Phosphatidylinositol 5 phosphate 4 kinase regulates phosphatidylinositol 3,4 bisphosphate levels in vivo

Phosphatidylinositol 5 phosphate 4 kinase regulates phosphatidylinositol 3,4 bisphosphate levels in vivo | 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 Phosphatidylinositol 5 phosphate 4 kinase regulates phosphatidylinositol 3,4 bisphosphate levels in vivo Aishwarya Venugopal , Shreya Varma , View ORCID Profile Padinjat Raghu doi: https://doi.org/10.1101/2025.11.24.690171 Aishwarya Venugopal 1 National Centre for Biological Sciences -TIFR , GKVK Campus, Bangalore, 560065, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shreya Varma 1 National Centre for Biological Sciences -TIFR , GKVK Campus, Bangalore, 560065, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Padinjat Raghu 1 National Centre for Biological Sciences -TIFR , GKVK Campus, Bangalore, 560065, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Padinjat Raghu For correspondence: praghu{at}ncbs.res.in Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract In Drosophila , loss of phosphatidylinositol 5 phosphate 4 kinase (PIP4K) results in a reduction in larval salivary gland cell size. Previous studies have shown that this reduction in cell size is not correlated with the levels of phosphatidylinositol 5 phosphate (PI5P), the canonical substrate of PIP4K but to the levels of phosphatidylinositol 3 phosphate (PI3P) a substrate that is used less effectively by the PIP4K enzyme in vitro . The phosphorylation of PI3P by PIP4K generates phosphatidylinositol 3,4 bisphosphate [PI(3,4)P 2 ]. Using a biosensor for PI(3,4)P 2 , surprisingly, we find that depletion of PIP4K leads to an elevation of intracellular PI(3,4)P 2 punctae in salivary gland cells. This elevation in PI(3,4)P 2 punctae was not dependent on the catalytic activity of dPIP4K. Rather, we found that the elevation of PI(3,4)P 2 was dependent on the catalytic activity of Class II phosphatidylinositol 3 kinase (Class II PI3K). Thus, the PIP4K protein regulates an intracellular pool of PI(3,4)P 2 via Class II PI3K activity in Drosophila cells. Introduction The response of cells to changes in the environment is mediated by communication mechanisms that involve the use of chemical messengers which convey information across the plasma membrane. Cell surface receptors that detect extracellular stimuli lead to the generation of chemical signals within cells, that tune cellular biochemistry to meet ongoing needs. One such class of chemical signals generated in cells are phosphoinositides, generated by differential phosphorylation of the hydroxyl group at the 3 rd , 4 th , and 5 th positions on the inositol headgroup. This combinatorial modification produces a family of seven phosphoinositides, namely three mono-phosphorylated regioisomers PI3P, phosphatidylinositol 4 phosphate (PI4P) and PI5P, three bis-phosphorylated regioisomers - PI(4,5)P 2 , PI(3,4)P 2 and PI(3,5)P 2 and one tris-phosphorylated isomer- PI(3,4,5)P 3 ( Posor et al., 2022 ). Each of the species functions as a discrete second messenger in eukaryotic cells. Individual phosphoinositides bind specifically to cellular proteins thereby relaying information during cell signalling. Phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P 2 ] is one of the phosphoinositides whose resting levels in cells is very low [( Ray et al., 2024 );( Dickson & Hille, 2019 )]. The majority of cellular PI(3,4)P 2 is produced downstream of receptor mediated PI3K activation in which phosphatidylinositol (3,4,5)-trisphosphate [PIP₃] serves as the precursor for PI(3,4)P₂[( Hawkins et al., 1992 );( Stephens et al., 1991 )]. PI(3,4)P 2 was for long considered as a minor and inconsequential by-product of PIP 3 metabolism. However, a growing body of evidence has demonstrated that PI(3,4)P 2 itself performs signalling functions. It regulates critical cellular and physiological processes such as clathrin mediated endocytosis (CME)( Posor et al., 2013 ), metabolism ( Dong et al., 2019 ) and tumour metastasis [( Gewinner et al., 2009 );(S. Ghosh et al., 2018 )]. As with all signalling molecules the ability of PI(3,4)P 2 to control cellular physiology depends on its generation in a precise spatial and temporal profile. This itself is controlled by the activity of precisely localized lipid kinases and phosphatases that receive upstream signals and are activated to add or remove phosphate groups ( Balla, 2013 ) from the the inositol headgroup. Thus, the activity of the lipid kinases and phosphatases that control PI(3,4)P 2 is central to its signalling functions in cells. Two pathways primarily responsible for PI(3,4)P 2 production have been reported. The first involves the sequential action of a 3-kinase (Class I PI3K) on phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ] to convert it into PIP 3 and the subsequent action of a 5-phosphatase (e.g., SHIP1/2) to convert PIP 3 to PI(3,4)P 2 [( Damen et al., 1996 );( Pesesse et al., 1997 )].This mechanism predominantly generates PI(3,4)P 2 at the plasma membrane. A recent study has suggested that this pool of PI(3,4)P 2 can be endocytosed via CME to give rise to PI(3,4)P 2 at early endosomes ( Liu et al., 2018 ). An alternate pathway for PI(3,4)P 2 production is regulated by the Class II PI3Ks. The mammalian genome contains three genes encoding these large multidomain enzymes (PI3K-C2α, PI3K-C2β, and PI3K-C2γ) [( Domin et al., 1997 ),( Rozycka et al., 1998 ) ( Misawa et al., 1998 )] whereas less complex metazoans such as Drosophila have only one gene (Pi3K68D) ( MacDougall et al., 2004 ). Class II PI3Ks can synthesize both PI3P by phosphorylation of PI and PI(3,4)P 2 by phosphorylation of PI4P in vivo [( Braccini et al., 2015 );( Velichkova et al., 2010 )]. Recently, much attention has focused on the cellular functions of PI(3,4)P 2 produced from PI4P by class II PI3K [( Marat et al., 2017 );( Posor et al., 2013 )]. On the other hand, PI(3,4)P 2 can be metabolised by selective degradation through inositol polyphosphate 4-phosphatases (INPP4A and INPP4B) [( Norris & Majerus, 1994 );( Norris et al., 1995 );( Gewinner et al., 2009 )] or the 3-phosphatase (PTEN) [( Malek