Indeed, in contrast to fibroblasts bearing the I382M mutation, which displayed mild mitochondrial network fragmentation, fibroblasts with the p.R445H and p.S545R mutations were marked by the disappearance of filamentous mitochondria in favour of fragmented and punctuated mitochondria (patients 1–3) (Fig. ​(Fig.2B2B and Fig. S1). Quantification of the ratio of mitochondrial to cellular volume showed a 20% decrease of mitochondrial content in fibroblasts with the dominant‐negative p.R445H and p.S545R mutations (patients 1–3), compared to control fibroblasts, whereas no significant loss of mitochondrial volume was observed in the haploinsufficient OPA1 mutated fibroblasts (patients 4–7) (Fig. ​(Fig.2C).2C).

The effects of a previously identified T364M mutation and R619G mutation on IDUA activity were examined by transfection into COS-7 cells of the IDUA cDNA construct. When compared to that of wild type cDNA, the two point mutations showed only trace amounts of IDUA activity (1.2 and 1.5% respectively, Table 2). Table 2. Expression of α- l-iduronidase in transfected COS-7 cells Transfected vector α- l-Iduronidase activity (nmol mg−1 protein−1 h−1) Total (% of wild type) pcDNA3 20.9 0.0 pcDNA3-IDUA 90.2 100.0 pcDNA3-IDUA/T364M 21.7 1.2 pcDNA3-IDUA/R619G 21.9 1.5 The activity of endogenous α- l-iduronidase in COS-7 cells was subtracted before the percentage of wild type was calculated. Efficiency of transfection was similar in all dishes by 3′ IDUA cDNA PCR amplification of transfected genomic DNA (data not shown).

Mucopolysaccharidosis type I: identification of novel mutations that cause Hurler/Scheie syndrome in Chinese families.

While the Q785R mutation presented normal mtDNA and partially affected mitochondrial morphology, similar to the D603H mutation, the S545R mutation had an intermediate behavior, borderline across phenotypes. Indeed, the complete fragmentation of the mitochondrial network was associated with only a slight, non-significant reduction of mtDNA amount, supporting a borderline nature of this variant, causing either DOA or DOA “plus” possibly depending on modulation of other genetic and/or environmental modifiers. MEFs analysis suggests that a complete fragmentation of the mitochondrial network, in association with mtDNA depletion, hallmarks the DOA “plus” phenotype, whereas if these two parameters are mildly affected the final outcome is expected to be pure DOA. This hypothesis is represented in the scheme of Fig. 10.

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Download : Download high-res image (1MB)Download : Download full-size imageFig. 3. Mutated forms of fusion genes modify the shape of the mitochondrial network. Skin fibroblasts from patients with dominant mutations: MFN2:p.R94Q, OPA1:p.S545R and OPA1:p.R733X, show altered organization of the mitochondrial networks. (A) Representative images of controls and fibroblasts with OPA1:p.S545R and MFN2:p.R94Q mutations. Individual mitochondrial tubules are shown distinctively coloured. (B) Histograms indicate the number of individual mitochondrial tubules per total mitochondrial length; (C) the number of network branch points per total mitochondrial length; (D) the mitochondrial volume per cellular volume (convex hull algorithm); (E) and the bar diagram shows the distribution of mitochondrial lengths. Values of mutated skin fibroblasts are the mean ± s.d. of at least 40 measurements from two independent acquisitions. Control values are the mean ± s.d. of at least 160 measurements (X, XL or XXL cell size) from four healthy control cell lines. *P < 0.01, **P < 0.001, Mann–Whitney U-test. Scale bar: 10 μm.Dominant OPA1 mutations are associated with fragmented mitochondrial networks in fibroblasts from ADOA patients (Amati-Bonneau et al., 2005), and in HeLa cells transfected with OPA1 mutants (Olichon et al., 2007) but no reduction of mitochondrial volume has been described. In our study, the OPA1:p.S545R and OPA1:p.R733X mutations were associated with a higher ratio of individual mitochondrial tubules to total mitochondrial length (Fig. 3B). Unconnected mitochondria consisted of small tubular structures (Fig. 3E) with few dichotomous branches and a small number of connection points (Fig. 3C). Significantly, mitochondrial fragmentation in the OPA1:p.S545R cells was associated with a 20.5% reduction of mitochondrial volume compared to controls (P < 0.001; Mann–Whitney U-test) (Fig. 3D). The mitochondrial fragmentation observed in OPA1:p.R733X cells did not affect the total mitochondrial volume since the length of each individual tubule in these cells is longer than in OPA1:p.S545R cells.This fragmentation was associated with a disorganization of the mitochondrial network that did not affect cell size or shape. Interestingly, in OPA1:p.S545R cells, most mitochondrial tubular structures were found close to the MTOC (Fig. 3, Fig. 4) whereas isolated mitochondria were mainly found in the three extension arms of the triangular cell and around the nucleus. It would have been interesting to perform a statistical analysis of the variation of mitochondrial volume according to the various cell regions but the precise definition of these subregions for automatic anaylsis presented considerable difficulties. However, our visual analysis indicated that each region of the cell – central, perinuclear, and periplasmic – was equally affected by the reduction of mitochondrial volume in OPA1:p.S545R cells.Download : Download high-res image (848KB)Download : Download full-size imageFig. 4. Mitochondrial network of OPA1:p.S545R fibroblasts. (A) Mitochondrial network (in black) in control fibroblast (MitoTracker Green staining). (B) Mitochondrial network fragmentation induced by CCCP 10 μM 24 h. (C1–4) In OPA1:p.S545R fibroblasts, the major portion of the mitochondrial network was fragmented except for mitochondria emerging from the MTOC (white spots are supposed to indicate the position the MTOC). Representative images of at least 160 cells (X, XL or XXL cell size) from three control cell lines, acquired in two independent times. Scale bars: 10 μm.We noted that no tubular structures were preserved when mitochondrial fragmentation was induced by the mitochondrial uncoupler CCCP (Fig. 4B) or by siRNA OPA1 (supplementary material Fig. S4B).The expression of the GTPase-domain mutant of MFN2 (MFN2:p.K109T) in HeLa cells was shown to induce mitochondrial fragmentation (Detmer and Chan, 2007). However, standard fluorescent imaging revealed no mitochondrial network defects in heterozygous MFN2 CMT2A fibroblasts (Loiseau et al., 2007, Amiott et al., 2008). In contrast, our standardized technique of mitochondrial imaging showed that the missense mutation MFN2:p.R94Q induced mitochondrial biogenesis and the over-fusion of mitochondria. Reticulated mitochondria were distributed all over the cell (Fig. 3A) with a 24% higher number of mitochondrial network branch points compared to controls (P < 0.01; Mann–Whitney U-test) (Fig. 3C) and a 14.8% increase of mitochondrial content per cell compared to controls (P < 0.01; Mann–Whitney U-test) (Fig. 3D).In contrast to the mitochondrial fragmentation observed after neuroblastoma cell lines were transfected with GDAP1 mutants (Niemann et al., 2009), no alterations in mitochondrial network morphology were found in fibroblasts with the GDAP1:p.R120W mutation (Fig. 3).

