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2.628 Treffer

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  2. Detailansicht für Treffer 62 öffnen
    Figure 1. Endogenous VCP regulates mitochondrial fusion via negative regulation of Mfn protein levels. ; (A–C) MitoGFP localization to mitochondria serves as a marker for healthy mitochondria and their morphology in the indirect flight muscle. VCP overexpression (VCP OE, B) results in small mitochondria compared to wildtype (WT) flies (A); VCP RNAi expression results in elongated mitochondria (C). Scale Bar: 5 µm. (A’–C’’) Electronic microscopic (EM) images show that VCP OE (B’–B’’) results in smaller mitochondria as compared with WT (A’–A’’). VCP RNAi generates elongated mitochondria with intact cristae (outlined with white dashed lines in C’ and C’’). (A’–C’) Lower magnification; (A’’–C’’) Higher magnification of mitochondria outlined with black solid lines in A’–C’. Scale bar: 1 µm. (D): Schematic diagram of nurse cell mosaic analysis in Drosophila female germline. A stage 10A egg chamber has three types of cells. The monolayer follicle cells coat the surface of the egg chamber; 15 nurse cells provide proteins and RNAs to the oocyte during oogenesis. Heat shock induction of mitotic recombination during larvae stages results in the creation of a mosaic pattern in the adult nurse cells. Ubi-mRFP.NLS (Red) is used as a clone marker. Wildtype (WT) cells are labeled with two copies of RFP, +/+; heterozygous mutant cells have one copy of RFP and one copy of vcp loss-of-function mutant, vcpK15502/+; homozygous mutant cells are RFP negative and have two copies of vcp loss-of-function mutant, vcpK15502/vcpK15502. (E): A stage 10A egg chamber with a mosaic pattern of nurse cells. Red signal is Ubi-mRFP.NLS. Scale bar: 20 µm. (E’) Anti-ATP5A antibody staining is used to visualize mitochondrial morphology in nurse cells. Scale bar: 20 µm. (F–I) Higher magnification view of mitochondrial morphology in E’ (outlined in white solid lines). Mitochondria appear as discrete and punctate structures in wildtype (G) and heterozygous vcp mutant cells (F), but becomes elongated and clumped in homozygous vcp loss-of-function mutant cells (H and I). Scale bar: 5 µm. (J) A stage 10B egg chamber with a mosaic pattern of nurse cells. Red signal is Ubi-mRFP.NLS; Green signal is anti-GFP staining of pCasper-Mfn-eGFP. Scale bar: 20 µm. (J’–J’’) Higher magnification of the egg chamber in J (outlined in white solid lines). Wildtype cells are RFP positive and homozygous vcp loss-of-function mutant cells are RFP negative (J’). pCasper-Mfn-eGFP levels significantly increase in homozygous vcp loss-of-function mutant cells (J’’). Scale bar: 10 µm.
    2017
    unknown
    Wie komme ich dran?
  3. Detailansicht für Treffer 63 öffnen
    Figure 5. VCP disease mutants are hyperactive in downregulating Mfn protein levels and inhibiting mitochondrial fusion. ; (A–B’) Electronic microscopic images of muscle of different genotypes. Compared to WT (IFM-Gal4 control, A and A’), mitochondria are smaller with intact cristea in VCP WT (B and B’) 6 days after eclosion. Scale bar: 1 µm. (C–E’) Expression of VCP RH and AE leads to distorted fiber structure, and small mitochondria with broken or empty cristae. mfn RNAi flies have similar morphological defects in mitochondria (E and E’). Scale bar: 1 µm. (F) Expression of VCP RH and AE lead to a further reduction in Mfn as compared to VCP WT. Mfn levels in VCP WT are reduced to 0.73 ± 0.21 as compared with Gal4 control, which is set as 1 (p=0.037, independent t test, N = 3). Mfn levels in VCP RH and AE are reduced to 0.52 ± 0.14 (p=0.037, independent t test, N = 3) and 0.32 ± 0.16 (p=0.003, independent t test, N = 3) as compared with Gal4 control. Mfn levels are normalized with those of Hsp60, a mitochondria marker. Expression of VCP ATPase defective mutant E2Q (p=0.097, independent t test, N = 3) does not significantly change the Mfn level in fly thoraxes as compared with Gal4 control. n.s.: no statistical significance, p>0.05. Normalized Mfn levels are shown as mean±½SD. (G–L) MitoGFP localization assay shows expression of VCP WT (H), RH (I) and AE (J) potently rescues the mitochondrial defects in parkin mutant (G). Expression of the ATPase defective mutant E2Q (K) or VCP RNAi (L) does not. Scale bar: 20 µm. (M–R) MitoGFP assay shows expression of VCP WT (N), RH (O) and AE (P) potently rescues the mitochondrial defects in parkin mul1 double mutants (M). Expression of VCP E2Q (Q) and VCP RNAi (R) do not. Scale bar: 20 µm. (S) Western blot shows that increased Mfn levels normally present in a parkin null mutant are significantly decreased in VCP WT, RH and AE flies, but not in VCP E2Q flies. Mfn levels are further decreased in a parkin mutant expressing VCP RH (p=0.0228, independent t test, N = 3) or AE (p=0.00565, independent t test, N = 3), as compared to VCP WT. Samples are from 2-day-old fly thoraces. Normalized Mfn levels are shown as mean±½SD. *p
    2017
    unknown
    Wie komme ich dran?
