Figure 2.
EXC-2 C-terminal fragment is colocalized with EXC-9 to the canal apical membrane. (a) Scale diagram of domains of intermediate filament expressed in the excretory cell, including EXC-2, IFB-1, and IFA-4. The lamin-like intermediate filament domain allows homo- and hetero-dimerization of the filaments. (b–b’’’) Overexpression of EXC-2(1–767; N-terminal half) in BK590 (EXC-9 modified via CRISPR/Cas9 insertion of mKate2) reduced binding of mKate::EXC-9 to the apical surface. Overexpressing EXC-2(1–767) fluorescence (b); EXC-9 fluorescence (b’); merge (b’’); and fluorescence intensity profiles of EXC-2(1–767) (green) and EXC-9 (red) along white line indicated in b’’ (b’’’). n = 29 animals examined. (c–c’’’) Overexpressing EXC-2 (1112–1339) in BK590 (mKate2::EXC-9) showed colocalization of both proteins to the growing tip of the luminal canal surface; EXC-2(1112–1339) (C-terminal portion) fluorescence (c); EXC-9 fluorescence (c’); merge (c’’); and fluorescence intensity profiles of EXC-2(1–767) (green) and EXC-9 (red) at the canal tip along the white line indicated in c’’ (c’’’). (n = 25 animals). (d–d’’) EXC-2(1–767) overexpressed in exc-2(qp110) homozygotes forms a meshwork at the canal apical surface. EXC-2 fluorescence, inset at 2× magnification (d); DIC image showing cystic lumen (d’); merge (d’’). (n = 32 animals). (e–e’’’) Mutation of the LIM domain prevents EXC-9 localization to the apical surface. Diagram of EXC-9 showing conserved cysteines (C) and histidine (H) in loops of LIM domain. Amino acids in red indicate replacement by alanine (A) in mutant strain (e); EXC-9 fluorescence in cysteine-substituted mutant is evenly expressed throughout the cytoplasm surrounding an enlarged lumen (e’); fluorescence intensity profile along three lines in e’ (n = 26 animals, e’’); comparison of canal length ± SD in the exc-9(qp128) mutant (3 Cys→Ala) to that in wild-type (e’’’). ***, P < 0.001 (n = 50). All scale bars, 5 µm.

EXC-2 C-terminal fragment is colocalized with EXC-9 to the canal apical membrane. (a) Scale diagram of domains of intermediate filament expressed in the excretory cell, including EXC-2, IFB-1, and IFA-4. The lamin-like intermediate filament domain allows homo- and hetero-dimerization of the filaments. (b–b’’’) Overexpression of EXC-2(1–767; N-terminal half) in BK590 (EXC-9 modified via CRISPR/Cas9 insertion of mKate2) reduced binding of mKate::EXC-9 to the apical surface. Overexpressing EXC-2(1–767) fluorescence (b); EXC-9 fluorescence (b’); merge (b’’); and fluorescence intensity profiles of EXC-2(1–767) (green) and EXC-9 (red) along white line indicated in b’’ (b’’’). n = 29 animals examined. (c–c’’’) Overexpressing EXC-2 (1112–1339) in BK590 (mKate2::EXC-9) showed colocalization of both proteins to the growing tip of the luminal canal surface; EXC-2(1112–1339) (C-terminal portion) fluorescence (c); EXC-9 fluorescence (c’); merge (c’’); and fluorescence intensity profiles of EXC-2(1–767) (green) and EXC-9 (red) at the canal tip along the white line indicated in c’’ (c’’’). (n = 25 animals). (d–d’’) EXC-2(1–767) overexpressed in exc-2(qp110) homozygotes forms a meshwork at the canal apical surface. EXC-2 fluorescence, inset at 2× magnification (d); DIC image showing cystic lumen (d’); merge (d’’). (n = 32 animals). (e–e’’’) Mutation of the LIM domain prevents EXC-9 localization to the apical surface. Diagram of EXC-9 showing conserved cysteines (C) and histidine (H) in loops of LIM domain. Amino acids in red indicate replacement by alanine (A) in mutant strain (e); EXC-9 fluorescence in cysteine-substituted mutant is evenly expressed throughout the cytoplasm surrounding an enlarged lumen (e’); fluorescence intensity profile along three lines in e’ (n = 26 animals, e’’); comparison of canal length ± SD in the exc-9(qp128) mutant (3 Cys→Ala) to that in wild-type (e’’’). ***, P < 0.001 (n = 50). All scale bars, 5 µm.

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