Research Article 2239 Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process Satoshi Tadokoro, Mamoru Nakanishi and Naohide Hirashima* Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan *Author for correspondence (e-mail: [email protected]) Accepted 18 February 2005 Journal of Cell Science 118, 2239-2246 Published by The Company of Biologists 2005 doi:10.1242/jcs.02338 Summary Recent studies have shown that soluble N-ethyl maleimide- distributed throughout the cytoplasm before antigen sensitive factor attachment protein receptor (SNARE) stimulation. However, the distribution of complexin II proteins are involved in exocytotic release in mast cells as changed dramatically with stimulation and it became in neurotransmitter release. However, the roles of the localized on the plasma membrane. This change in the proteins that regulate the structure and activity of SNARE intracellular distribution was observed even in the absence proteins are poorly understood. Complexin is one such of extracellular Ca2+, while exocytotic release was inhibited regulatory protein and is involved in neurotransmitter almost completely under this condition. The degranulation release, although ideas about its role are still controversial. induced by phorbol 12-myristate 13-acetate and A23187 In this study, we investigated the expression and role depended on the extracellular Ca2+ concentration, and its of complexin in the regulation of exocytotic release sensitivity to Ca2+ was decreased in knockdown cells. These (degranulation) in mast cells. We found that complexin II, results suggest that complexin II regulates exocytosis but not complexin I, is expressed in mast cells. We obtained positively by translocating to the plasma membrane and RBL-2H3 cells that expressed a low level of complexin II enhancing the Ca2+ sensitivity of fusion machinery, and found that antigen-induced degranulation was although this translocation to the plasma membrane is not suppressed in these cells. No significant changes in the sufficient to trigger exocytotic membrane fusion. Ca2+ response or expression levels of syntaxins and synaptotagmin were observed in knockdown cells. An Journal of Cell Science immunocytochemical study revealed that complexin II was Key words: Mast cell, Complexin, Exocytosis, SNARE, Allergy, Rat Introduction higher order structure that is required for the fusion of synaptic It has been shown that soluble N-ethyl maleimide-sensitive vesicles. They also showed that peptides that prevent factor attachment protein receptor (SNARE) proteins play an complexin from binding to the SNARE complex inhibit evoked essential role in exocytotic release in both neuronal cells transmitter release in a squid giant synapse. Double-knockout (Sollner et al., 1993; Calakos and Scheller, 1996; Brunger, mice for complexins I and II show reduced neurotransmitter 2001) and non-neuronal secretory cells (Wheeler et al., 1996; release (Reim et al., 2001). These results suggest that Nagamatsu et al., 1999; Reed et al., 1999; Flaumenhaft, 1999). complexin acts as a positive regulator of exocytosis. However, In addition to SNARE proteins, several proteins that regulate the injection of complexin into Aplysia nerve terminal the conformation and activity of SNARE complexes are suppressed transmitter release, while the injection of anti- involved in exocytosis. Complexin (also called synaphin), a complexin antibody stimulated neurotransmitter release (Ono small soluble protein (18-19 kD), is a regulatory protein in the et al., 1998). The overexpression of complexin in PC12 mammalian brain (McMahon et al., 1995; Takahashi et al., reduced exocytotic release (Itakura et al., 1999). As these 1995; Ishizuka et al., 1995). Complexin interacts with ternary findings show, it is still unclear how complexin functions in SNARE complex and is thought to stabilize the SNARE exocytotic release, even in neuronal cells. Furthermore, it is not complex (Pabst et al., 2000; Pabst et al., 2002). Based on clear whether or not complexin is involved in exocytosis in studies of the three-dimensional structure of the complexin/ non-neuronal cells. If complexin regulates exocytosis in non- SNARE complex, it has been suggested that complexin neuronal secretory cells, it would be very interesting to stabilizes the fully assembled SNARE complex (Bracher et al., investigate how complexin regulates exocytosis in such cells. 