Fig 1: PO relocation in response to ACBD5 expression does not depend on the interaction with VAPB or acyl-CoA binding in neurons.(a, b) Localization of VAPB and (a) EGFP-SKL and (b) myc-ACBD5 in hippocampal neurons. MAP2-staining (in blue) was used as a neuronal dendrite marker (scale bar: 10 μm). (c, d) PO localization in neurons co-transfected with cytosolic mPlum-N1 and (c) FLAG-ACBD5-WT or (d) FLAG-ACBD5-FFAT or (e) FLAG-ACBD5-ACB expressing ACBD5 FFAT and ACB mutant proteins deficient in VAP or acyl-CoA binding, respectively. Both mutant proteins induce comparable PO relocations as compared to the wildtype ACBD5. Insets: Higher magnification of POs in close proximity to the plasma membrane (arrowheads) of dendrites (scale bar: 10 μm).
Fig 2: ER-mitochondria and ER-peroxisome contacts are required for HCMV-driven organelle remodeling.A Model for ER-mitochondria contact remodeling during the HCMV infectious cycle. Contact is decreased early to evade STING-TBK1-IRF3 signaling induced by increased ER-mitochondria tethering, a shared function with HSV-1 and likely Infl. A. Late in infection, contact is increased and restructured into ER-mitochondria encapsulations (MENCs) that recruit VAP-B and then PTPIP51, which increase in tethering interactions, and PTPIP51 is required for HCMV production. B Model for ER-peroxisome contact functions during HCMV infection. Early in infection, increased ACBD5-mediated contact prevents virus-induced peroxisome proliferation and restricts HCMV, HSV-1, Infl. A, and HCoV-OC43 production. As infection progresses, ER-peroxisome contact is increased and enriched at the enlarged peroxisomes formed by infection, which require ACBD5-VAP-B tethering to form.
Fig 3: PO morphology and distribution in primary hippocampal neurons in response to the overexpression of myc-ACBD5.(a, b) POs in neurons co-transfected with plasmids encoding cytosolic mCherry as well as (a) EGFP-SKL and (b) myc-ACBD5 respectively. (c, d) PO distribution in astrocytes of the primary hippocampal cultures with (d) or without (c) myc-ACBD5 expression. Astrocytes were identified by GFAP immunostaining. Note that POs in astrocytes unlike in neurons are similarly distributed in both conditions. (e) PO numbers and (f) area distribution (μm2) in the soma and neurites (proximal 30 μm). (g, h) Percentage areas of POs in soma (g) and neurites (h) (proximal 30 μm). (i) Total PO number and (j) area covered by POs in transfected neurons. Representative immunofluorescence images are presented as maximum intensity projections (scale bar: 10 μm).
Fig 4: Autophagy inhibitors do not restore peroxisomal import of PTS1- and PTS2-targeted peroxisomal matrix proteins in primary PEX1-G843D fibroblasts. Immunoblot analysis of homogenates of primary fibroblasts from two PBD-ZSD patients homozygous for the PEX1-G843D mutation [PEX1-G843D(1) (A) and PEX1-G843D(2) (B)] treated as described in Figure 1A. The protein levels of the PMPs ACBD5 and ABCD3 and the ratio of the processed over unprocessed forms of PTS1-targeted ACOX1 and PTS2- targeted thiolase were determined using specific antibodies. Below the ACOX1 bands, the corresponding ratios between the intensity of processed (50 kDa) over unprocessed (70 kDa) ACOX1 bands are indicated. Below the thiolase bands, the corresponding ratios between the intensity of processed (41 kDa) over unprocessed (44 kDa) thiolase bands are indicated. Below the ACBD5 and ABCD3 bands, the corresponding signal intensity relative to the tubulin band intensity, which was used as a loading control, is indicated. Homogenates of non-treated control fibroblasts and PEX1-null fibroblasts (PEX1–/–) were used for reference. The effect of the different treatments on autophagy was assessed by determining the relative protein levels of the autophagy adaptor protein SQSTM1/P62 (P62; relative to tubulin levels) and the ratio of MAPILC3B-II (LC3-II) over MAPILC3B-I (LC3-I). Cells were incubated for 7 days with the different compounds, except for bafilomycin (24 h incubation). Experiments were repeated twice; shown are representative blots from one experiment.
Fig 5: Live confocal imaging analysis of POs and mitochondria in hippocampal neurons.(a) POs and mitochondria were identified by expression of EGFP-SKL and mCherry-mito-7, respectively (Scale bar: 10 μm). Images are displayed as maximum intensity projections. (b-d) Representative kymographs illustrating peroxisomal (b, c) and mitochondrial (d) movements. Kymograph (b) represents the region of the neurite highlighted in (a). The kymographs were generated from the proximal 30~40 μm of neurites, straight vertical lines show static POs and mitochondria; Fig 2B includes a rapid saltatory movement across approximately 10 μm. (e, f) Classification of PO and mitochondria motility into long range movements (> 10 μm), short range movements (5 μm– 10 μm), very short range movements (1 μm– 5 μm), and static organelles (< 1 μm) in myc-ACBD5 expressing and control neurons (total analysis time = 8 min). (g, h) Maximum and average speed (μm/s) of all measured POs and mitochondria in neurites of ACBD5 expressing and control cells. (j, k) Averaged total travelled distance (μm) of measured POs and mitochondria in ACBD5 expressing and control neurons. (i, l) Cumulative distribution of PO travelled distance and speed. (m) Directionality of PO movements in the neurites. Box plots show the PO motility as cumulative net displacement (μm) of anterograde and retrograde movements of all POs in response to ACBD5 expression. Proportions of anterograde (positive values) and retrograde (negative values) movements remain stable after ACBD expression. (n) Comparison of the maximal actual speed and average speed of POs with long range movement between the two groups. Number of organelles analyzed: in control neurons 323 POs, 307 mitochondria; in myc-ACBD5 expressing neurons 360 POs, 267 mitochondria.
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