Journal: Molecular Biology of the Cell

Article Title: Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

doi: 10.1091/mbc.E09-10-0914

Figure Lengend Snippet: BAPTA, but not EGTA, stimulates COPII vesicle fusion. (A) Homotypic COPII vesicle fusion measured using the in vitro VSV-G heterotrimer cargo mixing assay. The no myc control includes radioactive VSV-G* vesicles, but not VSV-G-myc vesicles, to control for the specificity of the anti-myc immunoprecipitation. BAPTA and EGTA were included in fusion reactions at the indicated concentrations. The on ice reactions had the indicated chelators added after the fusion incubation, before detergent solubilization, to control for possible nonfusion-related effects of the chelators. (B) Titration of fusion reactions with BAPTA in the presence and absence of excess free Ca 2+ . Because the BAPTA stimulation curve is pushed to the right, it seems that only free BAPTA, and not the Ca 2+ -bound chelator, has a stimulatory effect on heterotrimer formation. (C) Structures of EGTA (black) and BAPTA (black plus red). (D) Effects of aminomethoxy (/AM) derivatives of BAPTA and EGTA on homotypic COPII vesicle fusion. Both /AM esters were able to partially mimic the effects of BAPTA, suggesting that the selective effects of BAPTA were due to its ability to rapidly chelate escaping luminal Ca 2+ . Fusion assay data are presented as means of duplicate determinations with error bars representing SE where larger than symbol size.

Article Snippet: The fluorescence resonance energy transfer-based Ca 2+ sensor D1ER was excited at 440 ± 21 nm (440AF21; Omega Optical, Brattleboro, VT), and emission was collected simultaneously at 535 and 480 nm with a single camera using an optical beam splitter (535 and 480 nm, Dual-View MicroImager; Optical Insights, Visitron Systems, Puchheim, Germany).

Techniques: In Vitro, Immunoprecipitation, Incubation, Titration, Single Vesicle Fusion Assay

Journal: Molecular Biology of the Cell

Article Title: Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

doi: 10.1091/mbc.E09-10-0914

Figure Lengend Snippet: ALG-2 regulates in vitro COPII vesicle homotypic fusion in a Ca 2+ -dependent manner. (A) Homotypic COPII vesicle fusion measured using the in vitro VSV-G heterotrimer cargo mixing assay. Purified GST, GST-ALG-2 wild-type, or GST-ALG-2 E47,114A mutant proteins were included in fusion incubations at the indicated concentrations. The ice control includes radioactive VSV-G* vesicles and VSV-G-myc vesicles, but the low temperature prevents a specific fusion signal. (B) PAGE gel on purified proteins used in A and C, stained with Coomassie Blue. Proteins were used in fusion assays in the same proportions as on the gel. (C) Fusion experiment using partially inhibitory doses of the proteins indicated along the bottom. The left panel experiment was conducted in the absence of BAPTA. The right panel experiment was conducted, in the same experiment, in the presence of 2 mM BAPTA. Fusion values for both panels are normalized to the positive control signal in the absence of BAPTA (set to 100%). BAPTA negates the specific inhibitory effect of GST-ALG-2 but not that of anti-syntaxin 5 antibody. Fusion assay data are presented as means of duplicate determinations, with error bars representing SE where they are larger than symbol size.

Article Snippet: The fluorescence resonance energy transfer-based Ca 2+ sensor D1ER was excited at 440 ± 21 nm (440AF21; Omega Optical, Brattleboro, VT), and emission was collected simultaneously at 535 and 480 nm with a single camera using an optical beam splitter (535 and 480 nm, Dual-View MicroImager; Optical Insights, Visitron Systems, Puchheim, Germany).

