Complex regulation and multiple developmental functions of misfire, the Drosophila melanogasterferlin gene

Michelle K Smith,Barbara T Wakimoto

doi:10.1186/1471-213x-7-21

Michelle K Smith, Barbara T Wakimoto

Open Access

https://doi.org/10.1186/1471-213x-7-21

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Abstract

BackgroundFerlins are membrane proteins with multiple C2 domains and proposed functions in Ca2+ mediated membrane-membrane interactions in animals. Caenorhabditis elegans has two ferlin genes, one of which is required for sperm function. Mammals have several ferlin genes and mutations in the human dysferlin (DYSF) and otoferlin (OTOF) genes result in muscular dystrophy and hearing loss, respectively. Drosophila melanogaster has a single ferlin gene called misfire (mfr). A previous study showed that a mfr mutation caused male sterility because of defects in fertilization. Here we analyze the expression and structure of the mfr gene and the consequences of multiple mutations to better understand the developmental function of ferlins.ResultsWe show that mfr is expressed in the testis and ovaries of adult flies, has tissue-specific promoters, and expresses alternatively spliced transcripts that are predicted to encode distinct protein isoforms. Studies of 11 male sterile mutations indicate that a predicted Mfr testis isoform with five C2 domains and a transmembrane (TM) domain is required for sperm plasma membrane breakdown (PMBD) and completion of sperm activation during fertilization. We demonstrate that Mfr is not required for localization of Sneaky, another membrane protein necessary for PMBD. The mfr mutations vary in their effects in females, with a subset disrupting egg patterning and causing a maternal effect delay in early embryonic development. Locations of these mutations indicate that a short Mfr protein isoform carries out ferlin activities during oogenesis.ConclusionThe mfr gene exhibits complex transcriptional and post-transcriptional regulation and functions in three developmental processes: sperm activation, egg patterning, and early embryogenesis. These functions are in part due to the production of protein isoforms that vary in the number of C2 domains. These findings help establish D. melanogaster as model system for understanding ferlin function and dysfunction in animals, including humans.

Highlights

Ferlins are membrane proteins with multiple C2 domains and proposed functions in Ca2+ mediated membrane-membrane interactions in animals
We found that the D. melanogaster ferlin gene misfire is expressed in the testis and ovaries of adult flies, and has tissue-specific transcription initiation sites, alternatively spliced mRNAs, and multiple developmental functions
Our cDNA study confirmed this ferlin structure for the predicted protein isoform T1, which we suggest from genetic data is the Mfr protein required for male fertility

Summary

Introduction

Ferlins are membrane proteins with multiple C2 domains and proposed functions in Ca2+ mediated membrane-membrane interactions in animals. Caenorhabditis elegans has two ferlin genes, one of which is required for sperm function. Drosophila melanogaster has a single ferlin gene called misfire (mfr). We analyze the expression and structure of the mfr gene and the consequences of multiple mutations to better understand the developmental function of ferlins. C2 domains are independently folded units of ~130 amino acids that form parallel β-sheets connected by surface loops They are present in many proteins involved in signal transduction and membrane trafficking, such as protein kinase C, phospholipase, and synaptotagmin, and have the capacity to bind substrates that include Ca2+, phospholipids, inositol polyphosphates, and phosphotyrosines [4,5,6]

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