Fusome morphogenesis is sufficient to promote female germline stem cell self-renewal in Drosophila

Fusome morphogenesis is sufficient to promote female germline stem cell self-renewal in Drosophila | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Fusome morphogenesis is sufficient to promote female germline stem cell self-renewal in Drosophila View ORCID Profile Amanda M. Powell , Anna E. Williams , View ORCID Profile Elizabeth T. Ables doi: https://doi.org/10.1101/2025.03.10.642432 Amanda M. Powell 1 Department of Biology, East Carolina University , Greenville, NC, 27858 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amanda M. Powell Anna E. Williams 1 Department of Biology, East Carolina University , Greenville, NC, 27858 2 Biochemistry, Cell and Developmental Biology Graduate Program, Emory University , Atlanta, GA, 30322 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elizabeth T. Ables 1 Department of Biology, East Carolina University , Greenville, NC, 27858 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elizabeth T. Ables For correspondence: ablese{at}ecu.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Many tissue-resident stem cells are retained through asymmetric cell division, a process that ensures stem cell self-renewal through each mitotic cell cycle. Asymmetric organelle distribution has been proposed as a mechanism by which stem cells are marked for long-term retention; however, it is not clear whether biased organelle localization is a cause or an effect of asymmetric division. In Drosophila females, an endoplasmic reticulum-like organelle called the fusome is continually regenerated in germline stem cells (GSCs) and associated with GSC division. Here, we report that the β-importin Tnpo-SR is essential for fusome regeneration. Depletion of Tnpo-SR disrupts cytoskeletal organization during interphase and nuclear membrane remodeling during mitosis. Tnpo-SR does not localize to microtubules, centrosomes, or the fusome, suggesting that its role in maintaining these processes is indirect. Despite this, we find that restoring fusome morphogenesis in Tnpo-SR -depleted GSCs is sufficient to rescue GSC maintenance and cell cycle progression. We conclude that Tnpo-SR functionally fusome regeneration to cell cycle progression, supporting the model that asymmetric rebuilding of fusome promotes maintenance of GSC identity and niche retention. Summary Statement Regeneration of the fusome, an ER-like organelle in Drosophila female germline stem cells, relies on Tnpo-SR-dependent reorganization of the microtubule network during interphase. Introduction Stem cells maintain cellular diversity in tissues undergoing regeneration, repair, or remodeling ( Das et al., 2020 ; de Morree and Rando, 2023 ). The long-term maintenance of stem cells within tissues requires molecular mechanisms that effectively balance stem cell self-renewal with differentiation to a committed cell fate ( Knoblich, 2008 ; Li and Xie, 2005 ). Among these, asymmetric cell division (ACD) is a conserved process in which two cells of unequal fates are produced at each round of mitosis ( Chen and Yamashita, 2021 ; Sunchu and Cabernard, 2020 ). This allows a self-renewing population of stem cells to be retained in tissues even as differentiated daughters are continually produced. ACD can be achieved by differential reception of extrinsic factors or by asymmetric localization of intrinsic cell fate determinants like proteins, mRNA, or organelles. Although many stem cells display unequal partitioning of organelles at mitosis, the extent to which organelle localization promotes self-renewal remains unclear. Drosophila female germline stem cells (GSCs) are an excellent genetic and cell biological model of ACD ( Hinnant et al., 2020 ; Venkei and Yamashita, 2015 ). GSCs reside at the anterior end of ovarioles (the oocyte-producing units of the ovary) and are established during development in a somatic niche composed of cap cells and terminal filament cells ( Figure 1A ). GSCs divide perpendicular to the niche, such that each mitotic division creates one daughter cell destined for differentiation (called the cystoblast) and one stem cell, which remains anchored to the cap cells via adherens junctions. The GSC and the cystoblast remain connected through an intracellular bridge following mitosis through G 2 phase of the next cell cycle, when the ring canal constricts and abscission occurs ( Figure 1B ) ( Ables and Drummond-Barbosa, 2013 ; Villa-Fombuena et al., 2021 ). The cystoblast goes on to divide exactly four times with incomplete cytokinesis forming 16-cell cysts, from which, one oocyte will be specified. Thus, each mitotic division of the GSC has the potential to create one differentiated oocyte. Download figure Open in new tab Figure 1: Tnpo-SR promotes GSC fusome regeneration. (A) Schematic of Drosophila germarium. Somatic cap cells (grey) are juxtaposed to GSCs (green). Asymmetric division of a GSC yields a cystoblast (blue) which divides four times through incomplete cytokinesis to produce a 16-cell interconnected cyst. One cyst cell becomes the oocyte (yellow). The fusome (red) interconnects germ cells. (B) Fusome morphogenesis parallels progression through the cell cycle. (C-F) Max projections of germaria from Scrib::GFP (green, C-D) or Rtnl1::GFP (green, E-F) control females (C, E) or combined with Tnpo-SR RNAi (D, F), immunostained for fusome core proteins ⍺-Spectrin (C-D, magenta) and Hts (E-F, magenta; with LamC to visualize cap cells). Asterisks (*) denote cap cells. Scale bars = 5µm. (G-H) Quantification of fusome area in round (G) and stretched (H) fusome morphologies. Numbers in bars represent number of GSCs analyzed. Error bars represent s.e.m. (***)p<0.00001, (**)p<0.0001; Student’s two-tailed T-test. Previous studies demonstrate that at least three distinct organelles (nucleolar/ribosome complexes, centrosomes, and fusomes) are asymmetrically distributed in GSCs ( de Cuevas and Spradling, 1998 ; Fichelson et al., 2009 ; Yamashita et al., 2007 ). Among these, the fusome is arguably the most well-characterized, in large part due to a preponderance of available molecular markers for its visualization ( Hinnant et al., 2020 ; Lighthouse et al., 2008 ; Williams and Ables, 2023 ). The fusome (also called the spectrosome in GSCs) is a cytoplasmic structure composed of endoplasmic reticulum-like membranous vesicles and