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, Control, 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, Control, 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, Membrane, 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: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 1 Expression of the ER- resident GPx8 attenuates palmitate (PA)- mediated perturbation of intraorganellar Ca2+ dynamics. (A) Average trace of time-dependent changes in cytosolic free Ca2+ in control and GPx8 expressing INS-1E cells perifused under basal conditions (KR + 3 mmol/L glucose + 1% FFA-free BSA), followed by 0.5 mmol/L PA-containing KR solution for 5 min. Bars on the right represent the average peak amplitude of cytosolic free Ca2+ in the presence of PA. (B) Average curve of ER luminal and (C) mitochondrial Ca2+ dynamics in control and GPx8 cells transfected with the site-specific FRET-based Ca2+ sensors D1ER or 4mtD3cpv before and after perifusion with 0.5 mmol/L PA. The respective bars show the average depletion of ER luminal Ca2+ and the average peak amplitude of mitochondrial Ca2+ levels in the presence of PA, respectively. Data are means ± SEM of 7–10 independent experiments; *p < .05, **p < .01, ***p < .001 compared with INS-1E control cells (Student's t-test, unpaired, two-tailed).

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Expressing, Control, Transfection, Two Tailed Test

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 2 Oxidative metabolism of palmitate (PA) is required for the disruption of ER Ca2+ homeostasis. (A) Average trace of ER Ca2+ dynamics upon continuous perifusion with 0.5 mmol/L 2-BromoPA, followed by 0.5 mmol/L PA in INS-1E cells, and (B) after co-stimulation with 0.5 mmol/L 2-BromoPA +0.5 mmol/L PA. Data are means ± SEM of 3 independent experiments.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Disruption

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 3 Expression of the ER-resident GPx8 has no impact on carbachol (CCH) or thapsigargin (TG)-evoked ER Ca2+ depletion. (A and C) Average trace of time-dependent changes in cytosolic free-Ca2+ in control and GPx8 expressing INS-1E cells perifused for 5 min under basal conditions (3 mmol/L glucose-containing KR), followed by 5 min perifusion with 0.25 μmol/L TG or 0.1 mmol/L CCH. Bars on the right represent the average peak amplitude of cytosolic free-Ca2+ in the presence of TG or CCH. (B and D) show the average curves of ER luminal free-Ca2+ under the same perifusion conditions. The respective bars show the average depletion of ER luminal Ca2+ in the presence of TG or CCH. Data are means ± SEM of 5–6 independent experiments.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Expressing, Control

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 4 Expression of the ER-resident GPx8 diminishes palmitate (PA)-mediated reduction of the Ca2+-ATPase activity. (A) Effects of PA pre-exposure on cytosolic free-Ca2+ in control and GPx8 expressing INS-1E cells. Control and GPx8 expressing INS-1E cells were pre-incubated with 0.5 mmol/L PA for 24 h, loaded with the ratiometric Ca2+ indicator Fura-2/AM, and then subjected to perifusion with 0.25 μmol/L TG for 5 min. Each point on the curve represents means ± SEM of 4–6 independent experiments. (B) INS-1E control and GPx8 expressing cells were incubated under control conditions or with PA (0.5 and 1.0 mmol/L). After 24 h, RNA was isolated and relative gene expression of SERCA2b was analyzed by RT-qPCR and normalized to the housekeeping genes ß-actin, peptidylprolyl isomerase A, and ribosomal protein L32. Data are means ± SEM of 4 independent experiments. (C) Representative trace of ER luminal Ca2+ upon perifusion under basal conditions, 1 μmol/L ionomycin for 5 min, 100 μmol/L EGTA for 3 min, followed by 5 min perifusion with 1 mmol/L Ca2+- containing KR in INS-1E control- and GPx8 expression before and after pre-incubation with PA for 4 and 24 h. (D) Half-time (t1/2) of ER Ca2+ reuptake in INS-1E control- and GPx8 expressing cells treated under basal conditions or with PA for 4 or 24 h. Data are means ± SEM of 4 independent experiments; *p < .05, compared with INS-1E control cells (ANOVA/Dunnett's-test).

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Expressing, Activity Assay, Control, Incubation, Isolation, Gene Expression, Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 5 An intact GPx8 catalytic activity is required for protection against palmitate (PA)-induced ER Ca2+ dysregulation and toxicity. (A) Scheme depicting the Site-Directed Mutagenesis of GPx8 cysteine 79 by serine and cysteine 108 by alanine, respectively. (B) Immunoblot showing the expression of GPx8 mutant variants in stably transfected INS-1E cells compared to control cells. (C) Control INS-1E, GPx8-WT, GPx8C79S, and GPx8C79S/C108A transfected INS-1E cells were incubated under control conditions or with increasing PA concentrations for 24 h. Thereafter, viability was determined by MTT assay. Data are means ± SEM of 4–6 independent experiments; *p < .05, **p < .01, compared with control cells incubated under the same conditions (Student's t-test, unpaired, two-tailed). Average trace of time-dependent changes in ER- (D) and mitochondrial Ca2+ (E) in control and INS-1E cells expressing either GPx8C79S or GPx8C79S/C108A upon perifusion with PA. Data are means ± SEM of 3 independent experiments.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Activity Assay, Mutagenesis, Western Blot, Expressing, Stable Transfection, Transfection, Control, Incubation, MTT Assay, Two Tailed Test

Journal: The FASEB Journal

Article Title: Luminal H ₂ O ₂ promotes ER Ca ²⁺ dysregulation and toxicity of palmitate in insulin‐secreting INS‐1E cells

doi: 10.1096/fj.202201237r

Figure Lengend Snippet: FIGURE 8 Summary illustration showing the role of luminal H2O2 in the dysregulation of ER Ca2+ homeostasis unteder lipotoxic conditions. Elevated palmitate concentrations contibuted to increased luminal H2O2 levels that promoted ER Ca2+ depletion by inhibiting SERCA pump actvity. Excessive ER Ca2+ effux affected mitochondrial function manifested by depolarized mitochondrial membrane potential and lowered ATP production. Scavenging of luminal H2O2 by ER-specific antioxidative enzyme GPx8 attanuated palmitate-induced pertubations of intraorganellar Ca2+ dynamics preserved insulin synthesis and mitochondrial function under lipotoxic conditions.

Article Snippet: ER- luminal and mitochondrial Ca2+ fluctuations were measured by site- specific genetically encoded FRET- based Ca2+ biosensor Camelon D1ER and 4mtD3cpv (kind gift from Amy Palmer & Roger Tsien (Addgene plasmid #36325; http://n2t.net/addge ne:36325; RRID: Addgene_36325 and Addgene plasmid #36324; http:// n2t.net/addge ne:36324; RRID: Addgene_36324)).

Techniques: Membrane

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