Prestin is an anion transporter dispensable for mechanical feedback amplification in Drosophila hearing.

In mammals, the membrane-based protein Prestin confers unique electromotile properties to cochlear outer hair cells, which contribute to the cochlear amplifier. Like mammals, the ears of insects, such as those of Drosophila melanogaster, mechanically amplify sound stimuli and have also been reported to express Prestin homologs. To determine whether the D. melanogaster Prestin homolog (dpres) is required for auditory amplification, we generated and analyzed dpres mutant flies. We found that dpres is robustly expressed in the fly's antennal ear. However, dpres mutant flies show normal auditory nerve responses, and intact non-linear amplification. Thus we conclude that, in D. melanogaster, auditory amplification is independent of Prestin. This finding resonates with prior phylogenetic analyses, which suggest that the derived motor function of mammalian Prestin replaced, or amended, an ancestral transport function. Indeed, we show that dpres encodes a functional anion transporter. Interestingly, the acquired new motor function in the phylogenetic lineage leading to birds and mammals coincides with loss of the mechanotransducer channel NompC (=TRPN1), which has been shown to be required for auditory amplification in flies. The advent of Prestin (or loss of NompC, respectively) may thus mark an evolutionary transition from a transducer-based to a Prestin-based mechanism of auditory amplification.

Regulation of rho family GTPases is required to prevent axons from crossing the midline.

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. Our data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho.

Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos.

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.