QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 May 2005
doi: 10.1242/jcs.02338

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tadokoro, S. Articles by Hirashima, N. Search for Related Content PubMed PubMed Citation Articles by Tadokoro, S. Articles by Hirashima, N. Social Bookmarking                         What's this?

Complexin II facilitates exocytotic release in mast cells by enhancing Ca²⁺ sensitivity of the fusion process

Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan

View larger version (51K): [in a new window]   Fig. 1. Expression of complexin II in RBL-2H3 cells. (A) RT-PCR products amplified with specific primer pairs for complexins I and II were electrophoresed in agarose gel (1.5%). Far left and far right lanes are for the 100 bp ladder marker (M). PCR products amplified with primer pairs of complexin I (CPX I) and II (CPX II) using templates derived from rat brain (positive control) and RBL-2H3 cells are shown. The expected size of the product is about 400 bp for both isoforms. A single band in the lane for complexin II of RBL-2H3 cells was detected, and the product was identified as complexin II by DNA sequencing. (B) Western blot analysis for complexin. Cell lysates were prepared from RBL-2H3 cells, P815 cells and rat cerebrum and were electrophoresed by SDS-PAGE. After the samples were transferred to a PVDF membrane, blots were probed with primary antigens specific for complexins I and II. Blots were visualized with anti-mouse IgG conjugated with horseradish peroxidase using chemiluminescence methods. Complexin II was detected at about 19 kD (lower panel), but complexin I was not detected (upper panel) in mast cells. (C) Intracellular distribution of complexin II. Complexin II was detected with anti-complexin II antibody and visualized with FITC-conjugated anti mouse IgG. Fluorescent images were collected with a confocal laser scanning microscope. Complexin II was detected in the cytoplasm and the nucleus.

View larger version (42K): [in a new window]   Fig. 2. Characterization of complexin II-knockdown cells. (A) Western blot analysis for complexin II, syntaxins 3, syntaxin 4 and synaptotagmin II in four knockdown clones (kd-1 to kd-4). Expression of complexin II was reduced in knockdown cells, while expression of syntaxins and synaptotagmin II was not affected. (B) Intracellular distribution of complexin II in a kd cell. The fluorescence image was obtained as described in Fig. 1C. (C,D) Timecourse of the intracellular Ca2+ concentration in wild-type (C) and kd cells (D), respectively. Cells were sensitized with IgE and loaded with Fura2-AM, and stimulated with antigen (DNP-BSA) at the time indicated by an arrow. Average fluorescence intensity ratios (F340/F360) were plotted against time. Values are obtained from four independent preparations of wild-type cells and four independent knockdown clones [mean±s.e.m. (n=4)]. No significant differences in the Ca2+ response were detected between wild-type and kd cells.

View larger version (10K): [in a new window]   Fig. 3. Degranulation in complexin II knockdown cells. (A) Wild-type or knockdown cells were sensitized with IgE and stimulated with antigen (100 ng/ml DNP6-BSA). The quantity of ß-hexosaminidase in the supernatant is expressed as a percentage of total ß-hexosaminidase. Values were obtained from four independent preparations of wild-type cells () and four independent knockdown clones () [means.e.m. (n=4)]. (B) Cells were stimulated with A23187 (1 µM) and PMA (50 ng/ml). Values were obtained and plotted as mentioned above.

View larger version (30K): [in a new window]   Fig. 4. Translocation of complexin II after stimulation. Complexin II in RBL-2H3 was visualized with anti-complexin II antibody or anti-myc antibody, using FITC-conjugated anti-mouse IgG, as in Fig. 1C. (A) Distribution of complexin II before antigen stimulation. The left image shows the distribution of complexin II in wild-type cells using anti-complexin II antibody. The right image shows the distribution of myc-tagged complexin II in cells transfected with myc-tagged complexin II using anti-myc antibody. (B) Distribution of complexin II at 5 minutes after antigen stimulation. Complexin II was translocated to the plasma membrane. Left and right images show the distribution of complexin II and myc-tagged complexin II, respectively. (C) Distribution of complexin II at 5 minutes after antigen stimulation in the absence of extracellular Ca2+. Complexin II was translocated to the plasma membrane. (D) Timecourse of antigen-induced degranulation in the absence of extracellular Ca2+. Values were obtained from four independent preparations of wild-type cells [mean±s.e.m. (n=4)]. (E) Distribution of complexin II at 5 minutes after thapsigargin (50 nmol/l) stimulation in the absence of extracellular Ca2+. Complexin II was translocated to the plasma membrane. (F) Timecourse of thapsigargin-induced degranulation in the absence of extracellular Ca2+. Values are obtained from four independent preparations of wild-type cells [mean±s.e.m. (n=4)].

View larger version (16K): [in a new window]   Fig. 5. Effects of the extracellular Ca2+ concentration on Ca2+-dependent degranulation. Mast cells were stimulated with PMA and A23187 at various extracellular Ca2+ concentrations. (A) Degranulation activity at 20 minutes after stimulation is plotted against extracellular Ca2+ concentration. Values are expressed as the percentage of total ß-hexosaminidase as shown in Fig. 3, but ß-hexosaminidase at [Ca2+]ex=0 was subtracted. Each point was obtained from four independent preparations of wild-type cells () and four independent knockdown clones () [mean±s.e.m. (n=4)]. (B) Degranulation activity is re-plotted against intracellular Ca2+ concentration, which was estimated from Ca2+ concentration at plateau phase after stimulation using Fura-2 as shown in the inset. (Inset) Timecourses of intracellular Ca2+ concentration in wild-type cells induced by PMA and A23187 at various extracellular Ca2+ concentrations, indicated on each line. Cells were stimulated at the time indicated by the arrow. Ca2+ concentration was converted from ratio values and shown as a second ordinate.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter