Protein Phosphatase 1ß Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

Shinya Yamamoto,Change Tan,Vafa Bayat,Hugo J Bellen,Shree Ram Singh

doi:10.1371/journal.pone.0070502

Abstract

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.

Highlights

The first step in sexual reproduction is the formation of functional male and female gametes
We found that Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with Drosophila myosin binding subunit of myosin phosphatase (DMYPT)
We reasoned that a mutation interfering with incomplete cytokinesis (IC) would cause oogenesis failure, leading to infertility, and the resultant ring canals would be small, as seen in DMYPT mutations, or be oversized, opposite to the phenotype seen in DMYPT mutants

Summary

Introduction

The first step in sexual reproduction is the formation of functional male and female gametes. A key feature of gamete formation in many organisms is incomplete cytokinesis (IC), in which contractile rings during cytokinesis constrict, but do not fully close and generate cysts (groups of interconnected cells) [1,2,3,4,5,6,7,8]. A germline stem cell (GSC) divides asymmetrically via complete cytokinesis to form another GSC and a cystoblast (Figure 1B). Anillin localizes to the contractile ring, ring canal, and/or nuclei, with levels and distribution dependent on the cell cycle and the cyst age [9,10,11,12,13,14,15]. A-spectrin, an actin-crosslinking/scaffolding protein, localizes to membranous organelles called fusomes that are part of the continuous ER network, and is required for their formation [16,17,18,19] Anillin localizes to the contractile ring, ring canal, and/or nuclei, with levels and distribution dependent on the cell cycle and the cyst age [9,10,11,12,13,14,15]. a-spectrin, an actin-crosslinking/scaffolding protein, localizes to membranous organelles called fusomes that are part of the continuous ER network, and is required for their formation [16,17,18,19]

Methods

Results

Conclusion