et al., 2017 );( Goulden et al., 2019 )]. The first indication of a PI3P to PI(3,4)P 2 conversion pathway came from studies in Swiss 3T3 fibroblasts, where oxidative stress induced selective PI(3,4)P 2 accumulation alongside a proportional decrease in PI3P, pointing to the involvement of a PI3P-specific 4-kinase ( Van Der Kaay et al., 1999 ). The critical gap that still remained at the time was the identification of the kinase responsible for this activity. Subsequent work implicated phosphatidylinositol 4 phosphate 5 kinase (PIP4K) in this process. PIP4K is a metazoan specific lipid kinase( Krishnan et al., 2025 ) that efficiently phosphorylates PI5P and generates PI(4,5)P 2 . Consistent with this, experiments in multiple species and cells has shown that depletion of PIP4K leads to an increase in PI5P levels [reviewed in ( Krishnan et al., 2026 )]. Zhang et al. ( Zhang et al., 1997 ) showed that PIP4K could convert PI3P into PI(3,4)P 2 , a finding further confirmed by Rameh et al ( Rameh et al., 1997 ). Complementary studies in mammalian cells showed that double knockdown of PIP4K2A/B reduced PI(3,4)P 2 levels ( Emerling et al., 2013 ), whereas overexpression of PIP4K2B increased PI(3,4)P 2 in the context of p110Caax expression( Carricaburu et al., 2003 ), thus reinforcing the idea of a direct conversion of PI3P to PI(3,4)P 2 . A study in Drosophila has also reported that PI3P levels are elevated in flies depleted of PIP4K in a kinase dependent manner (A. Ghosh et al., 2023 ). However, the ability of PIP4K to directly phosphorylate PI3P to generate PI(3,4)P 2 in cells remains to be unequivocally established. In this study, we have developed tools to determine the levels and distribution of PI(3,4)P 2 in Drosophila cells. For this purpose, we have used the C-terminal PH domain of TAPP1 ( Dowler et al., 2000 ) protein repeated thrice in tandem fused to eGFP (cPHx3::eGFP), a high avidity probe for PI(3,4)P 2 detection, as developed by Goulden et al ( Goulden et al., 2019 ). Using this probe, we find that Drosophila cells show both plasma membrane and endomembrane pools of PI(3,4)P 2 . We find that the endomembrane pool of PI(3,4)P 2 is virtually undetectable in wild type cells but accumulates at high levels in cells depleted of dPIP4K. Interestingly this elevation of PI(3,4)P 2 levels could be rescued by a kinase dead version of dPIP4K indicating a non-catalytic mechanism of regulation of PI(3,4)P 2 levels. Further, we find that the elevated levels of PI(3,4)P 2 likely arises from the catalytic activity of a Class II PI3K driven pathway. Thus, our studies define a role for dPIP4K in the control of a Class II PI3K dependent pool of PI(3,4)P 2 in Drosophila cells. Results cPHx3::eGFP reports levels of PI(3,4)P 2 in Drosophila cells We expressed the cPHx3::eGFP probe in Drosophila S2R+ cells [ Fig 1A ] . As, expected, the probe predominantly enriched at the plasma membrane with very few punctate structures [ Fig 1B(a) ] . In contrast, a non-binding version of the cPHx3::eGFP probe (R211LcPHx3::eGFP) [ Fig 1A ] in which a critical residue Arginine (R) is mutated to Leucine (L) in all the three cPH domain, is diffused throughout the cytosol [ Fig 1B(c) ] . This highlights that the probe is specific to PI(3,4)P 2 levels. Download figure Open in new tab Figure 1: cPHx3::eGFP reports levels of PI(3,4)P 2 in Drosophila cells ( A ) Immunoblot analysis was performed on S2R+ cells, probing with anti-GFP antibody to assess the expression of the binding probe cPHx3::eGFP and the non-binding probe R211LcPHx3::eGFP in the following genotypes: Act>cPHx3::eGFP and Act>R211LcPHx3::eGFP. The cPHx3::eGFP and R211LcPHx3::eGFP fusion protein migrates at approximately 80 kDa. Tubulin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( B ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP and R211LcPHx3::eGFP in the S2R+ cells from the following genotypes: (a) U - Act>cPHx3::eGFP Untreated, (b) T - Act>cPHx3::eGFP Starvation treated, (c) U - Act>R211LcPHx3::eGFP Untreated and (d) T - Act>R211LcPHx3::eGFP Starvation treated. The scale bar is indicated at 5 μm for spatial reference. ( C ) Quantification of PI(3,4)P 2 punctae using cPHx3::eGFP and R211LcPHx3::eGFP in the S2R+ cells from the following genotypes: (a) U - Act>cPHx3::eGFP Untreated (n=10), (b) T-Act>cPHx3::eGFP Starvation treated (n=10), (c) U - Act>R211LcPHx3::eGFP Untreated (n=9) and (d) T - Act>R211LcPHx3::eGFP Starvation treated (n=10). Statistical test : Kruskal-Wallis Test (***P value cPHx3::eGFP Untreated, (b) T* - Act>cPHx3::eGFP H 2 O 2 treated, (c) U - Act>R211LcPHx3::eGFP Untreated and (d) T* - Act>R211LcPHx3::eGFP H 2 O 2 treated. The scale bar is indicated at 5 μm for spatial reference. ( E ) Quantification of endomembranous PI(3,4)P 2 levels using cPHx3::eGFP probe and R211LcPHx3::eGFP probe in the S2R+ cells from the following genotypes: (a) Act>cPHx3::eGFP Untreated (n=18), (b) Act>cPHx3::eGFP H 2 O 2 treated (n=18), (c) Act>R211LcPHx3::eGFP Untreated (n=20) and (d) Act>R211LcPHx3::eGFP H 2 O 2 treated (n=13). Statistical test : Kruskal-Wallis Test (****P value0.9999). To explore the responsiveness of the cPHx3::eGFP probe in monitoring the changes in PI(3,4)P 2 levels, we starved S2R+ cells. Starvation in principle should reduce PIP 3 levels ( Schwarzer et al., 2006 ) and thus as a consequence reduce PI(3,4)P 2 on the plasma membrane. As expected, starving cells for 30 mins decreased plasma membrane associated cPHx3::eGFP fluorescence for the binding probe but not for the non-binding probe R211LcPHx3::eGFP [ Fig 1, B and C ] . Hydrogen peroxide (H 2 O 2 ) stimulation is known to increase plasma membrane PI(3,4)P 2 levels ( Dowler et al., 2000 ). On treating cells with H 2 O 2 , we observed a robust increase of cPHx3::eGFP on the plasma membrane along with an increase in discrete cPHx3::eGFP decorated endomembranous compartments [ Fig 1, D and E ] . These punctae were positive for the early endosomal protein Rabenosyn-5 (data not shown) suggesting that the punctae most likely originated from the plasma membrane through endocytosis. The increase in cPHx3::eGFP positive punctae were specific to PI(3,4)P 2 as the accumulation