A Ubiquitin Ligase Complex Regulates Caspase Activation During Sperm Differentiation in Drosophila
Figure 7
Mutations in klhl10 Block Caspase Activation and Spermatid Individualization, but Not Axonemal Tubulin Polyglycylation
(A–H) Visualization of active effector caspase with anti-cleaved caspase-3 antibody (CM1; green) and (A–F) axonemal tubulin polyglycylation with anti-glycylated tubulin monoclonal antibody (AXO 49; red) or (G–H) F-actin, which stains the ICs and the spermatids' tails (phalloidin; red). These figures are composed of combined green and red layers.
(A) Wild-type individualizing spermatids positively stain for cleaved caspase-3 and polyglycylated axonemal tubulin.
(B–F) In a variety of klhl10−/− alleles, elongated spermatids stain for polyglycylation but not for cleaved caspase-3.
(G and H) Transgenic klhl10 construct (tr-klhl10, composed of cul3Testis promoter and 5′ UTR, klhl10 coding region, and cul3Testis 3′ UTR) restores caspase activation, spermatid individualization, and fertility to klhl10−/− male flies.
(I) Schematic representation of the Klhl10 protein. The relative locations of the BTB, BACK, and Kelch domains are depicted by thick bars. Different colored stars depict the locations of the different mutations, and the colors correspond to the colored amino-acids in (J). The molecular nature of the mutations and their color code are as follows: klhl102 (Z2–1331) carries a G1801-to-A transversion that converts glutamic acid (E601, red) to lysine at repeat VI. klhl103 (Z2–0960) carries a G1119-to-A transversion that converts tryptophan (W373, purple) to stop codon, resulting in a deletion of most of the Kelch domain. klhl104 (Z2–2739) carries a C1237-to-T transversion that converts arginine (R413, yellow) to stop codon, which also deletes most of the Kelch repeats. klhl105 (Z2–3284) carries a G1486-to-A transversion that converts glycine (G496, blue) to arginine at repeat IV. klhl106 (Z2–4385) carries a C1439-to-T transversion that converts serine (S480, green) to phenylalanine at repeat IV. On the other hand, klhl107 (Z2–3353) carries a G508-to-A transversion that converts a highly conserved alanine (A170) to threonine in the BTB domain (gray star).
(J) Alignment of the six Kelch repeats of Klhl10. The alignment is based on the crystal structure of the Keap1 Kelch domain which folds into a β-propeller structure with 6 blades. The residue range for each blade is indicated at the left. The four conserved β-strands in each blade are indicated above the sequences by arrows. Residues conserved in all six blades are highlighted with dark gray and appear in upper case in the consensus line, whereas residues that are conserved in at least three blades appear in lower case. Any two and above conserved residues are highlighted with light gray. Color highlighted residues are mutated in the various klhl10−/− alleles and correspond to the stars in (I).