a spectrin/spectraplankin cytoskeletal core that elongates during the cell cycle and is partitioned to the cystoblast ( Figure 1B ) ( de Cuevas et al., 1996 ; de Cuevas et al., 1997 ; Lighthouse et al., 2008 ; Snapp et al., 2004 ). Fusome material accumulates at each mitotic division, concomitant with the disassembly of the mitotic spindle ( de Cuevas and Spradling, 1998 ; Diegmiller et al., 2023 ; Koch and King, 1966 ; Lin et al., 1994 ; Mahowald, 1971 ). For much of their cell cycle, GSCs have a large, round fusome anchored at the anterior margin of the cell, closest to the neighboring somatic cap cells ( de Cuevas and Spradling, 1998 ; Huynh, 2006 ; Ong et al., 2010 ; Villa-Fombuena et al., 2021 ). During GSC mitosis, a small plug of fusome material is deposited at the central spindle, foreshadowing placement of the intracellular bridge that forms between the GSC and the presumptive cystoblast. The fusome likely aids in the establishment and maintenance of this intracellular bridge, similar to other systems ( Chaigne and Brunet, 2022 ; Li et al., 2024 ; Ong et al., 2010 ; Spradling, 2024 ). As the GSC transitions from G 1 to S, the nascent fusome in the ring canal builds anteriorly to join with the fusome material that remained at the cap cell interface. The fusome is eventually severed between the GSC and the cystoblast when abscission occurs in G 2 (resulting in an “exclamation point” morphology; Figure 1B ). Following abscission, the GSC fusome retracts and condenses to a round morphology. The fusome is therefore asymmetrically inherited at mitosis, as the GSC always retains more organelle material than the cystoblast ( de Cuevas and Spradling, 1998 ; Huynh, 2006 ). Despite the well-characterized morphogenesis of the fusome, it is not clear whether the fusome per se is essential for GSC self-renewal. In germ cells lacking core cytoskeletal fusome components like β-spectrin or the adducin homolog hu li tai shao ( hts ), fusomes fail to form (as visualized by α-Tubulin and α-Spectrin localization) and germ cells largely fail to form cysts. However, it is unclear whether GSCs are retained in the niche, since Hts and α-Spectrin are also key tools for GSC identification ( de Cuevas et al., 1996 ; Röper, 2007 ). Similarly, loss of the recycling endosome protein Rab11, which is enriched in the GSC fusome, is essential for both fusome morphogenesis and maintenance of GSCs ( Bogard et al., 2007 ; Lighthouse et al., 2008 ). In contrast, loss of the fusome-enriched proteins Scribble or Tropomodulin do not impact fusome structure (as visualized by Hts localization) or GSC maintenance ( Lighthouse et al., 2008 ). Thus, while not every fusome-enriched protein is necessary for GSC self-renewal, the question of whether the fusome promotes GSC identity remains unresolved. Here, we take advantage of a genetic model in which GSC fusome morphogenesis is impaired to explore the connection between GSC maintenance and fusome/ER asymmetric inheritance. We previously demonstrated that the β-importin encoded by Transportin-Serine/Arginine rich ( Tnpo-SR ) is necessary for GSC self-renewal, development of 16-cell cysts, and timely oocyte specification ( Beachum et al., 2023 ). Extending these observations, we find that although Tnpo-SR does not localize to the fusome, it is essential to rebuild the fusome at each cell cycle, impacting the cytoskeletal core, ER-like membranes, and fusome-associated proteins. Consistent with the roles of other β-importins, our data suggests that Tnpo-SR promotes nuclear envelope remodeling at mitosis and suppresses microtubule growth from centrosomes during interphase. We also identify the microtubule binding protein encoded by abnormal spindle ( asp ) as a putative Tnpo-SR binding partner. Intriguingly, however, over-expression of Asp in Tnpo-SR -depleted GSCs does not rescue GSC self-renewal. Instead, we find that over-expression of the fusome component, Hts, the polarity protein Bazooka (Baz)/Par-3, or a Kinesin heavy chain fusion protein (Khc::nod::lacZ) are sufficient to restore fusome area and re-establish GSC self-renewal in Tnpo-SR -depleted GSCs. Taken together, these results suggest that rebuilding the microtubule-based fusome core at successive cell cycles builds an intracellular trafficking system that is essential to maintain the GSC fate. Results Tnpo-SR promotes regeneration of the GSC fusome during successive cell cycles Our previous work revealed that loss of Tnpo-SR reduced accumulation of Hts at the fusome at all phases of the cell cycle ( Beachum et al., 2023 ). Indeed, further analysis demonstrated that depletion of Tnpo-SR in GSCs reduced overall fusome core area, as measured by fluorescence of α-Spectrin ( Figure 1C-D , G-H) or Hts ( Figure 1E-F, G-H ). The reduction of core area in Tnpo-SR -depleted GSCs also impacted accumulation of fusome-associated proteins, such as the apicobasal scaffolding protein Scribble (Scrib; Figure 1C ’-D’, G-H), and the fusomal membrane protein, Reticulon-like1 (Rtnl1; Figure 1E ’-F’, G-H). Intriguingly, although overall fusome area was reduced, the dynamic movements of the fusome at different stages of the cell cycle were largely normal in the absence of Tnpo-SR ( Figure 2 ). Fusome morphologies consistent with all stages of the cell cycle could be readily identified ( Figure 2 and 3A ), and the overall cycling time (as measured by the percentage of GSCs labeled with the nucleoside analog EdU during a one-hour pulse; Figure 3B ) was not drastically altered. Measurements were taken on germaria collected six days after eclosion, and GSCs cycle every 15-16 hours ( Villa-Fombuena et al., 2021 ); therefore, we estimate that the fusome size was reduced ∼45% over about nine cell cycles. This suggests that Tnpo-SR is necessary to regenerate the fusome in GSCs at each mitotic division. Download figure Open in new tab Figure 2: Tnpo-SR maintains fusome asymmetry. (A-G) Control and (I-N) Tnpo-SR RNAi GSCs immunostained for the fusome (Hts, white), microtubules (⍺-Tub, magenta), polarity and cell membrane marker Scrib (Scrib::GFP, green), and DNA (DAPI, blue). Graphics represent the wild-type fusome morphological changes and the subsequent localization of Hts, Scrib and ⍺Tub as GSCs go through the cell cycle. Asterisks denote cap cells. Scale bar = 5µm. Arrowheads denote microtubule defects (M’, N’). Download figure Open in new tab Figure 3: Loss of Tnpo-SR