ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism

ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism | 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 ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism View ORCID Profile Andrew K.J. Boyce , Haifei You , Leigh E. Wicki-Stordeur , Leigh Anne Swayne doi: https://doi.org/10.1101/2025.09.11.675618 Andrew K.J. Boyce 1 Division of Medical Sciences; University of Victoria ; Victoria, British Columbia V8P 5C2, Canada 2 Department of Neurosciences, University of New Mexico School of Medicine , Albuquerque, USA , 87131 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrew K.J. Boyce For correspondence: anboyce{at}salud.unm.edu lswayne{at}uvic.ca Haifei You 1 Division of Medical Sciences; University of Victoria ; Victoria, British Columbia V8P 5C2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Leigh E. Wicki-Stordeur 1 Division of Medical Sciences; University of Victoria ; Victoria, British Columbia V8P 5C2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Leigh Anne Swayne 1 Division of Medical Sciences; University of Victoria ; Victoria, British Columbia V8P 5C2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: anboyce{at}salud.unm.edu lswayne{at}uvic.ca Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Macropinocytosis is an endocytic process that allows cells to respond to changes in their environment by internalizing nutrients and cell surface proteins, as well as modulating cell size. Here, we identify that adenosine triphosphate (ATP) triggers macropinocytosis in murine Neuro2a neuroblastoma cells, driving an increase in cell size, and internalizing the ATP release channel pannexin 1 (PANX1) to macropinosomes. Amiloride treatment and mutation of an extracellular tryptophan (W74) in PANX1 abolished ATP-evoked cell area enlargement, suggesting that PANX1 may itself regulate this form of macropinocytosis. Transient expression of the GTP-hydrolysis resistant ADP-ribosylation factor 6 GTPase (ARF6 Q67L) led to increased cell size, PANX1 internalization and localization to endosomal compartments, consistent with macropinocytosis. Inhibiting macropinocytosis-associated GTPases, phosphoinositide-3 kinase (PI3K), and disrupting actin polymerization abolished ATP-induced PANX1 internalization supporting a macropinocytic mechanism. Further, these inhibitors disrupted co-distribution of intracellular PANX1 with macropinosomal cargo. Several lipid-PANX1 interactions were identified with relevance to macropinocytic mechanisms. The role of PANX1 in ATP-mediated macropinocytosis could be particularly important for disease states implicating PANX1, such as cancer, where ATP can act as a purinergic regulator of cell growth/metastasis and as a supplementary energy source following internalization. INTRODUCTION Amongst the many ways that cells adapt to environmental changes, several key modifications include cell surface receptor density, nutrient acquisition, and cellular structure. Macropinocytosis, or “cell drinking,” is a non-canonical membrane internalization process that enables these tightly associated dynamic adaptations 1 . In some cell types, macropinocytosis enables recycling of the equivalent of the full cell surface area once every thirty minutes 2 . A more thorough appreciation of the regulation of macropinocytosis is important for understanding antigen presentation in phagocytic immune cells 3 and receptor-mediated signalling localized to endosomal compartments 4 , 5 , as well as many types of viral and pathogenic bacterial entry 6 – 9 and nutrient acquisition in tumorigenic cells (for reviews see Finicle et al. 10 and Bloomfield and Kay 11 ). In the context of cancer, macropinocytic mechanisms are hijacked by tumor cells for internalization of adenosine triphosphate (ATP) to promote cell growth and survival 12 , 13 . Here, extracellular ATP provides energy status information in the tumour microenvironment 14 by way of purinergic signalling through ATP-sensitive purinergic receptors and ATP release mechanisms, including the large pore channel pannexin 1 (PANX1) 15 . In our previous studies, we identified that PANX1 is internalized in response to elevated extracellular ATP via an undetermined non-canonical endocytic mechanism in murine neuroblastoma cells 16 , 17 . PANX1 is widely understood to promote tumor cell survival in various cancers 15 , including the proliferation and differentiation of murine neuroblastoma 18 , 19 and malignancy of human neuroblastoma 20 . In this report, we investigate the hypothesis that ATP-mediated PANX1 internalization occurs through macropinocytosis and drives a PANX1-dependent increase in cell size. Macropinocytosis is distinct from classical types of membrane internalization or endocytosis in that it does not rely on coat and scission proteins used in clathrin- and caveolin-mediated