Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: INF2 formin variants linked to human inherited kidney disease reprogram the transcriptome, causing mitotic chaos and cell death

doi: 10.1007/s00018-024-05323-y

Figure Lengend Snippet: INF2 R218Q induces the formation of multipolar spindles in MDCK cells. A Percentage of Cherry and INF2 R218Q cells in mitosis after 48 h of expression. More than 1000 cells were analyzed for each experimental condition in five independent experiments. B Distribution of Cherry and INF2 R218Q cells across different phases of the cell cycle. More than 200 cells were analyzed for each experimental condition, four independent experiments. C Percentage of mitotic Cherry and INF2 R218Q cells displaying multipolar spindles. 68 Cherry cells and 152 INF2 R218Q cells were examined; four independent experiments. D Images of INF2 R218Q mitotic cells stained for centrin, α-tubulin and γ-tubulin. Nuclei were visualized using DAPI. E Classification of INF2 R218Q mitotic cells based on the number of MTOCs expressed as percentage of total cells. MTOCs were identified by γ-tubulin and α-tubulin staining. Over 200 cells were examined; four independent experiments. F Top: Schematic of centriole arrangements. Bottom: centriole arrangement, as visualized with centrin, in INF2 R218Q mitotic cells categorized as clustered in two centrosomes (2 + 2), disengaged in one (2 + 1 + 1) or both (1 + 1 + 1 + 1) centrosomes, or with an abnormal number of centrioles, presented as a percentage of total cells. More than 170 cells were examined; three independent experiments. G Top panel: schematic of ER invasion of the spindle space in cells expressing pathogenic INF2. Bottom panels: INF2 L76P cells stably expressing GFP-sec61 and stained with SiR-DNA analyzed by videomicroscopy during mitosis. The arrowheads indicate regions of the mitotic spindle invaded by ER membranes. H The graph shows the percentage of wt INF2 and INF2 R218Q cells with the spindle space invaded by ER membranes. More than 80 cells were analyzed; three independent experiments. I Videomicroscopic analysis of INF2 R218Q cells stably expressing GFP-sec61 and stained with SiR-DNA during formation of multiple micronuclei. Scale bars, 5 μm. n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001

Article Snippet: The DNA constructs expressing GFP fused to centrin-1 (# 72641), centrin-2 (# 41147), and centrin-3 (# 69746), CaM (# 47602), H2B (# 11680), sec61β (# 62008), HEC1 (# 114049) and α-tubulin (# 58197) were obtained from Addgene.

Techniques: Expressing, Staining, Stable Transfection

Journal: The Journal of Neuroscience

Article Title: PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation

doi: 10.1523/JNEUROSCI.1691-18.2019

Figure Lengend Snippet: 52 kDa PINK1 interacts with the ERAD machinery. A, PINK1 and Sec61β interaction in WT PINK1 stably-expressing HEK293T cells transfected with Sec61β evidenced by IP using either an anti-PINK1 or anti-Sec61β antibody. *Denotes IgG heavy chain. B, Endogenous PINK1 and Sec61β interaction in SH-SY5Y cells evidenced by IP using an anti-Sec61β antibody. C, WT PINK1 and endogenous gp78 interaction in stably PINK1 expressing HEK293T cells evidenced by co-IP using either an anti-PINK1 or anti-gp78 antibody. D, Ub-PINK1 IB in PINK1 stably-transfected HEK293T cells. Endogenous Sec61β was efficiently knocked down by Sec61β shRNAs. E, Ub-PINK1 IB in PINK1 stably-transfected HEK293T cells. Endogenous gp78 was efficiently knocked down by gp78 shRNA validated by IB. F, Ub-PINK1 IB in Hrd1 deficient HEK293T cells generated by CRISPR that were transfected with WT PINK1. Experiment shown in F was repeated once independently with similar results while those shown in A–E are representative of 3–4 independent experiments performed under identical conditions.

Article Snippet: HA-Ubiquitin and GFP-Sec61β plasmids were from Addgene.

Techniques: Stable Transfection, Expressing, Transfection, Co-Immunoprecipitation Assay, shRNA, Generated, CRISPR