Techniques: In Vitro, Purification, Mutagenesis, Staining, Positive Control, Single Vesicle Fusion Assay

Journal: Molecular Biology of the Cell

Article Title: Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

doi: 10.1091/mbc.E09-10-0914

Figure Lengend Snippet: Luminal Ca 2+ and ALG-2 regulate the retention of select coat subunits on pre-Golgi fusion intermediates. (A) BAPTA specifically extracts select COPI and COPII subunits from forming pre-Golgi intermediates. Homotypic fusion in vitro assays were conducted in the absence or presence of 2 mM BAPTA or EGTA (indicated above), and immunoisolated using anti-myc antibodies. Vesicles were generated from VSV-G-myc transfected cells (+) or nontransfected cells (−) to demonstrate specificity of isolation. In addition, 1 μM purified sar1 T39N was included during the budding stage to demonstrate that the isolated intermediates are COPII derived. Immunoblotted proteins are indicated along left edge. IP3R3, a resident ER membrane protein, additionally demonstrates specificity of budding. (B) Quantitation of a similar immunoisolation experiment (see blot in Supplemental Figure S3). In this experiment, it can be seen that peripheral membrane protein ALG-2 is also sensitive to BAPTA, but p115 is not. (C) Similar immunoisolation experiment where purified ALG-2 is present during the fusion experiment. ALG-2 caused a dramatic retention of outer shell component sec31 but not the COPI component β-COP. The absence of ER resident luminal protein PDI demonstrates specificity of the intermediates isolated. A star indicates the position of cross-reactive antibody heavy chain bands. GMP-PNP shows an estimate of components present on a fully coated vesicle as opposed to components remaining on tethered/fused pre-Golgi intermediates (D) Quantitation of experiment from C. For quantitations (B and D), band intensities were normalized to the recovery of cargo VSV-G-myc in each condition before plotting them on a relative scale with the highest recovery set to 1. Differences in band intensities seen in the input cells lanes (A) were caused by using slightly denser cell suspensions for the nontransfected cell controls.

Article Snippet: The fluorescence resonance energy transfer-based Ca 2+ sensor D1ER was excited at 440 ± 21 nm (440AF21; Omega Optical, Brattleboro, VT), and emission was collected simultaneously at 535 and 480 nm with a single camera using an optical beam splitter (535 and 480 nm, Dual-View MicroImager; Optical Insights, Visitron Systems, Puchheim, Germany).

Techniques: In Vitro, Generated, Transfection, Isolation, Purification, Derivative Assay, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

doi: 10.1091/mbc.E09-10-0914

Figure Lengend Snippet: Luminal Ca 2+ regulates size of rbet1-positive pre-Golgi structures. (A) NRK cells were either mock treated, incubated at 15°C for 30 min, or treated with CPA to deplete luminal Ca 2+ (see Materials and Methods ), and then fixed and immunostained for rbet1 and the Golgi marker GM130. Shown are single focal planes from deconvolved widefield image stacks. (B) Cytosolic Ca 2+ dynamics during the CPA regimen to deplete luminal Ca 2+ . Fluorescence ratios are calibrated to free Ca 2+ values in Supplemental Figure S1A. (C) Luminal ER Ca 2+ dynamics during the CPA regimen. Fluorescence ratios are calibrated to free Ca 2+ values in Supplemental Figure S1B. (D–F) Quantitation of peripheral rbet1-positive objects in experiments such as described in A. Values represent per cell means derived from at least 20 randomly chosen cells. Error bars display SE. CX, cycloheximide; Tg, thapsigargin. In F, only objects that fall within the size bins indicated above each plot are included in each panel. Selected p values from two-tailed Student's t test are included. Areas of objects were calculated assuming that one image pixel width calibrates to 224 nm in the cell. Single-pixel objects (with calculated area 0.05 μm 2 ) are subresolution but were not eliminated from the size analysis; they did not contribute to the 0.2–0.45 μm 2 and >0.45 μm 2 size bins in F.

