A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis elegans Somatic Gonad.

Todd Starich,David Greenstein

doi:10.3390/biom10121655

Abstract

In Caenorhabditis elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. A strong loss-of-function mutation (T239I) affects the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. We conducted a genetic screen for suppressor mutations that restore germ cell proliferation in the T239I mutant background and isolated seven intragenic mutations, located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky channels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three of the suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. The mutations described here may be useful for elucidating the biochemical pathways that produce the active biomolecules transiting through soma–germline gap junctions.

Highlights

Gap junctions are nearly ubiquitous in multi-cellular animals
Because INX-8(T239I)::GFP localizes to compromised channels, it was an ideal candidate for a suppressor screen
The composition of hemichannels in soma and germline do not change, we expect that the specific requirements for molecules passing through gap junction channels changes with developmental progression from a mitotic state through the stages of meiosis and gametogenesis; components moving through distal tip cell (DTC)–germline junctions may differ from those moving through sheath–germline junctions

Summary

Introduction

The molecular constituents of gap junctions differ in chordates (connexins) and non-chordates (innexins), but their properties and biological functions are remarkably similar (recently reviewed in [1]). It is still unclear why a different class of gap junction molecule emerged within vertebrates, the ubiquity of gap junctions themselves suggests that being coupled is essential for most multi-cellular forms of life, and the functions of gap junctions are many. A somatic distal tip cell (DTC) occupies the leading edge of each expanding gonad arm, and the DTC establishes a stem cell niche supporting germ cell proliferation by producing Delta-class ligands LAG-2 and APX-1, which activate GLP-1/Notch receptors on germline

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Discussion

Conclusion