Journal: The Journal of Neuroscience

Article Title: PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation

doi: 10.1523/JNEUROSCI.1691-18.2019

Figure Lengend Snippet: PINK1 is localized in both the mitochondria and the ER. A, Colocalization between PINK1 and organelle markers. Cells were transiently transfected with PINK1 (red) and costained with cytochrome c (cyt c) or MitoTracker (mitochondria), Sec61β-GFP (ER), and GM130 (Golgi; green). Representative images were shown (left) and the quantification (right) was done by ImageJ software. Scale bar, 10 μm. **p < 0.001 difference from GM130, but p > 0.05 cyto C versus MitoTracker versus Sec61β (one-way ANOVA, F(3,8) = 48.13, p < 0.001, followed by a Holm-Sidak's post hoc test). B, COS-7 cells (ATCC) were transfected with PINK1 expression vectors and ER marker Sec61β-GFP or mitochondrial marker Mito-DsRed. Images were acquired in 3D-SIM mode using excitation at 488 and 561 nm and standard filter sets for green and red emission. Image z-stacks were collected with a z interval of 200 nm. SIM image reconstruction, channel alignment, and 3D reconstruction were performed using NIS-Elements AR and Fiji (Schindelin et al., 2012). SIM reconstructed images were threshold, using consistent thresholds for each channel. A binarized mask was created for each channel. Representative images of ER or mitochondria (left) PINK1 (center), and a composition of the PINK1 mask (yellow) on top of the ER/mitochondria mask (red) are shown. Unmasked overlays are shown in the first panel. Scale bar, 2 μm. C, PINK1 IB of purified subcellular fractions from HeLa cells (n = 3). Endogenous PINK1 in was enriched by MG132 treatment for 6 h, and then proceeded for IP using anti-PINK1 antibody. Note that 52 kDa PINK1 migrates slightly above the heavy chain of IgG (*).

Article Snippet: HA-Ubiquitin and GFP-Sec61β plasmids were from Addgene.

Techniques: Transfection, Software, Expressing, Marker, Purification

Journal: The Journal of Neuroscience

Article Title: PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation

doi: 10.1523/JNEUROSCI.1691-18.2019

Figure Lengend Snippet: Ub-PINK1 is found in both the ER and mitochondria. A, HeLa cells were cotransfected with PINK1 and IMS-APEX2, purified mitochondrial (mito) and ER fractions were used in the sequential pull-down. This experiment was repeated once independently with similar results. B, Mito-DsRed and Sec61β were used as mitochondrial and ER outer membrane markers, respectively. IMS-APEX2 specifically colocalized with mitochondrial marker Mito-DsRed in COS-7 cells. Scale bar, 10 μm. C, 293T cells were cotransfected with PINK1 and IMS-APEX2, labeled with biotin-phenol and processed for IP. Biotin-labeled protein was detected in elutes that was pulled-down by streptavidin magnetic beads using the total cell extract (Input). BIP, Sec61β, and calreticulin were used as ER markers; ATP5A, Smac, and Tom20 were used as mitochondrial markers; PKC and GAPDH were used as cytosolic markers. This experiment was repeated once independently with similar results. D, Ub-PINK1 IP followed by IB analysis of purified subcellular fractionation isolated from cotransfected PINK1 and HA-Ub HeLa cell exposed to MG132 (n = 3). E, F, Alkaline extraction (0.1 m Na2CO3, pH 11) of purified mitochondrial (E) or ER fractions (F) from the same cells as in Figure 3D followed by IP and IB analysis (n = 3). P, Particulate; S, supernatant. Tom70, Smac, and calnexin are markers of mitochondrial outer membrane, mitochondrial interspace and ER membrane integral proteins, respectively. G, Left, The Venus signal (yellow) was only observed in HeLa cells cotransfected with Vn-Ub and PINK1-Vc constructs. Middle, Ub-PINK1 signal (yellow) in HeLa cells coexpressing Vn-Ub and PINK1-Vc and immunostained with antibody against Tom20, Sec61β, KDEL, or GM130 (red). Scale bar, 10 μm. Right, Quantification of Ub-PINK1 and organelle marker colocalization index. *p < 0.005, **p < 0.001 difference from GM130 (one-way ANOVA, F(3,8) = 22.83, p < 0.001, followed by a Holm-Sidak's post hoc test). H, Top, PINK1 IB of purified subcellular fractions of HeLa cells expressing PINK1K137R. Bottom, PINK1 52 kDa:63 kDa ratio relative to WT PINK1 expressing cells. **p < 0.001, ***p < 0.0001 difference from WT controls (two-way ANOVA, interaction mutation × subcellular fraction: F(3,16) = 58.35, p < 0.001, followed by a Holm–Sidak's post hoc test). I, SIM imaging analysis was performed similar to that in Figure 3B, except that cells were transfected with PINK1K137R. Scale bar, 100 μm.

Article Snippet: HA-Ubiquitin and GFP-Sec61β plasmids were from Addgene.