delays exit from G 1 . Quantification of cell cycle progression via fusome morphology (A), incorporation of EdU in a one-hour pulse (B), or Fly-FUCCI fluorescent labeling (C) in control (solid) or Tnpo-SR RNAi (striped) GSCs. G 1 (yellow and white), S (purple), G 2 (blue), M (green). Mitotic GSCs were identified by chromosome morphology (labeled with DAPI). (B) Percentage GSCs that are EDU positive (purple) or negative (grey). Numbers below bars indicate number of GSCs analyzed. Fisher’s Exact test was used to compare the number of GSCs in G 1 (plug morphology, A; E2F1+, C) or the number of EdU+ GSCs (B) to the total number of GSCs. (**)p<0.001, (*)p<0.01. Tnpo-SR is dispensable for the assembly, maintenance, and function of the mitotic spindle Fusome regeneration in GSCs begins during mitosis, as new fusome material is formed from the microtubule remnants of the mitotic spindle ( Figure 1B ) ( Lin et al., 1994 ; Villa-Fombuena et al., 2021 ). β-importins have a variety of roles during mitosis, including regulation of mitotic spindle assembly, abscission, and nuclear membrane and nuclear pore assembly, at least in part via sequestration of spindle assembly factors ( Askjaer et al., 2002 ; Beaudet et al., 2017 ; Beaudet et al., 2020 ; Chen et al., 2015 ; Nachury et al., 2001 ; Wiese et al., 2001 ). Given the roles of β-importins in mitosis, we hypothesized that interphase fusome morphogenesis might be impaired in Tnpo-SR -depleted GSCs as a secondary consequence of disrupted mitotic spindle assembly. We labeled GSCs with a fluorophore-conjugated α-Tubulin, the mitosis marker phosphorylated Histone H3 (PHH3), and the centrosome core protein Asterless (Asl), using a light fixation protocol at room temperature to permit stabilization of microtubules and identification of GSCs and surrounding niche cells in control and Tnpo-SR RNAi germaria ( Figure 4A,C ) ( Grieder et al., 2000 ; Williams and Ables, 2023 ). Although some spindles appeared in a wavy shape, we found no significant difference in mitotic spindle length or width between control GSCs and GSCs lacking Tnpo-SR ( Figure 4A,C ,I-J). Since spindle stability relies on the activity of the spindle matrix ( Yao et al., 2018 ), we further tested whether localization or levels of two key Drosophila spindle matrix components, Chromator and Megator, were affected by depletion of Tnpo-SR . Congruent with previous studies, we found that Chromator localizes to the chromatin during interphase in wild-type GSCs, while Megator is localized to the inter-heterochromatin space, cytoplasm, and nuclear membrane (Figure S1A,C) ( Qi et al., 2004 ; Rath et al., 2004 ; Yao et al., 2012 ; Yao et al., 2018 ). We did not identify any clear differences in the localization of Chromator or Megator between wild-type and Tnpo-SR RNAi GSCs during interphase or at mitosis (Figure S1B,D). Download figure Open in new tab Figure 4: Tnpo-SR regulates nuclear lamina remodeling at mitosis and suppresses microtubule growth during interphase. (A-H) Control (A-B,E-F), and Tnpo-SR RNAi (C-D,G-H) germaria immunostained for a mitotic marker (PHH3, white) (A,C), the fusome (Hts, white) (B,D-H), microtubules (⍺-Tubulin, magenta), centrosomes (Asl, green), and DNA (DAPI) (A-D). GSCs outlined with white lines (solid, control) (dashed, Tnpo-SR RNAi ). Asterisks denote cap cells. Scale bars = 5µm. (I-L) Quantification of GSC mitotic spindle length (I) and width (J), LaminB fluorescence intensity (K), and interphase GSCs with microtubules emanating from centrosomes (L) in control (solid) or Tnpo-SR RNAi (striped) germaria. Numbers in/under bars represent number of GSCs analyzed. Error Bars represent s.e.m. (***)p<0.00001, (**)p<0.0001; Student’s two-tailed T-test (I-K) or Fishers Exact test (L). We then asked whether the minor spindle abnormalities in Tnpo-SR -depleted GSCs impacted chromosome separation. We analyzed localization of the centromeric protein Centromere Identifier (CID, the centromere-specific histone H34 variant), which is essential for proper assembly and function of centromeres and kinetochores, the primary drivers of spindle assembly ( Dattoli et al., 2020 ; Kochendoerfer et al., 2021 ). Consistent with the largely stable microtubule spindle, the number of CID foci was equivalent between Tnpo-SR -depleted and control GSCs (Figure S2A-C). Taken together, these data suggest that assembly, maintenance, and function of the mitotic spindle are largely unaffected by the loss of Tnpo-SR . Tnpo-SR is required for proper remodeling of the GSC nuclear lamina at mitosis In contrast to spindle morphology, loss of Tnpo-SR caused significant changes in organization of the nuclear lamina during mitosis. Wild-type GSCs undergo a non-canonical semi-closed nuclear envelope breakdown (NEB), retaining an intact but permeable nuclear membrane ( Duan et al., 2021 ). We visualized the fragmented nuclear membrane during mitosis in wild-type GSCs using antibodies raised against LaminB ( Figure 4B ). In GSCs lacking Tnpo-SR , high levels of LaminB localized around DNA during metaphase ( Figure 4D,K ). This suggests that in the absence of Tnpo-SR , the nuclear lamina collapses to surround the DNA, instead of fragmenting around the spindle. Abnormalities in nuclear lamina fragmentation did not appear to disturb centrosome duplication, maturation, or localization. In both controls and Tnpo-SR -depleted GSCs, centrosomes [visualized with antibodies against Asl or Pericentrin-like Protein (Plp)] were randomly localized until late-G 2 , at which time one centrosome became anchored at the niche adjacent to the round-shaped fusome in preparation for mitosis (Figure S2D-H). We did not detect overt differences in Asl or Plp accumulation. Thus, while we observed that loss of Tnpo-SR impacts nuclear lamina fragmentation, the biological significance of this phenotype remains unclear. Loss of Tnpo-SR causes aberrant interphase centrosomal microtubules Since our data did not support roles for Tnpo-SR in assembly or function of the mitotic spindle, we asked whether improper disassembly of the spindle and regeneration of the fusome in the GSCs caused the reduction in fusome size. Following mitosis, the GSC/cystoblast pair remains connected via a temporary intracellular bridge ( Ables and Drummond-Barbosa, 2013 ; Mathieu et al., 2013 ). During this time, fusome proteins (including Hts and the kinase Par-1) accumulate in the cytokinetic furrow ( Figure 2B-B ’), and condense anteriorly in the GSC towards the cap cells ( Figure 2C-E ’) ( Villa-Fombuena et al., 2021 ). As the central spindle disassembles, α-Tubulin becomes tightly associated with Hts, extending the fusome and likely serving as a framework for continued vesicular trafficking ( Figure 2D ). Following abscission, fusome material (both Hts and α-Tubulin) aggregates at the anterior side of the GSC, retracting away from the cystoblast and re-forming a round structure by mid-G 2 ( Figure 2F-G ). GSCs lacking Tnpo-SR still disassembled the bulk of microtubules during telophase and correctly formed the central spindle ( Figure 2H-I ); however, we saw abnormalities in α-Tubulin localization beginning as early as G 1 ( Figure 2I-N ). Microtubules appeared disorganized, oriented in random directions rather than coalesced towards the fusome or the anterior of the cell ( Figure 2I ’, M’, N’). Although α-Tubulin co-localized with Hts at the fusome, the levels of Hts were drastically reduced, particularly during S/G 2 when the fusome typically builds anteriorly. By late G 2 , when control cells had already condensed α-Tubulin into a spherical fusome, Tnpo-SR -depleted GSCs failed to condense α-Tubulin, and instead appeared to be building microtubules in random directions ( Figure 2N-N ’). These observations suggested that Tnpo-SR might suppress inappropriate microtubule growth during interphase. To test this hypothesis, we again co-localized α-Tubulin with Asl and imaged cells in interphase ( Figure 4E-H ). In control interphase GSCs, microtubules were observed either at the fusome or the cell cortex ( Figure 4E-F ). In contrast, microtubules in Tnpo-SR -depleted GSCs grew from centrosomes scattered throughout the cell, oriented intracellularly towards the somatic cap cells and occasionally extending to wrap around the cap cells ( Figure 4G-H ’, L). These results confirm that Tnpo-SR normally suppresses microtubule growth and suggest that loss of microtubule organization underlies the failure to regenerate the fusome properly in Tnpo-SR -depleted GSCs. We then asked whether the microtubule defects caused by Tnpo-SR depletion impacted progression through the cell cycle, using two independent assays to measure cell cycle phase length. First, since overall fusome structure remained largely intact in Tnpo-SR -depleted GSCs ( Figure 2 ), we used the morphology of the fusome as an indicator of cell cycle phase ( Figure 1B , 3A) ( Villa-Fombuena et al., 2021 ). Second, we employed the Fly-Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system to distinguish cell cycle phases using fluorochrome-tagged transgenes that are degraded in parallel with the cell cycle regulators Cyclin B and E2F1 ( Figure 3C ) ( Hinnant et al., 2017 ; Villa-Fombuena et al., 2021 ; Zielke et al., 2014 ). In both assays, and consistent with prior studies, control GSCs were most frequently found in G 2 , with a much smaller population in G 1 and M, which occur in exceedingly short windows of time ( Hinnant et al., 2017 ; Villa-Fombuena et al., 2021 ). In contrast, Tnpo-SR -depleted cells were more likely to be found in G 1 , suggesting that G 1 length is at least two times longer than normal ( Figure 3A,C ). Notably, in both Tnpo-SR -depleted and control GSCs, we observed accumulation and co-localization of the mitotic marker PHH3 with DNA during prophase and metaphase, and waning expression during telophase as chromatids separated to opposing poles ( Figure 4A,C ), suggesting that the length of mitosis is largely unaffected by loss of Tnpo-SR . Further, these results are not consistent with centrosome maturation defects, which typically arrest cells in G 2 ( Venkei and Yamashita, 2015 ). We conclude that Tnpo-SR -depleted cells delay exit from G 1 , perhaps as a consequence of inappropriate microtubule regulation or improper NEB. Tnpo-SR does not localize to centrosomes or the fusome Given that loss of Tnpo-SR caused defects in fusome regeneration and centrosome-dependent microtubule dynamics, we hypothesized that it might function in GSCs by directly binding proteins associated with these organelles, facilitating intracellular protein transport. We previously characterized two novel alleles to monitor Tnpo-SR localization ( Beachum et al., 2023 ). Of these, a transgenic allele encoding a hemagglutinin-tagged Tnpo-SR (HA::Tnpo-SR) permitted specific and robust expression in the germline and faithfully reproduced endogenous Tnpo-SR intracellular localization in differentiated germ cells. We used this allele to carefully monitor Tnpo-SR localization in GSCs at different points in the cell cycle. As expected, we observed HA::Tnpo-SR around the nuclear membrane, inside the nucleus, and, to lesser extent, in the cytoplasm ( Figure 5A-B ), consistent with its purported role in nucleocytoplasmic transport. At mitosis, HA::Tnpo-SR was evenly dispersed between the nucleus and cytoplasm, demonstrating that it can freely traverse the semi-permeable nuclear lamina during mitosis ( Figure 5C ’). We also noted the existence of small puncta of HA::Tnpo-SR that were difficult to discern with standard confocal microscopy ( Figure 5A ’). Indeed, higher resolution imaging (with image deconvolution to improve the signal to noise ratio) confirmed that some HA::Tnpo-SR localized to vesicles, frequently near the nucleus and of similar size as centrosomes ( Figure 5B ’). However, we did not observe HA::Tnpo-SR co-localized with α-Tubulin or the centrosome protein Asl, suggesting that it does not accumulate at centrosomes, fusomes, or the mitotic spindle. These data suggest that Tnpo-SR regulation of these organelles occurs indirectly. Download figure Open in new tab Figure 5: Tnpo-SR does not aggregate in fusomes or at centrosomes. Maximum intensity projections of interphase (A-B) or mitotic (C) UASz-HA::Tnpo-SR GSCs immunostained for Tnpo-SR (HA, green), microtubules (α-Tubulin, magenta), centrosomes (Asl, white), and DNA (DAPI, blue). A-B are the same germarium imaged with (B) and without (A) Airyscan high resolution imaging with deconvolution. Asterisks denote cap cells. Arrowheads denote centrosomes (Asl). Arrows denote HA-enriched foci. Scale bar = 5µm. Localization of Asp, but not other microtubule binding proteins, is regulated by Tnpo-SR Given its putative role as a nucleocytoplasmic transport protein, and the absence of co-localization at the