of R211LcPHx3::eGFP did not increase in response to H 2 O 2 treatment [ Fig 1, D and E ] . Together, these findings establish the robustness of the cPHx3::eGFP probe in reporting both plasma membrane as well as the endomembranous pools of PI(3,4)P 2 . To carry out in vivo experiments further, we generated transgenic flies expressing cPHx3::eGFP and R211LcPHx3::eGFP. dPIP4K regulates PI(3,4)P 2 levels in Drosophila salivary gland cells We expressed cPHx3::eGFP in Drosophila larval salivary gland cells and observed no obvious localization of the probe to either the plasma membrane or endomembranous structures [ Fig 2A (a) ] . To test the role of dPIP4K in converting PI3P to PI(3,4)P 2 , we overexpressed dPIP4K in salivary glands but found no increase in PI(3,4)P 2 levels [Supplementary Fig 1, A and B] . On the other hand, in dPIP4K 29 (loss of function allele), we observed that PI(3,4)P 2 levels were strongly upregulated in the salivary glands [ Fig 2, A and B ] . Immunoblotting of salivary gland extracts showed equivalent expression of cPHx3::eGFP between control and dPIP4K 29 confirming that variations in probe expression was not the underlying cause for elevation in the number of PI(3,4)P 2 punctae in dPIP4K 29 [Supplementary Fig 1C] . Our finding of increased PI(3,4)P 2 punctae in dPIP4K 29 was recapitulated on RNA interference (RNAi) mediated dPIP4K downregulation in salivary glands [ Fig 2, C and D ] despite equivalent levels of probe expression in control and RNAi glands [Supplementary Fig 1D] . The punctae were specific to PI(3,4)P 2 as a non-binding version of the probe (R211LcPHx3::eGFP) failed to pick up any PI(3,4)P 2 punctate signal [Supplementary Fig 3, B and C]; expression levels of both the binding and non-binding probes were comparable across genotypes, ruling out probe expression differences as a confounding factor [Supplementary Fig 3A] . Download figure Open in new tab Figure 2 : dPIP4K regulates PI(3,4)P 2 levels in Drosophila salivary gland cells ( A ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP and (b) AB1>cPHx3::eGFP; dPIP4K 29 . The scale bar is indicated at 20 μm for spatial reference. ( B ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP probe in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP (N=12, n = 34) and (b) AB1>cPHx3::eGFP; dPIP4K 29 (N=13, n = 48). Statistical test : Mann Whitney U test (****P value<0.0001). ( C ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP and (b) AB1>cPHx3::eGFP;dPIP4K RNAi . The scale bar is indicated at 20 μm for spatial reference. ( D ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP (N=12, n = 36) and (b) AB1>cPHx3::eGFP; dPIP4K RNAi (N=14, n = 43). Statistical test : Mann Whitney U test (****P value<0.0001). ( E ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP , (b) AB1>cPHx3::eGFP; dPIP4K 29 and (c) AB1>cPHx3::eGFP,dPIP4K Wild Type ;dPIP4K 29 .The scale bar is indicated at 20 μm for spatial reference. ( F ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP (N=9, n = 28), (b) AB1>cPHx3::eGFP; dPIP4K 29 (N=8, n = 33) and (c) AB1>cPHx3::eGFP; dPIP4K Wild Type ,dPIP4K 29 (N=7, n = 29). Statistical test : Kruskal-Wallis Test (****P value 0.9999). ( G ) Representative confocal z-projections illustrating levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP , (b) AB1>cPHx3::eGFP; dPIP4K 29 and (c) AB1>cPHx3::eGFP, dPIP4K D271A ;dPIP4K 29 .The scale bar is indicated at 20 μm for spatial reference. ( H ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP (N=6,n=24), (b) AB1>cPHx3::eGFP; dPIP4K 29 (N=9, n=36) and (c) AB1>cPHx3::eGFP,dPIP4K D271A ;dPIP4K 29 (N=10, n = 36). Statistical test : Kruskal-Wallis Test (****P value 0.9999). To determine if the observed elevation in PI(3,4)P 2 levels in dPIP4K 29 was specifically due to loss of PIP4K, we reconstituted full length dPIP4K (dPIP4K Wild Type ) in dPIP4K 29 and observed a full reversal of the phenotype [ Fig 2, E and F ] . Expression levels of the probes were comparable across genotypes [Supplementary Fig 1E] . Furthermore, to understand if the observed upregulation in PI(3,4)P 2 levels required the kinase activity of the enzyme, we reconstituted a kinase dead (non-ATP binding) dPIP4K (dPIP4K D271A ) in dPIP4K 29 . Remarkably and unexpectedly, PI(3,4)P 2 levels were restored back to those seen in controls [ Fig 2, G and H ] suggesting that regulation of PI(3,4)P 2 by dPIP4K does not depend on its kinase activity; expression levels of the probes were comparable across genotypes [Supplementary Fig 1F] . Endomembranous PI(3,4)P 2 accumulates in dPIP4K 29 independent of Class I PI3K activity dPIP4K 29 mutants show elevated PIP 3 levels on the plasma membrane( Sharma et al., 2019 ). The activity of a 5-phosphatase on PIP 3 can lead to accumulation of PI(3,4)P 2 on the plasma membrane, followed by endocytosis onto endomembrane. This hypothesis would predict that PI(3,4)P 2 on the plasma membrane would be elevated in dPIP4K 29 . However, we did not find evidence for such an elevation of PI(3,4)P 2 at the plasma membrane [Supplementary Fig 2, A and B] , suggesting that in dPIP4K 29 , plasma membrane PI(3,4)P 2 might not be the source for elevated PI(3,4)P 2 at the endomembrane. To validate this further, we stimulated control salivary glands with 10μM insulin, a treatment known to increase PIP 3 in the Drosophila salivary glands ( Sharma et al., 2019 ). This should give rise to PI(3,4)P 2 in a sustained manner but with a temporal delay at the plasma membrane [( Liu et al., 2018 );( Goulden et al., 2019 )] and if this pool were to be the source for endomembranous pool of PI(3,4)P 2 , we should see the punctate population of cPHx3::eGFP, with some temporal delay after stimulating glands with insulin ( Liu et al., 2018 ). As expected, a time course experiment using insulin stimulation of wild type glands led to an increase in PI(3,4)P 2 at the plasma membrane but there was no corresponding increase of endomembranous levels of PI(3,4)P 2 [ Fig 3A ] . As an alternative approach to test the involvement of PIP 3 as a precursor for the increased endomembranous PI(3,4)P 2 in dPIP4K 29 , we treated salivary glands with 100nM wortmannin ( Wymann et al., 1996 ), a concentration at which this compound inhibits Class I PI3K thus reducing PIP 3 levels at the plasma membrane. If PIP 3 was indeed the source for the elevated PI(3,4)P 2 in dPIP4K 29 , then pre-treatment with wortmannin is predicted to lead to a reduction in the levels of PI(3,4)P 2. However, pre-treatment of dPIP4K 29 glands with 100nM of wortmannin did not result in a reduction in PI(3,4)P 2 punctae [ Fig 3, B and C ] . These findings suggest that the accumulation of PI(3,4)P 2 in dPIP4K 29 are likely independent of Class I PI3K activity. Download figure Open in new tab Figure 3 : Endomembranous PI(3,4)P 2 accumulates in dPIP4K 29 independent of Class I PI3K activity (A) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the genotype AB1>cPHx3::eGFP, insulin stimulated (10 uM) for the following time points - (a) 0 (Unstimulated), (b) 5 mins (c) 10 mins (d) 15 mins and (e) 30 mins. The scale bar is indicated at 20 μm for spatial reference. (B) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP; dPIP4K 29 (DMSO) and (b) AB1>cPHx3::eGFP;dPIP4K 29 (Wortmannin - 100 nM). The scale bar is indicated at 20 μm for spatial reference. (C) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP; dPIP4K 29 (DMSO) (N=8, n =36) and (b) AB1>cPHx3::eGFP;dPIP4K 29 (Wortmannin -100 nM) (N=9, n =31). Statistical test : Mann Whitney U test (ns – P value = 0.8563). (D) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP; dPIP4K 29 (DMSO) and (b) AB1>cPHx3::eGFP;dPIP4K 29 (Wortmannin - 400 nM). The scale bar is indicated at 20 μm for spatial reference. (E) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP; dPIP4K 29 (DMSO) (N=11, n =57) and (b) AB1>cPHx3::eGFP;dPIP4K 29 (Wortmannin - 400 nM) (N=16, n =70). Statistical test : Mann Whitney U test (***P valuecPHx3::eGFP; dPIP4K 29 (DMSO) and (b) AB1>cPHx3::eGFP;dPIP4K 29 (Wortmannin- 1000 nM). The scale bar is indicated at 20 μm for spatial reference. (G) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: : (a) AB1>cPHx3::eGFP; dPIP4K 29 (DMSO) (N=9, n =33) and (b) AB1>cPHx3::eGFP;dPIP4K 29 (Wortmannin - 1000 nM) (N=10, n =32). Statistical test : Mann Whitney U test (****P value<0.0001). Class II PI3K dependent endomembranous PI(3,4)P 2 accumulates in dPIP4K 29 During our studies, we noted that pre-treating salivary glands with 400nM [ Fig 3, D and E ] and 1000nM [ Fig 3, F and G ] of wortmannin led to a noticeable and dose dependent reduction [Supplementary Fig 2C] in PI(3,4)P 2 punctae. One possible explanation for this differential response (compared to 100nM wortmannin) could be wortmannin’s inhibition profile; at 100 nM, Class I PI3K is completely inhibited and class II is only partially inhibited, whereas at 400 nM and 1000nM, Class I PI3K remains 100% inhibited while Class II PI3K is also inhibited to 50% and 100% respectively ( Domin et al., 1997 ). The Drosophila genome has a single gene encoding Class II PI3K (Pi3K68D)( MacDougall et al., 2004 ). To determine if the PI(3,4)P 2 punctae in dPIP4K 29 are dependent on Class II PI3K activity, we downregulated Pi3K68D using RNAi [ Fig 4A ] in dPIP4K 29 salivary glands. Under these conditions ( Pi3K68D RNAi in dPIP4K 29 ), we observed that the elevated PI(3,4)P 2 punctae in dPIP4K 29 were significantly rescued [ Fig 4, B and C ] ; the PI(3,4)P 2 probe was expressed at equal levels in all the genotypes [Supplementary Fig 2D] . These findings predict that the elevation of Class II PI3K function in salivary gland cells should increase PI(3,4)P 2 punctae; i.e., overexpression of Pi3K68D should phenocopy elevated PI(3,4)P 2 levels in dPIP4K 29 . Indeed, we observed elevated levels of endomembranous pool of PI(3,4)P 2 in glands overexpressing Pi3K68D [ Fig 4, D and E ] although expression levels of the probes were comparable across genotypes [Supplementary Fig 2E] . The punctae were specific to PI(3,4)P 2 as a non-binding version of the probe (R211LcPHx3::eGFP) failed to pick up any PI(3,4)P 2 punctate signal [Supplementary Fig 3, E and F] . Expression levels of both the binding and non-binding probes were comparable across genotypes, ruling out probe expression differences as a confounding factor [Supplementary Fig 3D] . Download figure Open in new tab Figure 4 : Class II PI3K dependent endomembranous PI(3,4)P 2 accumulates in dPIP4K 29 ( A ) qPCR measurements of Pi3K68D mRNA levels from genotypes : Control - Act> and Act> Pi3K68D RNAi . Pi3K68D expression levels were calculated as 2 −ΔCt normalized to the reference gene (ribosomal protein 49). This value was in turn normalised to the mean of the control genotype. X-axis represents the genotype and Y-axis represents 2 −ΔCt normalised to the average of the control. Statistical test : Mann Whitney U test (**P value = 0.0043). ( B ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP , (b) AB1>cPHx3::eGFP;dPIP4K 29 and (c) AB1> cPHx3::eGFP,Pi3K68D RNAi ;dPIP4K 29 . The scale bar is indicated at 20 μm for spatial reference. ( C ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1> cPHx3::eGFP (N=11, n =28), (b) AB1>cPHx3::eGFP;dPIP4K 29 (N=11, n =31) and (c) AB1> cPHx3::eGFP,Pi3K68D RNAi ;dPIP4K 29 (N=14, n =34). Statistical test : Kruskal-Wallis Test (****P value < 0.0001 and *P value = 0.0210). ( D ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) and (c) Control - AB1>cPHx3::eGFP,mCherry , (b) and (d) AB1>cPHx3::eGFP,mcherry::Pi3K68D . The scale bar is indicated at 20 μm for spatial reference. ( E ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) and (c) Control- AB1>cPHx3::eGFP,mCherry (N=5,n=14), (b) and (d) AB1>cPHx3::eGFP, mcherry::Pi3K68D (N=6, n =16). Statistical test : Mann Whitney U test (****P value<0.0001). Discussion The generation of plasma membrane PI(3,4)P₂ through the dephosphorylation of PIP 3 generated by Class I PI3K activity is well established as is its role in recruiting effector proteins that drive actin remodelling ( Montaño-Rendón et al., 2022 ) and cell migration ( Feng & Yu, 2021 ). By contrast far less is understood about the source of endomembrane PI(3,4)P₂ pools owing to its low concentration, rapid turnover and challenges in detection. A previous study had described the localization