Techniques: Purification, Marker, Labeling, Magnetic Beads, Fractionation, Isolation, Construct, Expressing, Mutagenesis, Imaging, Transfection

Journal: bioRxiv

Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

doi: 10.1101/2025.04.17.649323

Figure Lengend Snippet: (A) Schematic representation of the MERCdRED system. (B) Diagram of the MERCdRED-expressing vector. Two components of the MERCdRED system, Tomm20-GB and RA-Sec61β, were co-expressed from a single vector using a P2A sequence, which enables post-translational self-cleavage. (C) Diagram illustrating the establishment of the MERCdRED cell line. (D) Comparison of MERCdRED signals between live cells and cells fixed with 2% paraformaldehyde (PFA) and 0.5% glutaraldehyde (GA). (E) Representative live images of the MERCdRED cells (MERCdRED shown in magenta) transfected with plasmids encoding an ER marker GFP-Sec61β (yellow) or a mitochondrial marker Tomm20-iRFP (cyan). The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (F) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (E). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 29, 18 cells for the MERCdRED cells and the control cells (negative control) from five independent experiments. Statistical analysis was performed using two-tailed Student’s t-test. ****P < 0.0001

Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.

Techniques: Expressing, Plasmid Preparation, Sequencing, Comparison, Transfection, Marker, Control, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

doi: 10.1101/2025.04.17.649323

Figure Lengend Snippet: (A) Schematic representation depicting the depletion of the MERCS-tethering protein complex PDZD8 and FKBP8 in MERCdRED cells. Fkbp8 gene was knocked down using lentivirus-mediated shRNA infection, and Pdzd8 gene was knocked out by treating cells with 1 μM 4-hydroxy (4-OH) tamoxifen, which induces Cre/loxP-dependent conditional knockout. (B) Immunoblot analysis of Pdzd8 f/f ::Cre ERT2 MEFs infected with lentivirus carrying control shRNA or Fkbp8 shRNA, treated with or without 0.5 μM 4-OHT. Cell lysates were subjected to immunoblotting with antibodies to PDZD8, FKBP8, and α-tubulin. (C) Representative live images of MERCdRED cells with or without depletion of Pdzd8 and/or Fkbp8 . GFP-Sec61β (yellow) or Tomm20-iRFP (cyan) was used as an ER marker or a mitochondrial marker, respectively. Pdzd8 conditional knockout was indicated as Pdzd8 cKO, and Fkbp8 knockdown was shown as Fkbp8 KD. The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (D) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (C). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 17, 14, 15, 15 cells for control + control, Pdzd8 cKO + control, control + Fkbp8 KD, and Pdzd8 cKO + Fkbp8 KD, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. ****P < 0.0001, **P < 0.01, *P < 0.05.

Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.

Techniques: shRNA, Infection, Knock-Out, Western Blot, Control, Marker, Knockdown

Journal: bioRxiv

Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

doi: 10.1101/2025.04.17.649323

Figure Lengend Snippet: (A) Representative images from time-lapse imaging shown as Supplementary movie 1. Images were obtained at 0.5 Hz for 32 seconds (17 frames). MERCdRED-positive areas on mitochondria were shown in magenta, and Tomm20-iRFP (cyan) was used as a mitochondrial marker. Yellow arrowheads indicate newly emerging MERCdRED signals that appeared in the subsequent time frame relative to the previous representative frame. Data are representative of 20 cells from three independent experiments. (B) Representative tracking images of MERCdRED-positive puncta shown in Supplementary movie 1. Tracking images were generated using TrackMate (ImageJ plugin) across the images obtained in (A). The colors of trajectories indicate the speeds of MERCdRED-positive puncta (shown in white). (C) Relationship between the area and the speed of each MERCdRED punctum. The average speed and area of each punctum over the entire observation period were calculated from tracking images obtained in (B). (D) The average speed of small puncta (area < 0.05 μm 2 ; left side of the dotted line in (C)) and large puncta (area > 0.05 μm 2 ; right side of the dotted line in (C)) was calculated. Data are means ± s.e.m. of 762 small puncta and 300 large puncta in 20 cells from three independent experiments. Statistical analysis was performed using the two-tailed Mann-Whitney test. (E) Schematic representation depicting the nutritional starvation in the MERCdRED cells. The cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in fed condition, whereas they were incubated in EBSS for 5 hours in starved condition. (F) Representative images from live-cell imaging of MERCdRED cells in the fed or starved condition. GFP-Sec61β (yellow) or Tomm20-iRFP (cyan) was used as an ER marker or a mitochondrial marker, respectively. The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (G) Quantification of the mitochondrial area in images obtained as described (F). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 14, 13, 12, 12 cells for fed + control, starved + control, fed + Pdzd8 cKO, and starved + Pdzd8 cKO, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. n.s., not significant. (H) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (F). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 14, 13, 12, 12 cells for fed + control, starved + control, fed + Pdzd8 cKO, and starved + Pdzd8 cKO, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01.

Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.

Techniques: Imaging, Marker, Generated, Two Tailed Test, MANN-WHITNEY, Incubation, Live Cell Imaging, Control