fusome or centrosomes, we hypothesized that Tnpo-SR might function in the stability, transport, or translation of a protein essential for microtubule dynamics in germ cells. Previous studies identified Abnormal spindle (Asp), a microtubule binding protein that localizes to the poles of mitotic spindles and the minus ends of spindle microtubules, as a regulator of germ cell mitotic divisions ( Riparbelli et al., 2004 ; Saunders et al., 1997 ). Originally described as a regulator of centrosome organization, loss of asp altered germ cell mitotic divisions (by inducing ‘wavy’ mitotic spindles), disrupted microtubule organization, and induced defects in fusome structure, similar to loss of Tnpo-SR ( Beachum et al., 2023 ; Do Carmo Avides and Glover, 1999 ; Riparbelli et al., 2004 ; Saunders et al., 1997 ). Unfortunately, we were unable to source anti-Asp antibodies to visualize endogenous Asp. Instead, we confirmed Asp localization by driving a GFP-tagged Asp specifically in germ cells ( Figures 6A , S3A) ( Shao et al., 2010 ). Consistent with previous studies, Asp was enriched at the spindle poles and centrosomes during mitosis in GSCs and dividing cysts (Figure S3A). In interphase GSCs, however, Asp localized to the cytoplasm and was enriched at the fusome ( Figure 6A,C ). As germ cells differentiated, Asp remained localized to the cytoplasm until the 16-cell cyst stage, where it concentrated around the cortex of a single cyst cell (likely the presumptive oocyte) ( Figure 6A,D ). We then asked whether depletion of Tnpo-SR impacted Asp localization. In GSCs lacking Tnpo-SR , 40% of GSCs lost the cytoplasmic expression of Asp and instead concentrated it in the fusome ( Figure 6B,C ). Moreover, differentiated Tnpo-SR -depleted germ cells accumulated Asp in the nucleus, which was only rarely seen in controls ( Figure 6B,D ). These results suggest that Tnpo-SR is necessary to move or stabilize Asp in the cytoplasm of GSCs and their differentiating daughters. Download figure Open in new tab Figure 6: Asp intracellular localization is regulated by Tnpo-SR . (A-B) UASp-Asp::GFP (A) and Tnpo-SR RNAi ;UASp-Asp::GFP (B) germaria immunostained for the fusome (Hts, magenta), cap cells (LamC, magenta), and Asp (green). Asterisks denote cap cells, blue lines outline GSCs, yellow lines outline differentiated germ cells, arrow heads denote Asp localization in undifferentiated germ cells, arrows denote Asp localization in differentiated germ cells. Scale bar = 5µm. (C-D) Quantification of Asp localization in control (solid) and Tnpo-SR RNAi ;UASp-Asp::GFP (striped) GSCs (C) or differentiated germ cells (D). Numbers in bars represent numbers of GSCs (C) or cysts (D). (***)p<0.00001, Fishers Exact test. To determine whether the interaction between Tnpo-SR and Asp was specific, or a general property of effectors of spindle formation or disassembly, we examined localization of other regulators of microtubule dynamics. As with Asp, we used transgenic approaches to express a fluorescently-tagged version of each protein specifically in germ cells ( Figure 7A-H and S3B-D). We selected three proteins associated with microtubule binding: Mini-Spindles (Msps), a plus-end microtubule binding protein that regulates microtubule organization ( Brittle and Ohkura, 2005 ; Lu et al., 2023 ); Klp10A, a kinesin that induces microtubule depolymerization at plus ends ( Chen et al., 2016 ; Persico et al., 2019 ); and an engineered kinesin-like protein that binds preferentially to microtubule minus ends (Khc::nod-lacZ; ( Grieder et al., 2000 )). In each case, microtubule binding proteins co-localized with α-Tubulin, and this localization was equivalent between control and Tnpo-SR -depleted GSCs (representative images from Khc::nod-lacZ and Msps are shown in Figure 7A-D ). This suggests that Tnpo-SR specifically regulates Asp localization, rather than generally impacting localization of microtubule binding proteins. Download figure Open in new tab Figure 7: Restoration of fusome regeneration rescues GSC self-renewal and G 1 length in Tnpo-SR -depleted GSCs. Representative germaria from control (A,C,E,G) or Tnpo-SR RNAi (B,D,F,H) females carrying UASp-Khc::nod::LacZ (A-B), UASp-CFP::Msps (C-D), UASp-Hts::mCherry (E-F), or UASp-Baz::GFP (G-H), immunostained for α-Tubulin (αTub, magenta, A-F) and β-galactosidase (A-B), GFP (C-D, G-H), or mCherry (E-F) to visualize the tagged proteins. Asterisks denote cap cells. Scale bars = 5µm. (I-J) Quantification of fusome area in round (I) and stretched (J) fusome morphologies. (K) Quantification of the number of GSCs per germarium, as a readout of GSC self-renewal. (L) Quantification of cell cycle progression via fusome morphology. Numbers in bars represent number of GSCs (I-J,L) or germaria (K). Error bars represent s.e.m. Controls (solid bars) were compared to Tnpo-SR RNAi alone (white striped bars), while Tnpo-SR RNAi alone was compared to Tnpo-SR RNAi combined with over-expression of the indicated gene of interest (stripes with colors) using Student’s two-tailed T-test (I-K) [(***)p<0.00001, (**)p<0.0001] or Fishers Exact test (L) [(**)p<0.001, (*)p<0.01]. Restoration of fusome morphogenesis is sufficient to rescue GSC loss in the absence of Tnpo-SR If the fusome promotes asymmetry between the GSC and the cystoblast during division, then failure to regenerate the fusome at each cell cycle should decrease GSC self-renewal, resulting in fewer numbers of GSCs in older flies. To test this idea, we asked whether restoration of fusome regeneration could rescue GSC loss in germaria that lack Tnpo-SR . To rescue fusome regeneration, we first over-expressed a fluorescently-tagged version of Hts in Tnpo-SR -depleted GSCs ( Figure 7E-F ). Notably, over-expression of Hts alone was not sufficient to change the area of the fusome in control GSCs. In the absence of Tnpo-SR , however, over-expression of Hts was sufficient to rescue fusome area in G 2 /M (round fusomes) and G 1 /S/G 2 (stretched fusomes) ( Figure 7I-J ). Moreover, over-expression of Hts was also sufficient to rescue GSC number and the length of G 1 ( Figure 7K-L , Table 1 ). View this table: View inline View popup Download powerpoint Table 1: Summary of rescue phenotypes in Tnpo-SR -depleted GSCs. Original data ( Figure 7 ) is summarized, indicating the raw data for average (Avg.) GSC per germaria, pixelated area of round and stretched fusomes, and the percentage of GSCs