of a TAPP1 based probe to endocytic structures ( He et al., 2017 ). However, Goulden et al., suggested that this localisation of the probe to endocytic structures was due to the presence of a clathrin-binding motif in the C terminus end of the TAPP1 probe( Goulden et al., 2019 ). In this study, we report the development and use in vivo of a high avidity probe cPHx3::eGFP (originally described by Goulden et.al) in Drosophila ; this probe, based on the C-terminal PH domain of TAPP1 does not have a clathrin binding motif. However, we unequivocally find the presence of an endomembranous pool of PI(3,4)P 2 using this probe. This finding strongly suggests that in addition to plasma membrane pool, Drosophila cells also contain an endomembranous pool of PI(3,4)P 2 . This endomembranous pool of PI(3,4)P 2 is not readily detected in cells under resting conditions. However, in this study we noted that stimulation of cultured Drosophila S2R+ cells with H 2 O 2 resulted in an increase in the endomembranous pool of PI(3,4)P 2 . These findings suggest that the endomembrane pool of PI(3,4)P 2 may serve as a signalling lipid during response to extracellular stimuli. What might be the signalling function of an endomembrane pool of PI(3,4)P 2 Recent studies strongly suggest that the spatial and temporal dynamics of PI(3,4)P₂ signaling are critical determinants of its function. At the plasma membrane, PI(3,4)P₂ promotes cell growth and survival through the recruitment of PH-domain–containing effectors such as TAPP1 and TAPP2, consistent with studies showing enhanced AKT activation in TAPP mutant mice ( Landego et al., 2012 ). In addition, Liu at al., have demonstrated a new independent signaling function of PI(3,4)P₂; i.e., isoform- and site-specific membrane recruitment and activation of Akt2. They propose that PIP 3 is a transient signal confined at the plasma membrane, whereas PI(3,4)P₂ is a more sustained signal that operates at both the plasma membrane and early endosomes ( Liu et al., 2018 ). What are the biochemical regulators of endomembranous pool of PI(3,4)P 2 ? Our study identified dPIP4K as a novel regulator of PI(3,4)P₂ in Drosophila ; loss of dPIP4K results in the accumulation of PI(3,4)P₂ on endomembranes. Although PIP4K has been reported to phosphorylate PI3P and generate PI(3,4)P 2 , this enzymatic reaction cannot explain the elevation of PI(3,4)P 2 levels seen in cells depleted of the enzyme. Our finding that the elevated PI(3,4)P 2 levels in dPIP4K depleted cells could be rescued by reconstitution with a kinase dead dPIP4K transgene implies that the mechanism through which the enzyme controls PI(3,4)P 2 levels is non-enzymatic. Multiple studies have shown that depletion of PIP4K leads to an elevation of PIP 3 [(D. G. Wang et al., 2019 );( Sharma et al., 2019 )] raising the possibility that the elevated PI(3,4)P 2 may rise by dephosphorylation of PIP 3 . Our findings in this study indicate that this is unlikely to be the case (i) elevation of PIP 3 levels at the plasma membrane by insulin stimulation did not lead to a concomitant elevation of PI(3,4)P 2 at the endomembrane. This observation is consistent with Goulden et al., ( Goulden et al., 2019 ) who failed to detect endosomal PI(3,4)P 2 localization in serum-stimulated 293A cells or after insulin stimulation in HeLa cells. (ii) Inhibition of Class I PI3K by low nanomolar concentrations of wortmannin failed to decrease the elevated PI(3,4)P 2 levels in PIP4K depleted cells. In this study, we found evidence of a key role for Class II PI3K in regulating endomembrane PI(3,4)P 2 in salivary gland cells. (i) Overexpression of Class II PI3K increased PI(3,4)P 2 puncta Treatment with pharmacologically relevant concentrations of wortmannin that can inhibit Class II PI3K could reverse PI(3,4)P 2 puncta accumulated in PIP4K depleted cells (iii) RNAi depletion of Class II PI3K in PIP4K depleted cells could reverse PI(3,4)P 2 levels. Together, these findings suggest that increased Class II PI3K activity in dPIP4K depleted cells leads to elevated PI(3,4)P 2 levels. How PIP4K modulates Class II PI3K mediated PI(3,4)P 2 levels is an intriguing open question. Several possibilities exist: (i) PIP4K may regulate PI(4,5)P₂ and indirectly tune PI4P availability for Class II PI3K activity [ Fig 5 ] ; (ii) loss of PIP4K is known to elevate PI3P (A. Ghosh et al., 2023 ), providing an alternative precursor for PI(3,4)P₂ synthesis. If so, the enzyme that performs this 4 phosphorylation in vivo remains to be identified; (iii) PIP4K may function as an inhibitor of Class II PI3Ks, either directly or indirectly restraining its catalytic activity [ Fig 5 ] . Discriminating between these possibilities will be important for understanding how PIP4K shapes Class II PI3K activity and thus PI(3,4)P₂ pools at endomembranes. While the mechanism of action and the regulators of Class I PI3K has been extensively studied, the regulators of Class II PI3K has remained elusive. A recent study proposed an autoregulatory mechanism for PI3K-C2α involving coincidence detection at endocytic sites (H. Wang et al., 2018 ). In the cytosol, PI3K-C2α is maintained in an inactive state by folding of the PX-C2 module back onto its kinase domain. Upon membrane recruitment, the N-terminal region of PI3K-C2α interacts with assembled clathrin [( Gaidarov et al., 2001 );( Posor et al., 2013 );( Domin et al., 2000 )] and the C-terminal PX-C2 domain now binds to PI(4,5)P 2 . This conformational change relieves autoinhibition and allows the substrate to access the catalytic site, enabling localized PI(3,4)P 2 synthesis (H. Wang et al., 2018 ). In our study, we identified another potential regulator of Class II PI3K activity that is extrinsic to the enzyme, suggesting an additional layer of control. Recent analysis have shown that PIP4K is a metazoan specific enzyme ( Krishnan et al., 2025 ). Likewise, Class II PI3K is also a metazoan specific lipid kinase [as reviewed in ( Ray et al., 2024 )] leading to the possibility of an evolutionary link between the two to regulate phosphoinositide signalling in metazoans. Download figure Open in new tab Figure 5 : Schematic depicting the