Journal: The Journal of cell biology

Article Title: PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

doi: 10.1083/jcb.202407166

Figure Lengend Snippet: Figure 3. PITPβ promotes contact between the ER and COPI buds on the Golgi. Quantitative data are shown as mean ± SD, with the number of in- dependent experiments indicated. Statistics was performed using the two-tailed Student’s t test: ****P < 0.0001, ***P < 0.001, **P < 0.01, ns (non-significant) P > 0.05. (A) Colocalization of PITPβ with ER marker (Sec61β) and Golgi marker (giantin) as assessed by confocal microscopy using Airyscan, PITPβ (blue), Sec61β (green), giantin (magenta), n = 6. Representative images with scale bars are shown with inset highlighting PITPβ colocalizing with both Sec61β and Giantin (indicated by arrowheads). A reconstruction of this region is also shown, with a line scan along the dotted line providing quantitative information, as well as single-channel images. (B) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a Golgi marker (Giantin). Quantitation of a representative experiment is shown, n = 3. (C) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a TGN marker (TGN46). Quantitation of a representative experiment is shown, n = 3. (D) EM tomography showing COPI buds at the Golgi in close proximity to the ER membrane. A representative tomographic image slice is shown in the upper left panel with arrowheads pointing to COPI buds, bar = 200 nm. 3D reconstruction of the Golgi, COPI buds, and ER elements is shown in the lower left panel. Quantitation of a representative experiment is shown on the right, comparing the distance between COPI buds and ER membranes versus the distance between Golgi cisternal margins and ER membranes, n = 3. (E) Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and giantin. Quantitation of a representative experiment is shown on right, n = 3. Representative confocal images are shown on left, PLA signal (red),

Article Snippet: VAP-A (104447) and VAP-B (104448) in pEGFP-C1 and mEmerald-tagged Sec61β in pEGFP-C1 (90992) were obtained from Addgene.

Techniques: Two Tailed Test, Marker, Confocal Microscopy, Quantitation Assay, Tomography, Membrane, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture

doi: 10.1101/2023.12.21.572811

Figure Lengend Snippet: BAF controls rupture diffusion in a size-dependent manner. A) Measurements of the width of the NE rupture gap in BJ5ta cells expressing GFP-Sec61β. Number of cells analyzed: siControl, n=15; siBAF, n=14. Error bars indicate ± SEM from triplicate experiments. B) Representative images of BJ5ta cells expressing either Hsp90-GFP or α-Tubulin-GFP after laser-induced NE rupture. Scale bar; 10 µm. C) Quantification of the nuclear-to-cytoplasmic ratio of cells expressing either Hsp90-GFP or α-tubulin-GFP treated with either siControl (n = 29 and 14, respectively) or siBAF (n = 21 and 16, respectively) from triplicate experiments. Error bars indicate ± SEM. D) Initial rate of increase into the nucleus following NE rupture for cells in B. E) Representative images of BJ5ta cells expressing Hsp90-GFP or α-tubulin-GFP during FLIP. Green circle indicates area of photobleaching in the cytoplasm. Red circle indicates area of measurement. F) FLIP measurements showing relative mobility of Hsp90-GFP (n = 16 cells) or α-tubulin-GFP (n = 16 cells). The slope of each line x was used to factor relative mobility between Hsp90-GFP and α-tubulin-GFP. G) Mobility of Hsp90-GFP and α-tubulin-GFP (measured as the best line-of-fit slope for each individual cell). H) Initial rate of increase into the nucleus following NE rupture for cells in D normalized by mobility. Error bars indicate ± SEM. Statistical significance: *, P<0.05; **<0.005; ***, P<0.0005 using an unpaired student t-test.

Article Snippet: GFP-Sec61β in pBabe puro (a generous gift from Indra Chandrasekar) was created by PCR amplifying Sec61β (a gift from Gia Voeltz, Addgene plasmid #49154) and recombining into XhoI-SalI cut GFP pBabe puro.

