The short and long isoforms of DAAM and the localization of Profilin. (A) Domain structure of the two DAAM isoforms, PB and PD, expressed in the IFM. Note that the two isoforms are completely overlapping, while the PD isoform contains an N-terminal extension of 310 aa. The anti-DAAM (PD) serum was raised against an N-terminal, PD isoform–specific peptide, whereas the (R1) antibody was raised against a C-terminal region of the protein, present in both isoforms. (B and B′) Western blots show that, as expected, the (R4) antibody (raised against the same sequences as R1) recognizes the short (PB) and long (PD) isoform, while the anti-DAAM (PD) recognizes only the long isoform from S2 cell lysates transfected with DAAM-PB– or DAAM-PD–expressing constructs. (C and C′) Western blots with bacterially expressed proteins reveal that R1 and R4 both recognize the FH2 and DAD-CT regions but not the FH1 domain. It is of note that R1 appears to exhibit a much higher affinity for DAD-CT than R4. (D and E) Anti-DAAM (PD) staining in a 0–4 h IFM reveals a strong accumulation at the Z-disk and a weak signal at the M-line; conversely, (R1) displays a strong signal at the M-line and a very weak if any enrichment at the Z-disk. Thus, it appears that although (R1) recognizes both isoforms in denaturing conditions (i.e., after SDS-PAGE), when used for immunohistochemistry, it is specific for the short isoform. In addition, these results indicate that in these young IFMs, the PD isoform is enriched at the Z-disk, whereas the PB isoform accumulates at the M-line. Scale bar, 5 µm. (F) Comparison of the nanoscopic distribution of the two Fhos and two DAAM isoforms in the IFM demonstrates that the short isoforms (Fhos-PA and DAAM-PB) are enriched in a narrow stripe at the M-line, while the long isoforms (Fhos-PH and DAAM-PD) accumulate in two stripes along the Z-line. This differential distribution is likely to indicate an isoform specific in vivo function at the Z-disk versus the M-line, for example the short isoforms promote pointed end elongation while the long isoforms are involved in barbed end dynamics. Scale bar, 500 nm. (G and G′) Developmental analysis of Profilin accumulation in the IFM. Whereas former work suggested that Profilin accumulates at the Z-disk, we found that, in addition to a weak staining along the entire myofibril, the bulk of the protein is enriched at the M-line (indicated by the colocalization with Obscurin) in the IFM of 24-h-old adults. To rule out the possibility that Profilin expression displays a different pattern during development, we examined its distribution in two earlier (72 h after puparium formation [APF] and 0–4 h adult) and two later (48 h and 96 h adult) developmental time points. This analysis confirmed the M-line association at each developmental stage, although the enrichment looked somewhat less specific in 72-h-APF myofibrils. Scale bar, 5 µm. (H and I) Sarcomere association of DAAM depends on Mhc. (H) Confocal images show that DAAM localization in an Mhc-null mutant myofibril is virtually lost. (I) In Act88F-null mutant IFMs, a residual DAAM accumulation is clearly visible. In agreement with its nanoscopic protein distribution at the M-line, these data suggest that myofibril association of at least the short isoform of DAAM, recognized by R1 in 0–4 h IFM, primarily depends on Mhc and not on actin. (J–J″) Myofibrillar G-actin distribution was visualized with deoxyribonuclease I (DNaseI) staining, which revealed a nearly uniform localization along the entire myofibril but at the Z-disk, where the staining is much weaker than elsewhere. This result clearly confirms the presence of G-actin in the H-zone. Scale bar, 5 µm.