in G 1 when the cell cycle was quantified based on fusome morphology. P-value statistics were determine by comparisons made using Student’s two-tailed T-test For Avg. GSC and fusome measurements [(***)p<0.00001, (**)p<0.0001] or Fishers Exact test (%G 1 ) [(**)p<0.001, (*)p<0.01] for fusome morphology data. As an independent means to rescue fusome regeneration, we then over-expressed the polarity protein Baz ( Figure 7G-H ). Endogenous expression and localization of apical proteins (Baz, Scrib, or E-cadherin) was equivalent between Tnpo-SR -depleted and control cells (Figure S4) ( Beachum et al., 2023 ). Since Baz is sufficient to promote microtubule focusing to the oocyte in differentiated cells ( Cox et al., 2001 ; Huynh et al., 2001 ), we reasoned that over-expression of Baz might hyperpolarize GSCs, focusing microtubules to the apical border. Indeed, over-expression of a fluorescently tagged Baz alone or in Tnpo-SR -depleted GSCs significantly increased fusome area ( Figure 7I-J ). As with over-expression of Hts, over-expression of Baz in Tnpo-SR- depleted GSCs also rescued GSC number and the progression of these cells through G 1 ( Figure 7K-L , Table 1 ). Importantly, over-expression of other fusome-associated proteins was not sufficient to rescue fusome regeneration in the absence of Tnpo-SR . For example, fusome size in Tnpo-SR -depleted GSCs was not altered by over-expression of sticky ( sti ), a Drosophila ortholog of Citron Kinase that localizes to the GSC fusome and is essential for cytokinetic abscission ( D’Avino and Capalbo, 2016 ; Price et al., 2023 ), or over-expression of the microtubule binding protein Klp10A ( Figure 7I-L ). Moreover, even though Asp localization was concentrated in the fusome in Tnpo-SR -depleted cells, its over-expression was not sufficient to restore fusome regeneration or GSC number ( Table 1 ). This suggests that even though Tnpo-SR impacts Asp intracellular transport, this interaction is independent of the mechanism by which Tnpo-SR promotes fusome morphogenesis. We also observed that over-expression of Khc::nod-lacZ was sufficient to rescue fusome size in Tnpo-SR -depleted cells only in cells in G 1 /S/G 2 , when the fusome is back-building from the cytokinetic furrow to the apical side of the GSC ( Figure 7I-J ). This effect also restored GSC number and the length of G 1 ( Figure 7K-L ). Since Khc::nod-lacZ binds microtubule minus ends, we propose that its over-expression helps extend, focus, and/or stabilize microtubules at the building fusome, promoting regeneration. Surprisingly, although the over-expression of microtubule binding protein, Msps, did not rescue overall fusome integrity or G 1 progression in the absence of Tnpo-SR , we observed a significant increase in GSC number compared to loss of Tnpo-SR alone ( Figure 7K ). Previous studies showed that Msps is required to promote tubular ER formation through microtubule trafficking in later stage oocytes ( Pokrywka et al., 2009 ). We propose that the over-expression of Msps may similarly increase the tubular ER of GSCs. Thus, although the fusome core size (visualized by Hts) was not altered, fusome/ER membrane area may have been increased sufficiently to restore fusome function and GSC maintenance in the absence of Tnpo-SR . Taken together, these results suggest that the fusome, which must be rebuilt in GSCs at each cell cycle, facilitates the intracellular protein trafficking that promotes GSC self-renewal. Discussion Stem cells that undergo ACD often have organelles that are partitioned asymmetrically, but whether organelle asymmetry promotes and maintains self-renewal is unknown. Here, we further characterized the roles of the β-importin, Tnpo-SR, in the regeneration and asymmetric distribution of the ER-like fusome in GSCs. We confirmed that a primary defect of Tnpo-SR -depleted cells is the loss of fusome area due to insufficient fusome regeneration at successive cell cycles. We found that consistent with roles described for other β-importins, Tnpo-SR maintains cystoskeletal organization through the suppression of microtubules during interphase and described a novel role for Tnpo-SR in nuclear lamina remodeling during mitosis. We also identified the microtubule binding protein, Asp, as a putative binding partner of Tnpo-SR. Although the over-expression of Asp itself was not sufficient to restore Tnpo-SR loss of function, restoration of fusome regeneration compensated for loss of Tnpo-SR , preserving GSC number. These data suggest that the primary function of the fusome is to establish and maintain an intracellular trafficking network to the apical pole of GSCs. Moreover, this intracellular trafficking is essential for maintaining GSCs in the niche over time. We propose that asymmetric distribution of the fusome is not only a hallmark of GSC biology, but also an integral part of maintaining the undifferentiated fate of GSCs. Maintaining fusome integrity promotes GSC asymmetry In the interconnected germ cysts of the Drosophila ovary, the fusome acts to connect and coordinate germ cells via protein and organelle trafficking ( Bolívar et al., 2001 ; Cox and Spradling, 2003 ; Deng and Lin, 1997 ; Grieder et al., 2000 ; Mahowald, 1971 ; Mahowald and Strassheim, 1970 ; Röper and Brown, 2004 ). As germline cysts mitotically divide, the fusome acts as an anchor for centrosomes, polarizing the mitotic spindles ( Deng and Lin, 1997 ). In mutants lacking hts , mitotic divisions are randomized and cysts fragment ( Deng and Lin, 1997 ). Further, the asymmetry of the fusome in differentiated cysts is required to establish oocyte identity. The fusome must coordinate and polarize microtubules to concentrate pro-oocyte factors asymmetrically (BicD, Egl, Orb) ( Röper and Brown, 2004 ). Specialized structures like centromeres ( Mahowald and Strassheim, 1970 ), centrosomes ( Bolívar et al., 2001 ; Grieder et al., 2000 ), mitochondria ( Cox and Spradling, 2003 ), and Golgi elements ( Cox and Spradling, 2003 ) are then shuttled along the fusome and become concentrated in the oocyte. While the morphological changes of the fusome have been well described in asymmetric germ cells (GSC and oocyte), a direct role for the fusome in maintaining GSCs has not previously been described. The molecular mechanisms directing fusome asymmetry in GSCs may be paralogous to the distribution of organelles across cell types ( Barlan and Gelfand, 2017 ; Derivery et al., 2015 ; Jongsma et al., 2015 ; Ouellet and Barral, 2012 ). There are two evolutionary conserved principles that direct the separation of organelles. First, organelle inheritance must be spatially regulated. In yeast, this is regulated through interactions with microtubules ( Ouellet and Barral, 2012 ; Weisman, 2006 ). Second, this process must be regulated in tune with the mitotic cell cycle ( Ouellet and Barral, 2012 ; Weisman, 2003 ; Weisman, 2006 ). The latter of these rules seems to be much more complex among cell types because it relies on proper timing of organelle growth and cell cycle progression. In cells that undergo ACD, the process of organelle segregation is even more elaborate because the organelles themselves are asymmetrically segregated. For example, the distribution of ER in Drosophila neuroblasts relies on the regulation of both polarity and microtubules, suggesting a positive feedback loop between organelle and microtubules dynamics ( Smyth et al., 2015 ; Tikhomirova et al., 2022 ). We provide evidence suggesting that the ER-like fusome is asymmetrically segregated through a partnership with both microtubules and polarity factors. Coordinated with the mitotic cell cycle, the fusome relies on the stability of microtubules to asymmetrically segregate. In the absence of Tnpo-SR , interphase microtubule dynamics are disorganized and fusome integrity and asymmetry is diminished. Interestingly, the over-expression of Msps in the absence of Tnpo-SR restored GSC maintenance independently of fusome area. In Drosophila oocytes, Msps works in tandem with minus-end microtubule motor, dynein, to create a positive feedback loop that enhances microtubule transport, creating a small but stochastic difference in microtubule polarity ( Lu et al., 2023 ). Foundational studies on dynein and its transient presence in the fusome at mitosis suggest a role in polarizing the mitotic spindles of early germ cells ( McGrail and Hays, 1997 ). The over-expression of Msps in the absence of Tnpo-SR could enhance microtubule polarity at levels sufficient to promote GSC asymmetry, but not enough to fully regenerate fusome area. Importins are linked to several complex mitotic and meiotic regulatory events through their interaction with the RanGTPase cycle ( Cesario and McKim, 2011 ; Kalab et al., 1999 ; Kalab et al., 2002 ; Lonhienne et al., 2009 ; Mühlhäusser and Kutay, 2007 ). The most extensive studies on the regulation of cytoskeletal organization by importins focused on Importin-α and Importin-β. For example, Importin-β maintains the mitotic spindle through associations with Ran in C. elegans embryos, and competitively binds the microtubule associated protein, NuMa, during interphase ( Nachury et al., 2001 ; Wiese et al., 2001 ; Askjaer et al., 2002 ). Our analysis of Tnpo-SR -depleted GSCs suggests that Tnpo-SR shares some functional properties with Importin-β. While Tnpo-SR does not appear to influence mitotic spindle assembly or maintenance, it does play a significant role in the suppression of microtubules during interphase, similar to the interaction between Importin-B and NuMa. To date, Tnpo-SR has only been directly implicated in the binding of phosphorylated serine/arginine (SR) proteins in Drosophila ( Allemand et al., 2002 ; Lai et al., 2001 ). Yet protein conformation studies in the human ortholog of Tnpo-SR, Tnpo3, identified over one hundred potential binding partners, ranging from transcription factors to microtubule and cell cycle regulators ( Kimura et al., 2017 ). Although we cannot rule out the possibility that the effects we attribute to Tnpo-SR result from its interaction with a splicing factor (many of which are SR proteins), our data suggests Tnpo-SR may bind a variety of proteins that allow it to regulate multiple pathways. We observed that localization of the minus-end microtubule binding protein, Asp, is altered in the absence of Tnpo-SR , and that localization of Asp is different in GSCs than in differentiated cells. This suggests a direct role for Tnpo-SR in maintaining Asp localization. However, we found no obvious evidence that Tnpo-SR and Asp co-localize, suggesting that this interaction may not be direct or stable. Importin partner proteins largely fall into two categories. “Cargoes” are proteins that are destined for transport, whereas “binding partners” can be transient or competitive interactions ( Kuersten et al., 2001 ; Yang et al., 2023 ). Importin-β competitively binding microtubule associated protein, NuMa, during interphase is an example of an importin binding partner ( Nachury et al., 2001 ; Wiese et al., 2001 ). Importin-β acts to sequester NuMa during interphase, suppressing ectopic microtubule assembly, but also allowing the nuclear accumulation of spindle assembly factors during interphase to ensure they are accessible near chromatin once mitosis begins. We propose that Asp is a binding partner of Tnpo-SR, most similar to the interaction between Importin-ß and NuMa. Alternatively, Tnpo-SR could impact localization of a factor necessary for Asp localization, or Tnpo-SR and Asp could interact with a common set of microtubule regulators. Additional studies will be necessary to elucidate relevant molecular mechanisms. The changes in Asp localization depending on differentiation stage also suggests that importins like Tnpo-SR may regulate the developmental switches that occur during germ cell differentiation. Thus, while Tnpo-SR may serve to establish fusome asymmetry and thus maintain GSCs, it may likely play a different role in more differentiated germ cells, such as in establishment of oocyte identity or in the maintenance of fusome integrity necessary to keep differentiated cells interconnected. We posit that since Tnpo-SR promotes fusome morphogenesis, it may regulate GSC maintenance and oocyte identity at least in part by some common molecular mechanisms, though unique regulation is also likely. Indeed, the difference in Asp localization suggests that not all mechanisms will be shared by GSCs and differentiated oocytes. Further studies investigating the cargo loads of Tnpo-SR based on differentiation stage will help distinguish between these possibilities. Study Limitations While this data supports that rescuing fusome regeneration is sufficient to promote GSC maintenance there are a few important limitations. We relied on over-expression of tagged proteins