Article Snippet: The fluorescence resonance energy transfer-based Ca 2+ sensor D1ER was excited at 440 ± 21 nm (440AF21; Omega Optical, Brattleboro, VT), and emission was collected simultaneously at 535 and 480 nm with a single camera using an optical beam splitter (535 and 480 nm, Dual-View MicroImager; Optical Insights, Visitron Systems, Puchheim, Germany).

Techniques: Incubation, Marker, Fluorescence, Quantitation Assay, Derivative Assay, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

doi: 10.1091/mbc.E09-10-0914

Figure Lengend Snippet: Luminal Ca 2+ regulates the size of ERGIC but not ERES structures. (A) NRK cells were either mock treated or treated with CPA to deplete luminal Ca 2+ (see Materials and Methods ), then fixed and immunostained for p24 and the Golgi marker mannosidase II. Shown are single focal planes from deconvolved widefield image stacks. (B) Immunolabeling for rbet1 and sec16 under the same experimental conditions as described in A. (C) Quantitation of numbers of peripheral p24-positive objects in three size bins under the experimental conditions shown in A. (D) Quantitation of peripheral objects positive for rbet1 and sec16 from the experiment shown in B. All size bins were included in one analysis. The rbet1 and sec16 object data are from precisely the same set of double-labeled cells. Values represent means derived from at least 20 randomly chosen cells. Error bars display SE. Selected p values from two-tailed Student's t test are included.

Article Snippet: The fluorescence resonance energy transfer-based Ca 2+ sensor D1ER was excited at 440 ± 21 nm (440AF21; Omega Optical, Brattleboro, VT), and emission was collected simultaneously at 535 and 480 nm with a single camera using an optical beam splitter (535 and 480 nm, Dual-View MicroImager; Optical Insights, Visitron Systems, Puchheim, Germany).

Techniques: Marker, Immunolabeling, Quantitation Assay, Labeling, Derivative Assay, Two Tailed Test

Journal: PLoS Genetics

Article Title: Absence of the ER Cation Channel TMEM38B /TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

doi: 10.1371/journal.pgen.1006156

Figure Lengend Snippet: (A) Basal levels of [Ca 2+ ] i in control (C) and proband fibroblasts (P1, P2) and osteoblasts (P2). (B) Decreased Ca 2+ mobilization in TRIC-B deficient fibroblasts (red lines). ATP- and Ionomycin-stimulated Ca 2+ release, as well as the return to baseline levels, are decreased in proband (P2) cells. (C) Decreased Ca 2+ mobilization in TRIC-B deficient osteoblasts (red lines). (D) The intracellular Ca 2+ stores available for IP 3 R-mediated release are more rapidly depleted in Proband 1 fibroblasts (right) versus normal control cells (left). ATP-stimulated Ca 2+ release is abrogated within 10 minutes in proband cells compared to 30 minutes in control cells following inhibition of SERCA channels with thapsigargin (TG). (E) Measurement of ER luminal Ca 2+ using ER-localized Ca 2+ indicator. Each point represents the average of one measurement containing 1–7 cells. The difference between the steady-state and Ca 2+ -depleted FRET signal emitted by the D1ER chameleon class Ca 2+ sensor was equivalent in normal control and proband cells. (F) Quantitative RT-PCR in control (C) and proband (P1, P2, P3) cells. There is no significant difference in expression levels of SERCA2 ( ATP2A2 ) and IP3R1 ( ITPR1 ) in fibroblasts, but transcripts are significantly reduced in proband (P2) osteoblasts. (G) Immunoblots of control (C) and proband (P1, P2, P3) cell lysates demonstrate equivalent levels of SERCA2b and IP3R1 Ca 2+ channels. *, p < 0.05; **, p < 0.01.

Article Snippet: Normal and proband fibroblasts (5 x 10 5 cells) were transfected with 2 μg D1ER or with 1 μg of pmaxGFP plasmid by electroporation using a 4D-Nucleofector system (Lonza), seeded into covered chamber slides and cultured at 37°C in 5% CO 2 for 48 hr.

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Western Blot