non-catalytic regulation of PI(3,4)P₂ levels by dPIP4K on an endomembrane. ( A ) In an otherwise wild-type background, dPIP4K likely limits the substrate availability or inhibits (denoted by -) the activity of Class II PI3K/Pi3K68D thereby keeping the PI(3,4)P 2 levels in check. In dPIP4K 29 , this inhibition is relieved, leading to increased flux through Class II PI3K mediated pathway, thus giving rise to elevated PI(3,4)P 2 levels on the endomembrane. Looking forward, our findings open new directions for studying PI(3,4)P₂ signaling at the endomembrane. Future work will be needed to determine whether PIP4K similarly regulates PI(3,4)P₂ in other cell types and physiological contexts. It will be crucial to explore the downstream consequences of heightened PI(3,4)P₂ at endo-membranes in terms of the effector proteins it binds to as well as signalling pathways it maps onto. Given the central role of PI(3,4)P₂ in both growth-promoting and growth-restraining pathways, uncovering how PIP4K maintains its balance is likely to have broad implications for cell signaling, metabolism, and disease. Materials and Methods Fly husbandry Drosophila melanogaster was grown in a constant temperature laboratory incubator (25°C) on a standard fly media, the composition of which is mentioned in a table below. View this table: View inline View popup Download powerpoint Experimental fly crosses were set at 25°C in vials under non-crowding conditions with 50% relative humidity under no internal illumination in the incubator. The Drosophila strains used in the experiment is listed below – w 1118 (Wild type strain), dPIP4K 29 (homozygous null mutant of dPIP4K made by Raghu lab), UAS-cPHx3::eGFP/Tm6Tb (Lab generated), UAS-R211LcPHx3::eGFP/Tm6Tb (Lab generated), Act5C-Gal4/CyOGFP (BL 3953) , AB1Gal4 (BDSC 1824), UAS-dPlP4K RNAi (BDSC 65891), UAS-dPIP4K WildType (Lab generated), UAS-dPIP4K D271A (Lab generated),UAS-mCherry, UAS-Pi3K68D RNAi (kind gift from Amy Kiger, UCSD) and UAS-mCherry::Pi3K68D 2.1 /CyO (kind gift from Amy Kiger, UCSD). All experimental procedures, including animal care and use, were conducted under the approval of the Institutional Biosafety Committee (IBSC). Experimental designs incorporated Drosophila of both sexes to minimize potential sex-based variation and eliminate sex bias. The Gal4–UAS system ( Brand & Perrimon, 1993 ) was employed to achieve spatially and temporally controlled expression of the transgene. DNA constructs and transgenic fly generation The cPHx3::eGFP sequence was obtained from Goulden et al.,2019 ( Goulden et al., 2019 ). The sequence for R211LcPHx3::eGFP was obtained by changing the codon encoding arginine (R) to leucine (L) in all the three tandem repeats. Both the sequences were then provided to GenScript (RRID: SCR_002891) for custom gene/DNA synthesis followed by subsequent incorporation into a Drosophila expression vector pUAST-attB (RRID: DGRC_1419). Transgenic fly lines were generated using phiC31 integrase-mediated transgenesis in either attP40 (BDSC 25709) or attP2 (BDSC 25710) flies. S2R+ cells: culturing and transfection Drosophila S2R+ cells (RRID:CVCL_Z831) stably expressing Actin-Gal4 were maintained in Schneider’s insect medium (SIM) (HiMedia, Cat# IML003A) supplemented with 10% of a non-heat inactivated fetal bovine serum (FBS, Gibco, Cat# 16000044) and in the presence of glutamine and antibiotics such as penicillin and streptomycin (1:100). Effectene (Qiagen, Cat# 301425) was used to transfect 0.5m cells (for both imaging and western) according to the manufacturer’s instructions. After 36 hours of transfection, cells were fixed with 2.5% paraformaldehyde (PFA) (Electron Microscopy Sciences, Cat# 15710) and imaged to observe for GFP fluorescence using a 60X 1.4 NA objective in Olympus Confocal Laser Scanning Microscope Fluoview FV3000 (RRID:SCR_017015). Starvation treatment and H 2 O 2 treatment in S2R+ cells Following transfection with the cPHx3::eGFP and R211LcPHx3::eGFP probe, cells were dislodged and plated onto confocal dishes and allowed to settle for 2 hours at 25°C in 200 ul of fresh SCM. Post the attachment of cells to the dish surface, the existing media was taken out and the desired treatment was given. For starvation - Control cells were kept in SCM for 30 mins and treated cells were kept in PBS for 30 mins. For H 2 0 2 - Control cells were kept in SCM for 30 mins and treated cells were kept in H 2 0 2 (10 mM) in SCM for 30 mins. Post the desired treatment, cells were immediately fixed with 2.5% paraformaldehyde (PFA) (Electron Microscopy Sciences, Cat# 15710) and imaged to observe for GFP fluorescence using a 60X 1.4 NA objective in Olympus Confocal Laser Scanning Microscope Fluoview FV3000 (RRID:SCR_017015). qPCR Total RNA was isolated from 5 Drosophila 3 rd instar wandering larvae using the TRIzol reagent (Life Technologies, Cat# 15596018). 1000ng of the extracted RNA was then treated with DNase I (Amplification grade; Thermo Fisher Scientific, Cat# 18068015) to eliminate any genomic DNA contamination. First-strand cDNA synthesis was carried out using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Cat# 18064014) along with random hexamer primers (Thermo Fisher Scientific, Cat# N8080127). Primers targeting exon–exon junctions were designed based on standard qPCR design guidelines. Quantitative PCR (qPCR) was performed using cDNA samples and primers using Power SYBR Green PCR Master Mix (Applied Biosystems, Cat# 4367659) on an Applied Biosystems 7500 Fast Real-Time PCR System (RRID:SCR_018051). Expression levels were normalized to the ribosomal protein 49 (RP49) a housekeeping gene, thus serving as the internal reference control. Additionally, a no reverse transcription control and a non-template control was also included for each genotype to rule out amplification coming from genomic DNA or non-specific contaminants. View this table: View inline View popup Download powerpoint Table 1: List of primers used for qPCR measurement of Pi3K68D. Protein sample preparation Salivary glands - 6 stage-matched Drosophila larvae were dissected for salivary glands and the samples were sonicated in larval lysis buffer (3 ul/gland) with freshly