2002; Chen et al., 2002). However, the role of complexin in As non-neuronal secretory cells are often larger and have neurotransmitter release is not yet fully understood. Using a bigger secretory granules than nerve terminals, they could squid giant synapse, Tokumaru et al. (Tokumaru et al., 2001) provide a useful experimental system for investigating the reported that complexin facilitates the association of SNAREs mechanism by which complexin regulates exocytosis. into an intermediate complex that can oligomerize to give a In the present study, we examined the expression and role 2240 Journal of Cell Science 118 (10) of complexin in mast cells, which are typical non-neuronal (anti-sense) and 5′-ATGGACTTCGTCATGAAGCA (sense)/5′-TTA- secretory cells. In mast cells, cross-linking of high-affinity CTTCTTGAACATGTCCTGCA-3′ (anti-sense), respectively. PCR receptors for IgE (FcεRI) by multivalent antigen activates an products were extracted from agarose gel with Gene Clean (Bio 101) intracellular signaling cascade and leads to the exocytotic and subcloned into the TA cloning vector pCRII (Invitrogen). Cloned release of granular contents (degranulation), resulting in PCR products were sequenced with a DSQ1000 DNA sequencer allergic responses (Abraham and Malaviya, 1997; Swann et (Shimadzu, Kyoto, Japan) using FITC-labeled M13 universal primer. al., 1998; Turner and Kinet, 1999; Kinet, 1999). We have previously studied the mechanism of degranulation in mast Western blotting cells and identified the protein and lipid molecules that regulate RBL-2H3 or P815 cells (5 ϫ 106) were lysed with lysis buffer (10 this process (Hibi et al., 2000; Kato et al., 2002; Kato et al., mmol/l HEPES, 1% Triton-X100, 1 mmol/l EDTA, 50 mmol/l NaF, 2003). We and other groups have reported that SNARE 2.5 mmol/l p-nitrophenyl phosphate, 1 mmol/l Na3VO4, 1 mmol/l proteins are involved in degranulation in mast cells (Guo et al., PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin). 1998; Hibi et al., 2000; Paumet et al., 2000; Blank et al., 2001; After centrifugation at 23,000 g for 20 minutes, supernatant was mixed Blank and Rivera, 2004), although the active isoforms in mast with an equal volume of Laemmli sample buffer and boiled for cells are different from those in neuronal cells. However, there 5 minutes. For cellular lysate of rat brain, rat cerebrum lysate is little information available on the proteins that regulate was purchased from Transduction Laboratories. Samples were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. SNARE proteins in mast cells, except for Munc18-2 (Martin- After blocking with phosphate buffer containing 5% skimmed milk, Verdeaux et al., 2003). blots were probed with primary antibody for 1 hour. As primary In this study, we found that complexin II was expressed in antibodies, anti-complexin I antibody (dilution 1:200; Santa Cruz mast cells. In experiments using complexin II knockdown cells, Biotechnology), anti-complexin II antibody (dilution 1:500; we found that complexin II positively regulated exocytotic Transduction Laboratories), anti-syntaxin-3 antibody (dilution 1:250; release in mast cells. Immunocytochemical experiments Alomone Labs, Israel), anti-syntaxin-4 antibody (dilution 1:500; revealed that complexin II changed its localization from the Santa Cruz Biotechnology), anti-synaptotagmin II antibody (dilution cytoplasm to the plasma membrane, and this translocation 1:200 Santa Cruz Biotechnology), and β-actin antibody (dilution occurred in the absence of extracellular Ca2+, while 1:4000; Sigma) were used. After being washed with 0.1% Tween 20 degranulation was inhibited almost completely. We also found in PBS, membrane was treated with anti-mouse IgG conjugated with horseradish peroxidase. Immunoreactivity was detected by enhanced that the degranulation induced by phorbol 12-myristate 13- 2+ chemiluminescence (ECL, Amersham Pharmacia) with a LAS-1000 acetate (PMA) and A23187 depended on extracellular Ca (FUJI FILM, Tokyo Japan) and analyzed by Image Gauge (FUJI 2+ concentration, and its sensitivity to Ca was decreased in FILM). knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion Plasmid construction and transfection machinery, although the association of complexin II with Poly(A)+ RNA was obtained as described above. For the knockdown SNARE complex is not sufficient to trigger exocytotic of complexin II, 5′-GGATCCATGGACTTCGTCATGAAGCA-3′ ′ membrane fusion. (sense; BamHI site is underlined)/5 -GCGGCCGCTTACTTCTTG- Journal of Cell Science AACATGTCCTGCA-3′ (anti-sense; NotI site is underlined) was used as a primer pair. For the expression of myc-tagged complexin II, 5′- GGATCCATGGACTTCGTCATGAAGCA-3′ (sense; BamHI site Materials and Methods is underlined)/5′-GAATTCTTACTTCTTGAACATGTCCTGCA-3′ Chemicals (anti-sense; EcoRI site is underlined) was used as a primer pair. PCR PMA, 4-bromo-A23187, (±)-sulfinpyrazone, p-nitrophenyl-N-acetyl-