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

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A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis Elegans Somatic Gonad biomolecules Article A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis elegans Somatic Gonad Todd Starich * and David Greenstein * Department Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA * Correspondence: [email protected] (T.S.); [email protected] (D.G.) Received: 1 November 2020; Accepted: 8 December 2020; Published: 10 December 2020 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. Keywords: gap junctions; innexins; soma–germline interactions 1. Introduction Gap junctions are nearly ubiquitous in multi-cellular animals. 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]). Although 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. The sizes of connexin and innexin gene families within species further attest to a diversity of function. Gap junction coupling and the composition of junctional channels between cells can dramatically change over developmental time, though the rationale driving these changes is mostly unknown. We are studying the role that gap junctions play in the somatic control of germline development in C. elegans. Two symmetric gonad arms extend anterior or posterior of a central uterus and vulva (Figure1). Initially, germ cells proliferate in a single small pool which becomes partitioned by migration of somatic cells from each of the developing gonad arms late in the second larval stage; these somatic cells eventually divide and develop to form the gonadal sheath, spermatheca, and uterus [2]. 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 Biomolecules 2020, 10, 1655; doi:10.3390/biom10121655 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1655 2 of 15 Biomolecules 2020, 10, x 2 of 15 stemreceptors cells on [3– germline6]. At adulthood, stem cells the [3–6]. soma At ofadulthood a gonad, arm the soma includes of athe gonad DTC, arm five includes pairs of the sheath DTC, cells five andpairs the of spermatheca.sheath cells and The the Sh5 spermatheca. pair of sheath The cells Sh5 ispair the of most sheath proximal cells is the (closest most to proximal the uterus) (closest and to is connectedthe uterus) to and a constriction is connected of the to distala constriction portionof of the the spermatheca. distal portion A seriesof the ofspermatheca. sheath cell contractions, A series of coordinatedsheath cell contractions, with the dilation coordinated of the distalwith the constriction dilation of of the the distal spermatheca, constriction results of the in thespermatheca, ovulation ofresults a maturing in the oocyteovulation into of the a spermatheca, maturing oocyte where in fertilizationto the spermatheca, and the completion where fertilization of meiosis and occurs the (Figurecompletion1). of meiosis occurs (Figure 1). FigureFigure 1.1. Diagram of the symmetricsymmetric gonad arms inin C.C. eleganselegans (upper)(upper) andand photophoto ofof correspondingcorresponding gonadgonad armarm fromfrom thethe wild-typewild-type strainstrain N2.N2. DTC,DTC, distaldistal tiptip cell;cell; –1,–1, mostmost proximalproximal oocyte.oocyte. TheThe somasoma andand germlinegermline areare coupledcoupled byby twotwo classesclasses ofof gapgap junctionjunction channelschannels throughoutthroughout developmentdevelopment [7[7].]. TheThe somatic somatic DTC DTC and and sheath sheath express expressinx-8 inx-8and andinx-9 inx-9, a pair, a pair of recently of recently duplicated duplicated and redundantand redundant innexins, innexins, which which constitute constitute an operon an operon (a single (a innexinsingle innexin is found is at found this locus at this in C.locus briggsae in C.); eitherbriggsae) gene; either alone gene rescues alone the rescues double theinx-8(0) double inx-9(0) inx-8(0)knockout. inx-9(0) knockout. Germ cells Germ fail to cells proliferate fail to proliferate in inx-8(0) inx-9(0in inx-8(0)) mutants inx-9(0 (~4) mutants per gonad (~4 arm). per gonad In the arm). germline, In theinx-14 germline,, inx-21 inx-14, and inx-22, inx-21encode, and inx-22 innexins encode that assembleinnexins intothat twoassemble classes into of heteromeric two classes hemichannels, of heteromeric INX14 hemichannels,/INX-21 and INX14/INX-21 INX-14/INX-22. andinx-14(0) INX- and14/INX-22.inx-21(0) inx-14(0)null mutants and inx-21(0) resemble nullinx-8(0) mutants inx-9(0) resemblein failinginx-8(0) to inx-9(0) support in germfailing cell to support proliferation. germ Incell contrast, proliferation. the inx-22(0) In contrast,null mutant the inx-22(0) is fertile null and mutant its germline is fertile appears and its una germlineffected; appears however, unaffected; feminized inx-22(0)however,mutants feminized lacking inx-22(0) sperm mutants fail to inhibit lacking oocyte sperm meiotic fail maturationto inhibit oocyte and ovulation meiotic inmaturation the absence and of theovulation meiotic in maturation the absence signal of the (major meiotic sperm maturation protein, MSPsignal [8 ]).(major Therefore, sperm normal protein, germ MSP cell [8]). proliferation Therefore, isnormal likely germ to require cell proliferation the association is oflikely INX-8 to require and INX-9 the withassociation germline of hemichannelsINX-8 and INX-9 containing with germline INX-14 andhemichannels INX-21. Thus, containing two classes INX-14 of germlineand INX-21. hemichannels Thus, two classes (i.e., INX-14 of germline with either hemichannels INX-21 or (i.e., INX-22) INX- assemble14 with either into gapINX-21 junctions or INX-22) with INX-8assemble and into INX-9 gap in junctions the soma with to mediate INX-8 and multiple INX-9 soma–germline in the soma to interactionsmediate multiple required soma–germline for fertility—there interactions is currently requir noed evidencefor fertility—there suggesting is acurrently function no for evidence isolated hemichannels,suggesting a function independent for isolat of gaped hemichannels, junctions, in this independent system. of gap junctions, in this system. TheThe failurefailure of of germ germ cells cells to to proliferate proliferate in in the the absence absence of soma–germlineof soma–germline gap gap junctions junctions obscures obscures any roleany thatrole gapthat junctions gap junctions may play may in play later in germline later germ developmentalline developmental events. Toevents. address To thisaddress issue, this the issue,lag-2 promoterthe lag-2 promoter was used was to express used to INX-8 express in the INX-8 DTC, in but the not DTC, sheath but cells,not sheath in inx-8(0) cells, inx-9(0) in inx-8(0)mutants inx-9(0) [7]. Inmutants these animals,[7]. In these germ animals, cell proliferation germ cell isproliferation restored to ~1is /restored2 wild-type to ~1/2 levels, wild-type but few levels, progeny but (avg. few ~2)progeny and a (avg. limited ~2) numberand a limited of dead number embryos of dead (~20) embryos are produced (~20) are compared produced to compared the wild type to the (brood wild sizetype ~300). (brood This size phenotype ~300). This represents phenotype the represents ground state the for ground the complete state for absence the complete of sheath–germline absence of gap-junctionalsheath–germline coupling, gap-junctional and closely coupling, resembles and thatclosely observed resembles when that sheath observed cell precursors when sheath (but notcell DTC)precursors are ablated (but not from DTC) developing are ablated gonad from arms developing [9]. gonad arms [9]. ToTo exploreexplore moremore specificspecific rolesroles thatthat thethe somasoma playsplays inin nurturingnurturing thethe germline,germline, wewe focusfocus onon manipulatingmanipulating
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