Abstract

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the sessile adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actomyosin Mg2+-ATPase activity in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only <60% identity with each other. Northern blotting and whole mount in situ hybridization revealed that these isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.

Highlights

  • The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) D50867 and D85077

  • 1 The abbreviations used are: Tn, troponin; TnT, troponin T; TnI, troponin I; TnC, troponin C; Tm, tropomyosin; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); adTnT, H. roretzi TnT encoded by the cDNAs aTnT2 and aTnT19; embTnT, TnT encoded by eTnT11; NLS, nuclear localization signal

  • We cloned the TnT cDNAs by screening adult body wall muscle and tailbud embryo cDNA expression libraries with NT302

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Summary

The abbreviations used are

Troponin; TnT, troponin T; TnI, troponin I; TnC, troponin C; Tm, tropomyosin; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); adTnT, H. roretzi TnT encoded by the cDNAs aTnT2 and aTnT19; embTnT, TnT encoded by eTnT11; NLS, nuclear localization signal. Determination of the the primary and higher structures of the adult H. roretzi TnT and embryonic or larval counterparts, if present, may answer the questions how the adult TnT accelerates actin-myosin interaction, how its multiple isoelectric forms are generated, and how the embryonic or larval striated muscle contraction is regulated. To these ultimate ends, we cloned the TnT cDNAs by screening adult body wall muscle and tailbud embryo cDNA expression libraries with NT302. Forced expression of the adult TnT in cultured mammalian nonmuscle cells further suggests that complex formation with the other troponin subunits is required for the protein to associate with thin filaments or microfilaments in vivo

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