Techniques: Diffusion-based Assay, Expressing

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: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Sec61β protein and Gurken mRNA in ovaries from wild type and sec61β P1 germline clones . A) Ovaries were dissected from flies and lysed in buffer containing 2% SDS. Western blotting was performed using Sec61β antibody. A cross-reactive band serves as a loading control. B) Schematic representation of a stage 9–10 egg chamber with the oocyte abutting the 15 nurse cells and surrounded by a layer of somatic follicle cells. The dorsal/anterior corner is depicted in dark red. C-D) Drosophila egg chambers were processed for gurken RNA in-situ hybridization in wild type oocytes, C) and in oocytes mutant for Sec61β, D). n represents the oocyte nucleus.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Clone Assay, Western Blot, Control, RNA In Situ Hybridization, Mutagenesis

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Localization of Gurken Protein in Stage 10 Egg Chambers of Wild type and sec61β P1 Germline Clones . Drosophila egg chambers of the indicated genotype, wild type, (WT) or egg chambers mutant for sec61β, (sec61β), stained for actin using labelled Phalloidin (blue) and Gurken (red). A-B) Low magnification images of wild type (A) and sec61β mutant egg chamber (B) at stage 10; Gurken appears at the anterior-dorsal position. C-D) Magnification of the anterior-dorsal region of wild type and sec61β mutant oocytes. Gurken co-localizes with actin in the wild type oocytes (C), where as the sec61β mutant oocytes do not show this co-localization (D). E S1-S4 -F S1-S4 ) Optical sections of the egg chambers as shown in C and D, correspond to either wild type egg chambers (E S1-S4 ) or sec61β mutant egg chambers (F S1-S4 ). G-H) High magnification images of oocytes showing the region of the follicle cells from wild type (G) and sec61β mutant egg chambers (H). Gurken is seen in dot like structures within the follicle cells in the wild type egg chambers (indicated by arrows), these structures are absent in the mutant egg chambers.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Clone Assay, Mutagenesis, Staining

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Localization of Gurken Protein in Egg Chambers of Wild type and sec61β P1 Germline Clones during earlier stages of Oogenesis . Drosophila egg chambers of the indicated genotype, wild type, (WT) or egg chambers mutant for sec61β, (sec61β), stained for actin using labelled Phalloidin (blue) and Gurken (red). A -B) Staining for Gurken protein (A) in the stage 6 oocyte from wild type, co-staining with actin (B) to delineate the follicle cells. C-D) Staining for Gurken protein (C) in sec61β mutant oocytes shows protein distributed all over the oocyte, co-staining with actin to delineate the follicle cells. f, indicates the follicle cells, o, the oocyte and n, the oocyte nucleus.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Clone Assay, Mutagenesis, Staining

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Analysis of ER structure and function in sec61β mutant oocytes . A-D, Drosophila egg chambers of the indicated genotype, wild type, (WT) or egg chambers mutant for sec61β, (sec61β), stained for actin using labelled Phalloidin (blue), Gurken (red) and Boca (green). A and C) In oocytes from stage 10 egg chambers Boca stains a diffuse region below the plasma membrane that marks the ER in both the wild type (A) and the sec61β P1 germline clones (C). B and D) Gurken staining is observed either along the plasma membrane or in distinct punctate structures (B) or Gurken is excluded from the plasma membrane and is present exclusively in the cytoplam (D). f, indicates the follicle cells, o, the oocyte and n, the oocyte nucleus. E-J, Drosophila egg chambers of the indicated genotype, wild type, (WT) or egg chambers mutant for sec61β, (sec61β), stained for actin using labelled Phalloidin (blue) and Yolkless (red). In wild type egg chambers (E) or egg chambers mutant for sec61β (H) Yl is at a peripheral location in the oocyte. Co-staining with phalloidin (F and I) shows that both in wild type oocytes and oocytes from the germline clones Yl co-localizes at the plasma membrane (G and J). n indicates the position of the nucleus.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Mutagenesis, Staining, Clinical Proteomics, Membrane, Clone Assay