  4. Detailansicht für Treffer 64 öffnen
    Figure 4. Expression of VCP disease mutants and mfn RNAi knocking down lead to pathology in adult muscle tissue. ; (A) Diagram of Human p97/VCP protein domains and two disease mutants, VCP R155H and A232E. Their corresponding Drosophila homologues are VCP R152H and A229E, hereafter referred to as VCP RH and AE. (B) Expression levels of UAS-VCP WT, RH and AE disease mutants expressed under IFM-Gal4 are comparable. (C) Diagram of Drosophila indirect flight muscle structure. Mitochondria (Green) and nuclei (Orange) are densely packed in between myofibrils (which contain large amounts of actin, Black). (D–D''') MitoGFP (Green) and TUNEL staining (Red). 6-day-old WT (IFM-Gal4 control) flies and VCP WT flies have healthy muscles (no TUNEL staining) and are MitoGFP positive (D and D'). 6-day-old VCP RH and AE expressing flies show high levels of TUNEL staining. 97 ± 3.6% and 95.7 ± 5.1% of RH (p=0.03. RH v.s. WT, independent t test) and AE (p=0.015, AE v.s. WT, independent t test) and are MitoGFP negative (D'' and D'''). Scale bar: 10 µm. (E–E''') Toluidine Blue staining of muscle in WT, VCP WT, RH and AE flies. Myofibrils are well aligned with densely packed mitochondria in WT and VCP WT flies 6 days after eclosion (E and E'). In VCP RH and AE flies fiber structure is disrupted, and mitochondria are misaligned and lightly stained, with empty spaces in between (E'' and E'''). Scale bar: 40 µm. (F–F''') Anti-TDP43 antibody staining shows nuclear (white arrowhead) and sarcoplasmic localization of TDP-43 in WT and VCP WT adult fly muscle (F and F'). In VCP RH and AE flies the nuclear signal disappears and the signal is increased in muscle sarcoplasm (F'' and F'''). Scale bar: 5 µm. (G–G''') Anti-Ref(2)P/p62 antibody staining. The signal is weak and uniform in WT and VCP WT flies (G and G'), and punctate in VCP RH and AE flies (G'' and G'''). Scale bar: 5 µm. (H–H''') Anti-P4D1 ubiquitin antibody staining. Signal is weak and uniform in WT and VCP WT flies (H' and H'), and punctate in VCP RH and AE flies (H'' and H'''). Scale bar: 5 µm. (I–M) Effects of mfn RNAi in 8-day-old adult muscle tissue. (I–M) Wildtype muscle visualized with TUNEL and mitoGFP (I), anti-TDP43 (J), Toluidine blue (K), anti-p62 (L), and anti-ubiquitin (M). (I'–M') mfn RNAi muscle visualized with the same probes as above. Scale bar: 30 µm in K-K’, 5 µm in the rest.
    2017
    unknown
    Wie komme ich dran?
  5. Detailansicht für Treffer 65 öffnen
    Figure 5—figure supplement 2. Mfn expression can suppress mitochondrial defects observed in VCP RH and AE flies. ; (A) 2 copies of genomic rescue Mfn-HA (pCasper-Mfn-HA) lead to a heterogeneous mitochondrial phenotype in which some mitochondria are elongated. A’ shows a typical elongated mitochondrion. (B–E’) In 2-day-old flies, expression of 2 copies pCasper-Mfn-HA (C, C’ and E, E’) results in suppression of mitochondrial defects (fragmented mitochondria with abnormal cristae) in VCP RH (B and B’) and AE (D and D’) flies. Scale bar: 1 µm.
    2017
    unknown
    Wie komme ich dran?
