Involvement of a 25 kDa Tetrahymena Ca2+-binding Protein in Pronuclear Exchange

Abstract

ABSTRACT. The Tetrahymena Ca2+ -binding protein of 25 kDa (TCBP-25) is a calmodulin family protein containing four EF-hand type calcium-binding domains. TCBP-25 is localized in the whole cell cortex and around both the migratory and stationary pronuclei at the pronuclear exchange stage during conjugation. TCBP-25 is expected to play an important role in conjugation, though its function during sexual reproduction has not been elucidated. According to the localization of this protein and its timing, three possible roles of TCBP-25 are proposed. TCBP-25 may play a role in 1) differentiating the two functional pronuclei from the degenerative postmeiotic nuclei, 2) the process of pronuclear exchange and 3) pronuclear fusion. To test these hypotheses, the localization of TCBP-25 in conjugation mutants (cnj10, cnj7 and bcd2) was examined. The results ruled out the first and the third hypotheses and suggested that TCBP-25 may play a role in pronuclear exchange. In the next step we succeeded in reducing expression of the TCBP-25 gene using the antisense ribosome system, and we analyzed the phenotype of the transformants. The knock down of TCBP-25 function also suggests that TCBP-25 plays a role in the pronuclear exchange and in the maintenance of cell shape.

Role of the Cytosolic Heat Shock Protein 70 Ssa5 in the Ciliate Protozoan Tetrahymena thermophila

“…A calmodulin family protein (TCBP‐25) and a zinc finger protein (Zfr1), both of which localize at the conjugation junction (Nakagawa et al. ; Xu et al. ), are likely involved in the meshwork formation.…”

“…Defects in Tcbp‐25 and Zfr1 also abolish conjugation after post‐meiotic division due to a blockage of pronuclear exchange (Nakagawa et al. ; Xu et al. ).…”

“…Blocking conjugation at the possible checkpoint (Allen ,b; Kaney ; Nakagawa et al. ; Xu et al. ) may insure survival of parental clones in the case of failure of proper gamete exchange, which enables the cells to have another chance to produce progeny by mating with other strains that can complement the defects.…”

Amitosis requires γ‐tubulin‐mediated microtubule assembly in Tetrahymena thermophila

“…Cells were washed once with PiEM (10 mM Pipes, 10 mM EGTA, 5 mM MgSO 4 , pH 6.9) [Nakagawa et al, 2008] and permeated by incubation in PiEM with 0.5% TritonX-100 and 10 lM taxol for 3 min. After washing once with PiEM, cells were fixed in PiEM with 2% formaldehyde.…”

Solution NMR structures of the C‐domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium‐induced structural rearrangements

“…Tcb2 ( Tetrahymena calcium‐binding protein 2, UniProt/SwissProt P09226) is associated with the Tetrahymena membrane skeleton and has been hypothesized to play roles in the calcium‐dependent regulation of ciliary movement, in calcium‐mediated signaling processes in the cell cortex, in calcium‐dependent pronuclear exchange during conjugation, and in the maintenance of cell shape . Tcb2 has interesting contractile properties.…”