Figure 3: Protein yield and ATP-binding capacity of 37 naturally occurring ABCR variants produced in transiently transfected 293 cells.Membranes were analysed by immunoblotting with affinity-purified anti-ABCR antibodies (top) and photoaffinity labelling with α-32P azido-ATP (bottom). We loaded 1 μg (immunoblotting) or 2.5 μg (azido-ATP labelling) of total membrane protein, as determined by Bradford assay, per track. The mutations that reside in NBD-1 and NBD-2 are indicated above the corresponding lanes. The relative levels of the different variant proteins and the extent of azido-ATP labelling were observed to be highly reproducible in multiple independent experiments. ABCR (large arrowhead); an endogenous 55-kD protein (small arrowhead) serves as an internal control for azido-ATP labelling. Molecular mass standards are shown on the left in kD.Full size imageThe combination of immunoblotting and azido-ATP labelling revealed defects in more than 75% of the variants tested. Among the variants with reduced yield and/or ATP binding are G863A and delG863, the two protein products of a guanosine2588→cytosine mutation that both generates a glycine-to-alanine substitution at codon 863 and activates a cryptic splice acceptor site in exon 17 that results in the removal of codon 863 from approximately 50% of the transcripts10. This is the most common allele among STGD patients in Northern Europe, representing roughly 20% of disease-associated alleles. It is also present at a frequency of approximately 3% in the general population in Northern Europe and approximately 1% in the United States population7,9,10. Genotype-phenotype correlations suggest that it is a mild allele and that it leads to STGD only when paired with a more severe allele10. Relative to wild type, the G863A variant is subtantially impaired and the delG863 variant is mildly impaired (Fig. 3).

Variants S206R and L541P have reduced basal ATPase and S206R shows little, and L541P shows no, stimulation by retinal (Fig. 4a).

both the P and PV ABCA4 variants exhibited a dramatically reduced basal ATPase activity (∼30% of WT ABCA4), which did not increase upon the addition of all-trans-retinal. Thus, ABCA4 variants carrying the P mutation were functionally impaired.

Amounts of A2E in the eyes of Abca4PV/PV, Abca4−/− and WT mice at the ages of 1, 3, 6, 12 and 15 months were quantified by reverse-phase high-performance liquid chromatography (HPLC) (Fig. 9B). Mice were raised under a regular 12-h light (∼10 lux)/12-h dark cycle. Age-dependent A2E accumulation was noted in all genotypes, with Abca4PV/PV and Abca4−/− mice accumulating about 5-fold more A2E than WT mice. No statistically significant differences in A2E accumulation were found between Abca4PV/PV and Abca4−/− animals.

Your eyes do not deceive you: Sony's nearly decades-old console has received a new update. Version 4.93 of the PS3 firmware is now available for anyone still kicking around on the company's decidedly retro home system, though you shouldn't expect a lot from the update.This update is the latest in an annual trend. Once a year for the last several years, Sony has updated the PS3 firmware and every time, the system's patch notes have trotted out the same line: "This system software update improves system performance."