  6. Detailansicht für Treffer 66 öffnen
    Figure 5—figure supplement 1. Expression of VCP disease mutants leads to small mitochondria with abnormal cristae, phenotypes similar to mfn RNAi knocking down. ; (A–B’) 2 days after eclosion, EM shows that compared to WT (IFM-Gal4 control, A and A’), VCP WT expression results in smaller mitochondrial with intact cristae (B and B’). (C–E’) VCP RH and AE also have smaller and fragmented mitochondria, but the cristae are abnormal (C–D’), similar to the phenotype of mfn RNAi flies (E and E’). Scale bar: 1 µm.
    2017
    unknown
    Wie komme ich dran?
  7. Detailansicht für Treffer 67 öffnen
    Figure 5. p97 governs ER-OMM contact via the extraction of Mfn2 complexes. ; (A) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for the indicated time, separated by blue native- (BN-) and SDS-PAGE. (B, C) Immunoblot analysis of Mfn1- (B) and Mfn2- (C) containing complexes in NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT and C431S cells treated with 20 μM CCCP for four hours, separated by BN- and SDS-PAGE. (D) Mitochondria isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for one hour were, after solubilization in NP-40, incubated with 1 μM Usp2 for 30 min at 37°C prior to separation by SDS-PAGE. Red asterisks indicate ubiquitinated species of Mfn1 and Mfn2. Densitometry calculations for the Mfn1 and Mfn2 bands (shorter exposure) relative to CIII-core2 are shown under the respective immunoblots. (E) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP in the presence or absence of 25 μM NMS-873 for the indicated time, separated by blue native- (BN-) and SDS-PAGE. Red asterisks indicate ubiquinated Mfn species visible by SDS-PAGE, while the arrowhead denotes the unmodified band. (F) Representative TEM images of mitochondria in contact with ER (pseudocoloured blue) in U2OS:GFP-parkin cells treated with 20 μM CCCP (‘+CCCP’) for four hours in the presence or absence of 25 μM NMS-873. Scale bar, 500 nm. (G,H) Quantification of TEM from (F) in cells treated with (blue bars) or without (red bars) 20 μM CCCP for four hours. The percent of OMM per mitochondrion (G) and mitochondria per field (H) in contact with the ER was quantified. Bars represent mean ± SEM, n = 99 to 187 mitochondria in 12 to 14 fields per condition. n.s., not significant; *, p
    2018
    unknown
    Wie komme ich dran?
  8. Detailansicht für Treffer 68 öffnen
    Figure 3—figure supplement 3. Parkin recruitment kinetics in cells lacking both Mfns and other mitochondria-ER tethering factors. ; (A) Immunoblot analysis of whole-cell lysates from cells cultured in glucose or galactose transfected with control siRNA or siRNA targeting Mfn1 (‘siMfn1’), Mfn2 (‘siMfn2’) or both mitofusins (‘siMfn1 +2’). (B) Representative confocal images of GFP-parkin recruitment to mitochondria as a function of time in U2OS:GFP-parkin cells treated with 20 μM CCCP. Red asterisks indicate cells in which GFP-parkin has fully translocated to mitochondria. Scale bar, 20 microns. (C) Quantification of parkin recruitment in cells from (B). Data points represent mean ± SEM, n = 3 replicates cells per condition, with >100 cells counted per condition for each replicate. (D) Parkin recruitment at one hour CCCP in cells from (B) arranged as a histogram. Bars represent mean ± SEM. n.s., not significant; *, p100 cells counted per condition for each replicate. (I) Parkin recruitment at one hour CCCP in cells from (G) arranged as a histogram. Bars represent mean ± SEM. n.s., not significant; **, p
    2018
    unknown
    Wie komme ich dran?
  9. Detailansicht für Treffer 69 öffnen
    Pharmacophore-Based Design of Phenyl-[hydroxycyclohexyl] Cycloalkyl-Carboxamide Mitofusin Activators with Improved Neuronal Activity
    Xiawei Dang (8992400) ; Sidney B. Williams (11306623) ; et al.
    2021
    academicJournal
    Wie komme ich dran?
  10. Detailansicht für Treffer 70 öffnen
    Characteristics of primary antibodies used in this study.
    Aaron V. Bradshaw (11681739) ; Philip Campbell (9388052) ; et al.
    2021
    academicJournal
    Wie komme ich dran?
  11. Detailansicht für Treffer 71 öffnen
    Age and sex of PBMC donors.
    Aaron V. Bradshaw (11681739) ; Philip Campbell (9388052) ; et al.
    2021
    academicJournal
    Wie komme ich dran?