added PIC (Protease Inhibitor Cocktail) (Roche, Cat# 4693116001) and PS (PhosStop) (Roche, Cat# 4906845001), the detailed composition of lysis buffer is mentioned in Swarna et al., 2019 ( Mathre et al., 2019 ). Following lysis, the samples were boiled at 95°C for 10 min. S2R+ cells - Lysates of S2R+ cells post transfection expressing the desired construct were prepared by pelleting the cells at 1000 rpm/4°C/15 mins. The pellet was subsequently washed using ice cold 1X PBS (2 times). Following this, the cell pellet was lysed in the lamelli buffer, heated at 95°C for 10 min. Western Blotting Protein extracts were separated using an SDS-PAGE and electro blotted onto a nitrocellulose filter membrane [Hybond-C Extra; (GE Healthcare)] using a wet transfer apparatus (Bio-Rad).The membrane was blocked using 10% Blotto (Santa Cruz Biotechnology, Cat# sc-2325) in phosphate buffer saline (PBS) with 0.1%Tween 20 (Sigma-Aldrich, Cat# P1379) (0.1% PBST) for an hour at room temperature (RT) on an orbital shaker. Primary antibody incubation was done overnight at 4°C using appropriate antibody dilutions as listed in the table 2 . Following this, the membrane was washed in 0.1% PBST (3 times) at RT and incubated with 1:10,000 dilutions of appropriate secondary antibody (Jackson Immuno Research Laboratories) coupled to horseradish peroxidase (HRP) (Donkey anti rabbit, Cat # 711-035-152, RRID: AB_10015282),(Goat anti mouse, Cat# 115-035-003, RRID: AB_10015289) at RT for 2 h on an orbital shaker. The membranes were washed three times with PBST, developed using Clarity western ECL substrate (Bio-Rad Cat# 1705061), and imaged with the LAS 4000 ImageQuant system (GE Healthcare; RRID:SCR_014246). View this table: View inline View popup Download powerpoint Table 2: List of primary antibodies used for western blotting. Wortmannin and Insulin Treatment in salivary glands For Insulin time course Wandering third instar larvae were dissected one at a time and glands were immediately dropped in one well of a 4-well plate containing either only PBS or PBS+10uM insulin (Bovine Insulin, Sigma, Cat# I6634) and incubated for multiple time points such as 5, 10, 15 and 30 minutes at 25°C following which the glands were transferred to a well containing 4% PFA in PBS to fix for 15 minutes at room temperature and then washed with 1X PBS (2 times). Finally, the glands were mounted in 70% glycerol in PBS and imaged to observe for GFP fluorescence using a 60X 1.4 NA objective in Olympus Confocal Laser Scanning Microscope Fluoview FV3000 (RRID:SCR_017015). For Wortmannin treatment Wandering third instar larvae were dissected and glands were immediately dropped in one well of a 4-well plate containing either DMSO in Schneider’s insect media or Wortmannin (Sigma-Aldrich, Cat# W1628) at desired concentrations (100nM, 400nM or 1000nM) in Schneider’s insect media and incubated for 10 minutes at room temperature following which the glands were transferred to a well containing 4% PFA in PBS and fixed for 15 minutes at room temperature and then transferred to wells containing only 1X PBS for washes (2 times). Finally, the glands were mounted in 70% glycerol in PBS and imaged to observe for GFP fluorescence using a 60X 1.4 NA objective in Olympus Confocal Laser Scanning Microscope Fluoview FV3000 (RRID:SCR_017015). Analysis Punctae quantification The 3D images were stacked to give one 2D image using ZProject in ImageJ. The 2D images were then analysed for the number of punctae (counted manually). The number of punctae were normalised to the area of the cell. The value thus obtained was multiplied by 1000 and plotted for the respective genotypes. Plasma membrane to Cytoplasm mean fluorescence intensity (MFI) Quantification Confocal 3D slices were manually curated to generate maximum z-projections of middle few planes (2-3 slices) of cells. Thereafter, line profiles were drawn across clearly identifiable plasma membrane regions and their adjacent cytosolic regions and ratios of mean intensities for these line profiles were calculated for each cell. For salivary glands, about 5-6 cells from multiple glands were analysed. Sampling and Statistical Analysis Each experiment was performed at least twice with multiple biological replicates. All statistical analyses were conducted using GraphPad Prism (RRID:SCR_002798). Data normality or log-normality was assessed using the Shapiro–Wilk test ( p > 0.05). For comparisons between two groups, the Mann–Whitney U test was applied if the data did not follow a normal distribution, whereas an unpaired t -test with Welch’s correction was used for normally distributed data. When experiments involved more than two biological groups and the data were non-normal, the Kruskal–Wallis test was employed. Figure Legends Supplementary Figure 1 ( A ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP (Control) and (b) AB1>cPHx3::eGFP, dPIP4K OE .The scale bar is indicated at 20 μm for spatial reference. ( B ) Quantification of PI(3,4)P 2 levels using cPHx3::eGFP punctae and in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP (N=3, n =9) and (b) AB1>cPHx3::eGFP,dPIP4K OE (N=3, n =12).). Statistical test : Mann Whitney U test (ns - P value = 0.85). ( C ) Immunoblot analysis was performed on salivary glands from wandering third instar larvae, probing with anti-GFP antibody to assess the expression of cPHx3::eGFP and dPIP4K to confirm the mutant background in the following genotypes: (a) Control - AB1>cPHx3::eGFP and (b) AB1>cPHx3::eGFP; dPIP4K 29 . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. dPIP4K protein migrates at approximately 54kDa. Actin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( D ) Immunoblot analysis was performed on salivary glands from wandering third instar larvae, probing with anti-GFP antibody to assess the expression of cPHx3::eGFP and dPIP4K to confirm the mutant background in the following genotypes: (a) Control - AB1>cPHx3::eGFP and (b) AB1>cPHx3::eGFP; dPIP4K RNAi . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. dPIP4K protein migrates at approximately 54kDa. Actin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( E ) Immunoblot analysis was performed on salivary glands from wandering third instar larvae, probing with anti-GFP antibody to assess the expression of cPHx3::eGFP and dPIP4K to confirm the mutant background in the following genotypes: (a) Control - AB1>cPHx3::eGFP , (b) AB1>cPHx3::eGFP; dPIP4K 29 and (c) AB1>cPHx3::eGFP;dPIP4K Wild Type ,dPIP4K 29 . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. dPIP4K protein migrates at approximately 54kDa. Actin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( F ) Immunoblot analysis was performed on salivary glands from wandering third instar larvae, probing with anti-GFP antibody to assess the expression of cPHx3::eGFP and dPIP4K to confirm the mutant and rescue background in the following genotypes: (a) Control - AB1>cPHx3::eGFP , (b) AB1>cPHx3::eGFP; dPIP4K 29 and (c) AB1>cPHx3::eGFP; dPIP4K D271A ;dPIP4K 29 . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. dPIP4K protein migrates at approximately 54kDa. Actin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). Supplementary Figure 2 ( A ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using cPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP and (b) AB1>cPHx3::eGFP; dPIP4K 29 . The scale bar is indicated at 20 μm for spatial reference. ( B ) Quantification of PI(3,4)P 2 mean fluorescence intensity at the plasma membrane using cPHx3::eGFP probe in the salivary glands of wandering third instar larvae from the following genotypes: (a) Control - AB1>cPHx3::eGFP (N=8, n = 31) and (b) AB1>cPHx3::eGFP; dPIP4K 29 (N=7, n = 41). Statistical Test : Unpaired t test with Welch correction (ns - P value = 0.0898). ( C ) Quantification of PI(3,4)P₂ puncta in the genotype AB1>cPHx3::eGFP;dPIP4K 29 across increasing concentrations of wortmannin. The y -axis indicates the number of PI(3,4)P₂ puncta per unit area normalized to the control condition (0 nM wortmannin), while the x -axis represents the corresponding wortmannin concentration in nanomolar (nM). ( D ) Immunoblot analysis was performed on salivary glands from 6 wandering third instar larvae, probing with anti-GFP antibody to assess the expression of cPHx3::eGFP in the following genotypes: (a) Control - AB1> cPHx3::eGFP , (b) AB1> cPHx3::eGFP;dPIP4K 29 and (c) AB1> cPHx3::eGFP,Pi3K68D RNAi ;dPIP4K 29 . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. Actin was used as the loading control to ensure equal protein loading across the samples. Additionally, dPIP4K protein levels were assessed in the samples to verify dPIP4K 29 mutant background. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( E ) Immunoblot analysis was performed on salivary glands from 6 wandering third instar larvae, probing with anti-GFP antibody to assess the expression of cPHx3::eGFP in the following genotypes: (a) Control - AB1>cPHx3::eGFP,mCherry and (b) AB1>cPHx3::eGFP; mcherry::Pi3K68D . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. Actin was used as the loading control to ensure equal protein loading across the samples. Additionally, dPIP4K protein levels were assessed in the samples to verify the presence of the dPIP4K 29 mutant background. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). Supplementary Figure 3 ( A ) Immunoblot analysis was performed on salivary glands from wandering third instar larvae, probing with anti-GFP antibody to assess the expression of the binding probe - cPHx3::eGFP and the non-binding probe – R211LcPHx3::eGFP in the following genotypes: (a) AB1>cPHx3::eGFP;dPIP4K RNAi and (b) AB1>R211LcPHx3::eGFP;dPIP4K RNAi . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. Actin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( B ) Representative confocal z-projections illustrating PI(3,4)P 2 levels using the binding probe - cPHx3::eGFP and the non-binding probe – R211LcPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP; dPIP4K RNAi and (b) AB1>R211LcPHx3::eGFP; dPIP4K RNAi .The scale bar is indicated at 20 μm for spatial reference. ( C ) Quantification of PI(3,4)P 2 levels using the binding probe - cPHx3::eGFP and the non-binding probe – R211LcPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) AB1>cPHx3::eGFP; dPIP4K RNAi (N=6, n =27)and (b) AB1>R211LcPHx3::eGFP; dPIP4K RNAi (N=7, n =24). Statistical test : Mann Whitney U test (****P value<0.0001). ( D ) Immunoblot analysis was performed on salivary glands from wandering third instar larvae, probing with anti-GFP antibody to assess the expression of the binding probe - cPHx3::eGFP and the non-binding probe – R211LcPHx3::eGFP in the following genotypes: (a) AB1>cPHx3::eGFP;mcherry::Pi3K68D and (b) AB1>R211LcPHx3::eGFP; mcherry::Pi3K68D . The cPHx3::eGFP fusion protein migrates at approximately 80 kDa. Actin was used as the loading control to ensure equal protein loading across the samples. The molecular weight (M r ) of the ladder is indicated on the left; molecular weights are shown in kilodaltons (kDa). ( E ) Representative confocal z-Projections illustrating PI(3,4)P 2 levels using the binding probe - cPHx3::eGFP and the non-binding probe – R211LcPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) and (c) AB1>cPHx3::eGFP; mcherry::Pi3K68D , (b) and (d) AB1>R211LcPHx3::eGFP; mcherry::Pi3K68D . The scale bar is indicated at 20 μm for spatial reference. ( F ) Quantification of PI(3,4)P 2 levels using the binding probe - cPHx3::eGFP and the non-binding probe – R211LcPHx3::eGFP in the salivary glands of wandering third instar larvae from the following genotypes: (a) and (c) AB1>cPHx3::eGFP; mcherry::Pi3K68D (N=8, n =24), (b) and (d) AB1>R211LcPHx3::eGFP; mcherry::Pi3K68D (N=7, n =19). Statistical test : Mann Whitney U test (****P value<0.0001). Acknowledgements This work was supported by the Department of Atomic Energy, Government of India, under Project Identification No. RTI 4006. We thank the Imaging, DNA sequencing and Drosophila facility at NCBS for support. Funder Information Declared Department of Atomic Energy , RTI-0046 Footnotes ↵ @ Department of Biotechnology, Manipal Institute of Technology (MIT), Manipal Academy of Higher Education (MAHE), Manipal, 576104, Karnataka, India References ↵ Balla , T . ( 2013 ). 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