due to the low endogenous levels in GSCs, as this methodology allowed us to easily visualize proteins of interest. However, over-expression does not always reflect endogenous localization and could impact the localization or functionality of the protein. All studies were performed using fixed cell imaging. While we and others have demonstrated great reproducibility between fixed and live imaging of fusome dynamics, it remains formally possible that fixation of the samples alters intracellular localization of fusome-associated proteins. Materials and Methods Drosophila Strains and Husbandry Flies were maintained at 22-25°C in standard medium (cornmeal/ molasses/yeast/agar) (NutriFly MF; Genesee Scientific). Fly genotypes are listed in Table 2 . Female progeny were collected within 24 h of eclosion and maintained on wet yeast at 25°C. For RNAi experiments, germline knockdown of Tnpo-SR was facilitated by expressing the pValium22 -based transgene UAS-Tnpo-SR RNAi using the germline-specific nos-GAL4::VP16-nos.UTR (referred to throughout as nos-Gal4 ) ( Beachum et al., 2023 ; Doren et al., 1998 ). Females carrying the driver alone were used as controls. View this table: View inline View popup Table 2. Reagents. List of fly lines and antibodies used in this study. Tnpo-SR null GSCs (Figure S4) were generated by inducing genetic mosaics using Flippase (FLP)/FLP recognition target (FRT) mitotic recombination, as previously described ( Beachum et al., 2023 ). Immunofluorescence and Microscopy Ovaries were prepared for immunofluorescence microscopy as described ( Hinnant et al., 2017 ; Williams and Ables, 2023 ). Ovaries were dissected and teased apart in room temperature Grace’s medium without additives (Caisson Labs) and fixed in 5.3% formaldehyde in Grace’s medium for 10 min at room temperature. They were then washed extensively in phosphate-buffered saline (PBS, pH 7.4; Fisher) with 0.1% Triton X-100, permeabilized in PBS with 0.5% Triton X-100 for 30 minutes and blocked for 3 hours in blocking solution [5% normal goat serum (MP Biomedicals), and 0.1% Triton X-100 in PBS] at room temperature. Primary antibodies (listed in Table 2 ) were diluted in blocking solution and used overnight at 4°C. Secondary antibodies were followed by a 2 hour (or overnight) incubation at room temperature with AlexaFluor 488-, 568- or 633-conjugated goat species-specific antibodies (Life Technologies; 1:200). All ovary samples were stained with 0.5 μg/ml 40 -6-diamidino-2-phenylindole (DAPI; Sigma) in 0.1% Triton X-100 in PBS, and mounted in 90% glycerol mixed with 20% n-propyl gallate (Sigma). Confocal z-stacks (1 or 0.5 μm optical sections) were collected with the Zeiss LSM700 laser scanning microscope or Zeiss LSM800 laser scanning microscope with Airyscan detector both using Zen Black software. Airyscan images were subject to deconvolution protocols performed by the Zen program. Images were analyzed using Zeiss ZEN software or FIJI and minimally and equally enhanced via histogram using ZEN and Adobe Photoshop Creative Suite. Statistical Analysis GSCs were identified based on the juxtaposition of their fusomes to the junction of adjacent cap cells. GSC loss was measured as the average number of GSCs per germaria, and results were subjected to a Student’s two-tailed T-test comparing ( Figures 1 , 3 ,4) driver controls versus driver>UAS-RNAi or ( Figure 7 ) driver>UAS-RNAi versus driver>UAS-RNAi,UASp rescue model. Each experimental group was evaluated using Microsoft Excel and Prism (Graphpad). Fusome measurements were taken from the center-most z-stack of each GSC where the main bulk of the fusome was most highly localized. The average pixel area of the fusome was collected using the FIJI area tool measuring the pixel area of the identified fusome using α-Hts, α-alpha-Spec, or α-GFP (Scrib::GFP, Rtnl1::GFP). Measurements were transferred to Microsoft Excel and Student’s two-tailed T-tests were performed comparing driver controls versus driver>UAS-RNAi versus driver>UAS-RNAi,UASp rescue model. Mitotic spindle measurements were taken using the line tool in FIJI using a max z-stack projection of the mitotic GSC being analyzed (from the bottom of the cell to the top). Length measurements were collected from one centrosome to the other (identified by centrosome marker Asl). Width measurements were taken from the center of the mitotic spindle at its widest point. Measurements were transferred to Microsoft Excel and Student’s two-tailed T-tests were performed comparing driver controls versus driver>UAS-RNAi versus driver>UAS-RNAi,UASp rescue model. Interphase microtubules were identified based on the presence of alpha-tubulin emanating from centrosomes (identified by centrosome marker Asl). A Fishers Exact test analysis was performed using Prism (Graphpad) comparing driver controls versus driver>UAS-RNAi versus driver>UAS-RNAi,UASp rescue model. Intensity of LaminB accumulation at the DNA during mitosis measurements were taken using the line tool in FIJI. Each mitotic GSC was visualized in a z-stack max projection and a line was drawn surrounding the DNA (using DAPI to visualize). The intensity of LaminB was measured at that line and measurements were transferred to Microsoft Excel and Students two-tailed T-tests were performed comparing driver controls versus driver>UAS-RNAi . Competing Interests No competing interests declared. Funding This work was supported by National Institutes of Health R15-GM117502 (E. T. A.). A.M.P. was supported by the East Carolina University Department of Biology, Harriot College of Arts and Sciences, and Graduate School. A.E.W. was supported by the ECU Office of Undergraduate Research. Data and Resource Availability Data and resource availability: All relevant data and resource can be found within the article and its supplementary information. Acknowledgements Many thanks to the ECU Department of Biology Microscopy Core Facilities and the ECU School of Dental Medicine for microscopy support, and to members of the Ables and Anllo laboratories and our anonymous reviewers for helpful discussion and critical reading of this manuscript. Drosophila lines were obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) or Kyoto Drosophila stock center or kind gifts from V. Gefland, A. Spradling, D. Drummond-Barbosa, T. Harris, and B. Riggs. Antibodies were a kind gift from N. 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