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First published online 3 April 2007
doi: 10.1242/jcs.001016

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Functional importance of polymerization and localization of calsequestrin in C. elegans

J. H. Cho^1,*, K. M. Ko^1,*, Gunasekaran Singaruvelu¹, Wonhae Lee¹, Gil Bu Kang¹, Seong-Hwan Rho¹, Byung-Jae Park¹, Jae-Ran Yu², Hiroaki Kagawa³, S. H. Eom¹, D. H. Kim¹ and Joohong Ahnn^1,

¹ Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, 500-712, Korea
² Department of Parasitology, College of Medicine, Kon-Kuk University, Chungju, 380-710, Korea
³ Department of Biology, Faculty of Science, Okayama University, Okayama, 700-8530, Japan

View larger version (22K): [in this window] [in a new window]   Fig. 1. Schema of the csq-1-null mutant csq-1 (jh109). (A) The 1042 bp deletion, which removed several exons, is shown by a horizontal bar. (1) and (2) indicate primer sets used for nested PCR (indicated by arrows) performed for mutant isolation. (B) Agarose gel of PCR fragments showing bands obtained from single worm PCR. Wild type (+/+), heterozygote (+/–), and homozygote (–/–). Absence of a band in lane labelled (–/–) confirms that the worm is a homozygote. (C) Western blot showing the expression of CSQ-1 and PYP-1 (inorganic pyrophosphatase as control) in wild-type worms (lane 1) and csq-1(jh109) animals (lane 2).

View larger version (62K): [in this window] [in a new window]   Fig. 2. Phenotypes of wild-type and csq-1 mutant worms. (A) Analysis of motility assays in liquid medium showing the average number of waveforms measured in 1 minute (error bars give s.d., n=20). (B) Analysis of average brood size of the indicated strains (error bars give s.d., n=20). (C,D,E) Survival rate under high-[Ca2+] stress conditions, low-[Ca2+] stress conditions and DTT stress conditions. Young adult worms were grown on plates containing 20 mM calcium acetate, 20 mM EGTA or 1 mM DTT solution. Surviving worms of each strain were counted after 3 days. The unc-68 gene encodes the RyR and crt-1 encodes calreticulin. csq-1;crt-1 is a double mutant of calsequestrin and calreticulin and csq-1;unc-68 is double mutant of calsequestrin and RyR. (F-H) Electron micrographs of the transverse section showing body-wall muscle structure of wild-type worms (F), csq-1(jh109) mutant worms (G) and csq-1(jh109) mutant worms grown on EGTA plates (H).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Homology model and schematic diagram of mutated C. elegans calsequestrin. (A) Schematic prediction of polymerized CSQ-1. The monomeric model of CSQ-1 was built using the rabbit calsequestrin structure. The tetramer is shown as C-traces to display putative polymerizing interactions between monomers. Monomers are either green or yellow. N and C-termini of each molecule are shown as dashed lines because they are disordered in the template structure. Note the positively charged residues in the C-terminus specific to C. elegans calsequestrins. The location of the conserved back-to-back interface formed between SAH (Serine Acidic Hydrophbic) and DBH (Dibasic Hydrophobic) is boxed in red. Lys111 residues are shown as spheres. (B) Close-up view of the back-to-back interface of CSQ-1. The two interacting molecules are green and yellow. Three negatively charged residues (Glu247, Glu251 and Asp196) are placed in the vicinity of Lys111 and might participate in a salt bridge. (C) Amino acid sequence of C. elegans calsequestrin aligned with the rabbit skeletal calsequestrin and human cardiac calsequestrin CSQ2 amino acid sequences. Green box, signal sequence; first to fourth red and first and second yellow boxes indicate C26 and C17, N23-31, N32-43, DBH, SAH, respectively. (D) Each DNA construct represents wild-type (WT), N-terminal 9-amino-acid truncation, N-terminal 12-amino-acid truncation, C-terminal 17-amino-acid truncation and C-terminal 26-amino-acid truncation. Mutant CSQ-1 constructs K111A and Bedouin contain a Lys111 to Ala and Asp318 to His mutation, respectively. These constructs were introduced into the csq-1(jh109) mutant and their expression was detected by western blotting using anti-CSQ-1 and anti-PYP-1 (internal control) antibodies.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Cellular localization and functional rescue of calsequestrin. (A,B) Wild-type worms stained with (A) anti-CSQ-1 antibody and (B) anti-UNC-68 (RyR) antibody show punctate and mesh-like staining in body-wall muscles. (C) The csq-1(jh109) mutant stained with anti-UNC-68 antibody shows similar punctuate and mesh-like staining pattern. (D) The unc-68(e540) mutant stained with anti-CSQ-1 antibody show a dispersed punctate pattern; no mesh-like pattern was observed. (E) csq-1-null mutant csq-1(jh109) transformed by constructs encoding WT, N23-31 and Bedouin CSQ-1 show mesh-like pattern. (F) N32-43 shows aggregated pattern. (G) K111A shows diffused pattern. (H) C17 and C26 show dispersed pattern similar to that shown in D. (I) Survival rates of wild-type and transgenic animals expressing mutated CSQ-1 under high-[Ca2+] and low-[Ca2+] conditions. Young F1 transgenic worms were grown on NGM plates with 20 mM EGTA and 20 mM CaAc. Survival rates of worms with rescued CSQ-1 function was compared with that of wild-type animals at 20°C. Over 50 animals were tested for each data point of a single set of experiments, and each experiment was repeated three times.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Direct interaction between the C-terminal region of calsequestrin and the intraluminal loops of RyR. (A,B) In vitro binding assays were performed using (A) full-length CSQ-1 and (B) C-terminally deleted CSQ-1 (C17-CSQ-1). Intraluminal loops of RyRs [GST-RyR loop I (lanes 4, 5, 6) and GST-RyR loop II (lanes 7, 8, 9)] were tested for direct interaction in the presence of Ca2+ (lanes 4, 7; 0.5 mM Ca2+, lane 5, 8; 2 mM Ca2+) or absence of Ca2+ (lanes 6, 9; 1 mM EGTA). Lane 1, supernatant of CSQ-1 (A) or that of C17-CSQ-1 (B); lane 2, affinity beads alone; lane 3, GST control. (C) Simplified model of calsequestrin localization in C. elegans. Positively charged residues at the C-terminal end of C. elegans calsequestrin may directly interact with the negatively charged residues of the loops of RyRs to localize calsequestrin into the vicinity of the SR membrane. This could allow fast and localized release of Ca2+ through RyRs once the receptor is activated.