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Expression of Gurken in HeLa cells . A) Plasmids encoding Gurken protein alone or in combination with Myc tagged Star and/or HA tagged Rho were transfected into HeLa cells as indicated on top of the figure or left un-transfected. Aliquots of the cell lysates were applied to SDS-PAGE either directly or after being treated with EndoH. Western bloting was done using an antibody against Gurken. The numbers on the right indicate the molecular mass in kilo Daltons. B-E) HeLa cells were transfected with plasmid generating a double stranded RNA against Sec61β or a control plasmid together with Gurken, Myc-Star and HA-Rho encoding plasmids as indicated. Cells lysates were applied to SDS-PAGE and western blot and probed with an antibody against Sec61β(B). The lysates were also probed with antibodies against Myc and HA to detect expression of Star and Rho respectively (C). The same sets of cells growing on plates were used for a 15 min pulse with 35 S methionine and cell lysates were prepared. These lysates were used for western blots with or without EndoH treatment and probed with the Gurken antibody (D). The lysates after the pulse analysis were subjected to immuno-precipitation using anti-Gurken antibody and immuno-precipitated samples were applied to SDS-PAGE either directly or after being treated with EndoH (E). Single glycosylated (0 g) and nonglycosylated (1 g) forms of Gurken are indicated.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Expressing, Transfection, SDS Page, Western Blot, Plasmid Preparation, Control, Immunoprecipitation

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Co-localization of Gurken with Lva . Wild type egg chambers and egg chambers derived from germline clones of sec61β P1 were stained with Gurken (Magenta), Lva (green) and Phalloindin (red). A-C) Wild type egg chambers with Gurken localized to the anterior-dorsal region of the ooctyte and also along the plasma membrane (A) and Lva localized in the cytoplasm of the oocyte (B). Co-staining of Gurken shows frequent overlap of the signal for Gurken and Lva, as indicated by the arrows (C). D-F) Egg chambers derived from the germline clones of sec61β P1 with Gurken localized to the anterior-dorsal end of the oocyte (D) and Lva localized in the cytoplasm of the oocyte (E). Co-staining of Gurken shows frequent overlap of the signal for Gurken and Lva, as indicated by the arrows (F). Insets show whole of the co-stained oocyte.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Derivative Assay, Clone Assay, Staining, Clinical Proteomics, Membrane

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Co-localization of Gurken with a Golgi Marker . Wild type egg chambers and egg chambers derived from germline clones of sec61β P1 were stained with Gurken (Magenta) and Phalloidin (red). The ubiquitously expressed Golgi complex marker is in green. A-C) Wild type egg chambers expressing the Golgi marker (A) and stained with Gurken, which is localized to the anterior-dorsal region of the oocyte (B). A magnification of the anterior-dorsal region of the oocyte showing frequent co-localization of punctuate Gurken containing structures with the Golgi marker (C). The inset is same as C but shows the whole egg chamber. D-F) Egg chambers mutant for sec61β expressing the Golgi marker (D) and stained with Gurken, which is localized to the anterior-dorsal region of the oocyte (E). A magnification of the anterior-dorsal region of the oocyte showing frequent co-localization of punctuate Gurken containing structures with the Golgi marker (F). The inset is same as F but shows the whole egg chamber. Co-localization is indicated by white arrows.

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Marker, Derivative Assay, Clone Assay, Staining, Expressing, Mutagenesis

Journal: BMC Cell Biology

Article Title: Sec61β, a subunit of the Sec61 protein translocation channel at the Endoplasmic Reticulum, is involved in the transport of Gurken to the plasma membrane.

doi: 10.1186/1471-2121-10-11

Figure Lengend Snippet: Localization of Sec5 in stage 10 oocytes . A-F) Wild type egg chambers or egg chambers from sec61β P1 germline clones were stained with Sec5 (red) and Phalloidin (green). In wild type egg chambers (A) or from sec61β P1 germline clones (D) Sec5 is at a peripheral location in the oocyte. Co-staining with phalloidin (B and E) shows that both in wild type oocytes and oocytes from the germline clones Sec5 co-localizes at the plasma membrane. n indicates the position of the nucleus (C and F).

Article Snippet: Antibodies used for western blotting were rabbit anti- Drosophila Sec61β (peptide used corresponds to amino acid 2–10 of the Drosophila sequence, PAPPSSTSV-C), anti-human Sec61β [ ], anti-Gurken 1D12 (Hybridoma bank), anti-Myc and anti-HA (Santa Cruz Biotech).

Techniques: Clone Assay, Staining, Clinical Proteomics, Membrane