PFTAIRE Kinase L63 Interactor 1A (Pif1A Protein) Is Required for Actin Cone Movement during Spermatid Individualization in Drosophila melanogaster.

Harrison D Pravder,Deborah Frank,Przemysław Zakrzewski,Kaushik Roychoudhury,Marta Lenartowska,Dorota Grabowska,Betty Zhang,Kathryn G Miller

doi:10.3390/ijms23063011

Abstract

A useful model for determining the mechanisms by which actin and actin binding proteins control cellular architecture is the Drosophila melanogaster process of spermatogenesis. During the final step of spermatogenesis, 64 syncytial spermatids individualized as stable actin cones move synchronously down the axonemes and remodel the membranes. To identify new genes involved in spermatid individualization, we screened a collection of Drosophila male-sterile mutants and found that, in the line Z3-5009, actin cones formed near to the spermatid nuclei but failed to move, resulting in failed spermatid individualization. However, we show by phalloidin actin staining, electron microscopy and immunocytochemical localization of several actin binding proteins that the early cones had normal structure. We sequenced the genome of the Z3-5009 line and identified mutations in the PFTAIRE kinase L63 interactor 1A (Pif1A) gene. Quantitative real-time PCR showed that Pif1A transcript abundance was decreased in the mutant, and a transgene expressing Pif1A fused to green fluorescent protein (GFP) was able to fully rescue spermatid individualization and male fertility. Pif1A-GFP localized to the front of actin cones before initiation of movement. We propose that Pif1A plays a pivotal role in directing actin cone movement.

Highlights

To identify candidate genes required for actin organization and function during Drosophila spermatid individualization, we obtained extant male sterile lines from an ethyl methanesulfonate mutagenesis screen [11]
Z3-5009, had a strong phenotype in which actin cones formed in association with spermatid nuclei in the basal portion of the cyst (Figure 1a, inset) and looked similar to those in testes dissected from wild type (WT) males (Oregon R, Figure 1b, inset 1)
Quantitative analysis revealed that the testes from Z3-5009 mutants had significantly more nuclear bundles without actin cones and more nuclear bundles associated with actin cones than testes from WT flies (Figure 1c)

Summary

Introduction

Cells adopt a wide variety of shapes to allow them to function properly. A key component of cellular architecture is the actin cytoskeleton, made up of filaments (F-actin) formed via polymerization of globular actin. A useful model for determining the mechanisms by which actin and actin binding proteins control cellular architecture is the Drosophila melanogaster process of spermatogenesis. Incomplete cytokinesis and meiosis produce 64 interconnected spermatids [1,2] surrounded by two somatic cyst cells. The cysts elongate as they elaborate axonemes [3–5]. The actin remodels to form an actin cone ( known as an investment cone), and the 64 actin cones travel synchronously down the axonemes. The membrane is reorganized so as to encase each spermatid in its own membrane tightly juxtaposed to the axoneme, and the cytoplasmic components are pushed out ahead of the actin cones, forming a cystic bulge [4]

Objectives

Methods

Results

Discussion

Conclusion