo (Gallagher and Holzbaur50), GFP–MFF (Addgene, 49153), GFP-RAB7 (Addgene, 12605), GFP–RAB7 T22N (Addgene, 12660), RFP–RAB5 (Addgene, 14437), EGFP– DRP1 (subcloned from pcDNA 3.1-Drp1 (Addgene, 34706) into pEGFP-C1 vector), pCRISPRia-vs2 (Addgene, 84832), PGK 4xMito-mEmerald (Addgene, 200430), pUbC-OsTIR1-myc-IRES-scFv-sfGFP (Addgene, 84563), AID–SunTag–MFF (subcloned from GFP–MFF, MFF cDNA clone (Transomic BC000797) and pUbC-FLAG-24xSuntagV4-oxEBFPAID-baUTR1-24xMS2V5-Wpre (Addgene, 84561) into pEGFP-N1 vector with EGFP removed), EGFP–TWINKLE (subcloned from TWINKLE– APEX2-V5 (Addgene, 129705) into pEGFP-N1 vector), EGFP–TFAM (subcloned from pCellFree_G03 TFAM into pEGFP-N1 vector) and Halo–Stop (Cason et al. 51).

Techniques:

Journal: Nature cell biology

Article Title: FMRP regulates MFF translation to locally direct mitochondrial fission in neurons.

doi: 10.1038/s41556-024-01544-2

Figure Lengend Snippet: Fig. 4 | Endolysosomes contribute to FMRP–mitochondria dynamics through Rab7 GTP hydrolysis. a, Time series showing a LAMP1-positive vesicle tethering an FMRP granule to a mitochondrion in a DIV10 rat hippocampal neuron dendrite. b, Time series and kymographs showing a LAMP1 vesicle moving with FMRP around a mitochondrion in a DIV10 rat axon. c, Percent of motile FMRP granules associated with Rab5, Rab7 and LAMP1 in axons of rat neurons. n = 21 Rab5, 19 Rab7, 12 LAMP1 axons, three biological replicates. Data points are colour-coded to replicate, with smaller points representing axons. d, Time series showing a LAMP1 vesicle contacting an FMRP granule at a mitochondrial fission site in a DIV10 rat dendrite. The fission site is indicated by arrowheads. e, Percent of mitochondrial fission events marked by EGFP–FMRP and LAMP1– Halo, Halo–FMRP and GFP–Rab7 or Halo–FMRP and RFP-Rab5 in axons and dendrites of rat neurons. n = 3 Rab5, 5 Rab7 and 3 LAMP1 biological replicates. f,g, Time series and kymographs showing FMRP–mitochondria dynamics in axons of DIV10 rat neurons expressing GFP–Rab7 (f) or GFP–Rab7T22N (g). h, Duration of FMRP–mitochondria contacts in axons of rat neurons expressing

Article Snippet: The following plasmids were used: mito-DsRed2 (a gift from T. Schwarz, Harvard Medical School), EGFP–FMRP (gift from G. Bassell, Emory University), GFP-POLG2 (gift from W. Copeland, NIH), Halo–FMRP (subcloned from EGFP–FMRP into pFN21A-HaloTag-CMV vector from Promega), COX8A–BFP (Goldsmith et al.49), LAMP1–Halo (Gallagher and Holzbaur50), GFP–MFF (Addgene, 49153), GFP-RAB7 (Addgene, 12605), GFP–RAB7 T22N (Addgene, 12660), RFP–RAB5 (Addgene, 14437), EGFP– DRP1 (subcloned from pcDNA 3.1-Drp1 (Addgene, 34706) into pEGFP-C1 vector), pCRISPRia-vs2 (Addgene, 84832), PGK 4xMito-mEmerald (Addgene, 200430), pUbC-OsTIR1-myc-IRES-scFv-sfGFP (Addgene, 84563), AID–SunTag–MFF (subcloned from GFP–MFF, MFF cDNA clone (Transomic BC000797) and pUbC-FLAG-24xSuntagV4-oxEBFPAID-baUTR1-24xMS2V5-Wpre (Addgene, 84561) into pEGFP-N1 vector with EGFP removed), EGFP–TWINKLE (subcloned from TWINKLE– APEX2-V5 (Addgene, 129705) into pEGFP-N1 vector), EGFP–TFAM (subcloned from pCellFree_G03 TFAM into pEGFP-N1 vector) and Halo–Stop (Cason et al. 51).

Techniques: Expressing