  12. Detailansicht für Treffer 72 öffnen
    Uncropped, unadjusted images of western blots.
    Aaron V. Bradshaw (11681739) ; Philip Campbell (9388052) ; et al.
    2021
    academicJournal
    Wie komme ich dran?
  13. Detailansicht für Treffer 73 öffnen
    Reverse transcriptase qPCR analysis of PINK1 in LCLs.
    Aaron V. Bradshaw (11681739) ; Philip Campbell (9388052) ; et al.
    2021
    academicJournal
    Wie komme ich dran?
  14. Detailansicht für Treffer 74 öffnen
    Analysis of PINK1 expression in different cell types.
    Aaron V. Bradshaw (11681739) ; Philip Campbell (9388052) ; et al.
    2021
    academicJournal
    Wie komme ich dran?
  15. Detailansicht für Treffer 75 öffnen
    Figure 5—figure supplement 3. Mfn expression does not significantly rescue the pathology in VCP RH and AE fly muscles. ; 2 copies of the pCasper-Mfn-HA/eGFP transgene in VCP RH and AE flies at 6 days of age does not significantly alter muscle cell death assayed by TUNEL/MitoGFP (A, A’ and F, F’, Scale bar: 10 µm), overall tissue disintegration (B, B’ and G, G’, Scale bar: 40 µm), TDP43 mislocalization (C, C’ and H, H’ Scale bar: 5 µm), or the frequency of p62 (D, D' and I, I', Scale bar: 5 µm) and ubiquitin positive aggregates (E, E’ and J-J’, Scale bar: 5 µm).
    2017
    unknown
    Wie komme ich dran?
  16. Detailansicht für Treffer 76 öffnen
    Figure 7. VCP inhibitors promote mitochondrial elongation. ; (A–A’’) MitoGFP assay for mitochondrial morphology. Compared to DMSO-fed flies, 10 µM NMS-873 or ML240 feeding results in more fused mitochondria in 2-day-old flies. Scale bar: 5 µm. (B–B’’ and C–C’’) In 6-day-old flies fed with 30 µM NMS-873 (B’) or 30 µM ML240 (B’’), elongated mitochondria (outlined in dashed white lines) are observed in muscle. Mitochondria are small in VCP WT flies (C) as compared with WT (IFM-Gal4 control, B). This phenotype is reversed by 30 µM NMS-873 (C’) or 30 µM ML240 (C’’) feeding and shifted towards a pro-fusion direction, as the mitochondria are also elongated as compared with WT (B). (B’’’ and C’’’) Statistical analysis of mitochondrial size in EM cross sections. In wildtype flies, 30 µM NMS-873 or ML240 feeding results in a mitochondrial size increase to 6.46 ± 0.44 µm2 (p=0.007, independent t test, N = 45) or 5.75 ± 0.48 µm2 (p=0.032, independent t test, N = 39) as compared to the DMSO group (3.45 ± 0.27 µm2, N = 47). VCP WT expression results in small mitochondria (1.78 ± 0.05 µm2, N = 68) as compared with WT (B). Feeding of 30 µM NMS-873 (5.41 ± 0.40 µm2, p=0.000, independent t test, N = 44) or ML240 (4.90 ± 0.42 µm2, p=0.000, independent t test, N = 45) reverses these effects. Mitochondria size is shown as mean±½SEM. (D) pCasper-Mfn-eGFP levels accumulate in a dose dependent manner when treated with NMS-873 at 0.5 µM and 1 µM for 14 hr.
    2017
    unknown
    Wie komme ich dran?
  18. Detailansicht für Treffer 78 öffnen
    MFN Right (Remix) (ft. Lil Wayne)
    Moser, Jeffrey
    In: Fixation Database of Music Videos, 2016
    academicJournal
    Wie komme ich dran?
  19. Detailansicht für Treffer 79 öffnen
    Do Retail MFN Clauses Lead to Softening of Competition?
    Sim, Justina ; Poh, Lip Hang ; et al.
    In: Asian Journal of Law and Economics ; volume 7, issue 1 ; ISSN 2154-4611 2194-6086, 2016
    academicJournal
    Wie komme ich dran?
  20. Detailansicht für Treffer 80 öffnen
    MFN CLAUSES AND DISPUTE RESOLUTION IN INVESTMENT TREATIES: HAVE WE REACHED THE END OF THE ROAD?
    Hobér, Kaj
    In: International Investment Law for the 21st Century ; page 31-41 ; ISBN 0199571341 9780199571345 9780191705472; (2009)
    Online Buch
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