endocytosis. Instead, during macropinocytosis, discrete regions of cholesterol and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 )-rich plasma membrane and its associated receptors are engulfed via actin-dense membrane ruffles 21 . Initially, ADP-ribosylation factor 6 (ARF6), a small GTPase resident to the membrane ruffles, activates phosphatidyl inositol-4-phosphate 5-kinase 22 to generate PI(4,5)P 2 23 , 24 . Generation of PI(4,5)P 2 enables recruitment of the small GTPases Ras-related C3 botulinum toxin substrate 1 and cell division control protein 41 (more commonly known as CDC42) to the ruffling membrane 25 , 26 , which then activate p21-activated kinase 27 . Next, p21-activated kinase phosphorylates a protein called brefeldin A-dependent ADP ribosylation substrate to initiate membrane curvature 28 , which is enabled by conversion of lysophosphatidic acid to phosphatidic acid (for review, see Bohdanowicz and Grinstein 29 ), leading to the formation of large vesicles ranging in diameter from 0.2 to 5 μm, referred to as macropinosomes. Given the lack of coat protein, structural specificity, or unique membrane-bound molecules, macropinosomes are difficult to distinguish from other endocytic compartments of similar size, and consequently, are commonly identified using fluorescently labelled fluid-phase endocytosis markers ( i . e ., FITC-dextrans) 30 . Fortunately, the small GTPases that regulate macropinocytosis are uniquely sensitive to changes in sub-membranous pH; and for this reason, amiloride inhibition of the Na + /H + exchanger selectively targets macropinocytosis over other forms of endocytosis 31 . Depending on the cell type, macropinocytosis can be constitutive process or triggered by extracellular stimuli and metabolites 32 . Examples of cells that undergo constitutive macropinocytosis include immature dendritic cells (for sampling of soluble antigens) 33 and cancerous fibroblasts transformed with oncogenic K-Ras or v-Src 34 . Extracellular molecules that can stimulate macropinocytosis include growth factors ( e . g ., epidermal growth factor 35 , macrophage colony-stimulating factor 36 ), and chemokines 37 ; this breadth of stimuli enables context-dependent regulation of signalling at the cell surface. Although not a canonical growth factor, ATP plays a growth factor-like role in regulating many cellular functions through activation of purinergic receptors 19 , 38 – 41 and can trigger macropinocytosis in human lung cancer cells 13 , driving an increase in intracellular ATP and promoting drug resistance. In addition to ATP, other macropinosome cargo can include amino acids and other metabolites important for cellular function ( i . e ., growth, survival, etc.) 42 , 43 . As a part of autocrine and paracrine purinergic signalling, ATP is released via a number of mechanisms including through channels like pannexin 1 (PANX1) as well as by vesicular release 44 . PANX1 forms ubiquitously-expressed heptameric channels that are permeable to small (Cl - ) and large ( e . g ., ATP) anions 45 – 52 . Additionally, PANX1 may also regulate the flux of small cations (Ca 2+ ) 53 , 54 and release of large cations (spermidine) 55 . Selectivity for anions or cations has been proposed to depend on the mechanism of channel activation 56 . PANX1 anion selectivity, characteristic of certain open states 45 , 51 , 52 , has been attributed to extracellular tryptophan (W74) and arginine (R75) residues that form a molecular filter for size and charge, respectively. While their overall properties are still the focus of intense investigation, it has been well-established that PANX1 channels play a role in ATP release 48 , 57 – 60 . Once in the extracellular space, ATP and its metabolites arising from the activity of tissue-specific ectonucleotidases trigger an array of downstream signaling pathways that can lead to myriad cellular changes 61 . We and others have demonstrated that extracellular ATP can also directly impact the ATP release machinery 16 , 17 , 50 , 62 , inducing sustained changes in cell signalling. Exogenously added extracellular ATP can inhibit PANX1 channel activity 50 , 62 , 63 as well as stimulate PANX1 internalization 16 , 17 . Cell surface physical association of PANX1 with the ionotropic purinergic P2X7 receptor (P2X7R) preceded internalization 16 . Site-directed alanine substitution of the PANX1 extracellular loop tryptophan, W74, disrupted cell surface P2X7R - PANX1 association 16 , as well as PANX1 internalization 17 , suggesting this residue regulates both ionic selectivity and cell surface stability. In the course of this work, we noted that a considerable amount of internalized PANX1 localized to structures much larger than canonical endocytic vesicles. ATP-triggered internalization of PANX1 did not utilize the canonical clathrin-, caveolin-, and dynamin-associated machinery 17 , consistent with work from other groups 64 , 65 . Furthermore, ATP-evoked PANX1 internalization in N2a cells coincided with active filopodial dynamics, relied on cholesterol, and occurred in the absence of canonical endocytic effectors 17 , suggesting a non-canonical endocytic process, such as macropinocytosis. We first identified that ATP triggered an increase in cell size via a mechanism that was disrupted by the internalization-deficient PANX1 W74A mutant and amiloride, consistent with expected cell size changes following macropinocytosis 66 . Next, we examined the impact of the macropinocytosis blocker amiloride on ATP-induced PANX1 internalization. As anticipated, amiloride, prevented ATP-evoked PANX1 internalization. Expression of a constitutively-active ARF6 GTPase increased cell size and basal intracellular PANX1 levels and prevented further ATP-induced PANX1 internalization suggesting that the macropinocytosis effector plays a role in ATP-induced PANX1 internalization. In the presence of several macropinocytosis inhibitors, intracellular PANX1 did not increase when cells were stimulated with ATP. Further, these inhibitors disrupted co-distribution of PANX1 with the macropinosome cargo, 70 kDa dextran, to intracellular compartments consistent with the size of macropinosomes. Cryo-EM structures have highlighted putative PANX1-lipid interactions 67 , 68 , while several recent studies have even suggested that lipid subtypes can permeate the channel 69 , 70 and regulate gating 68 , 71 . Here, we identify several lipid interactors for the PANX1 C-terminus related to macropinocytosis and internalization mechanisms. Taken together, these results suggest that, in addition to undergoing macropinocytosis in response to extracellular ATP, PANX1 could also play a broader role in the regulation of ATP-regulated macropinocytosis to promote expansion of neuroblastoma cell size. METHODS Plasmids The PANX1-EGFP and PANX1-RFP plasmids 72 were generous gifts from Drs. Dale Laird and Silvia Penuela. The pARF6-CFP (Plasmid #11382) and pARF6 Q67L-CFP (Plasmid #11387) were acquired from Addgene, courtesy of Dr. Joel Swanson. Cell Culture Neuro2a (N2a) mouse neuroblastoma cells (procured from the American Type Culture Collection, ATCC, in 2011) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin (all obtained from Gibco/Life Technologies). Where indicated, N2a cells were transfected using jetPEI reagent (Polyplus transfection/VWR) according to the manufacturer’s protocol. N2a cells stably expressing PANX1-EGFP or PANX1-RFP were maintained in DMEM/F12 containing 10% FBS, and 100 units/mL penicillin, 100 μg/mL streptomycin, and 400 μg/mL geneticin 418 (all obtained from Gibco/Life Technologies). Stable cell lines were generated as follows: N2a cells were transfected with PANX1-EGFP or PANX1-RFP using the jetPEI reagent (Polyplus transfection/VWR) according to the manufacturer’s protocol. Cells were plated on poly-D-lysine (PDL, Sigma)-coated coverslips. For all internalization-related experiments, protein translation was briefly inhibited by treatment with 20 μg/mL cycloheximide (CHX; Sigma) for 8 h, coincident with other treatments. Where indicated, stable cell lines were transfected with ARF6-ECFP or ARF6-Q67L-ECFP, Cells were fixed with 4% paraformaldehyde (PFA) washed three time in phosphate buffered saline (PBS) prior to mounting for imaging or processing for immunostaining. We investigated the role of ATP (500 μM, Sigma) versus vehicle control (equal volume of water) on PANX1 cell surface stability and trafficking. We disrupted macropinocytosis by pre-treating cells with amiloride (300 μM; Sigma), EIPA (25 μM, Tocris) Latrunculin A (15 μM, Sigma), LY 294002 (25 μM, Tocris), or vehicle (DMSO) for 1 hour. To identify co-distribution with macropinosome cargo, where indicated, cells were treated with ATP (500 μM, Sigma) and 70 kDa TRITC-Dextran (100 μg/mL, Thermo Fisher) for 30 minutes prior to fixation. Immunocytochemistry Antibody labelling of PFA-fixed cultures was performed as previously described 17 . The primary antibody used was early endosome antigen 1(EEA1 – 1:200; Cell Signaling) and the secondary antibody was Alexa647 AffiniPure donkey anti-rabbit IgG (1:600; Jackson ImmunoResearch). Microscopy Confocal microscopy and analysis were performed blinded to the experimental conditions. Images were acquired with a Leica TCS SP8 confocal STED microscope. Quantification was performed using Leica Application Suite (version 3.1.3) and in the z-section displaying the largest plane of the nucleus (Hoechst 33342), where applicable. In the absence of Hoechst staining (where CFP-tagged constructs were expressed), a z-stack was captured over the entirety of the cells in the field of view and the middle z-plane was selected for ROI analysis. ROIs with a cross-sectional area of ≤ 60 mm 2 were excluded from analysis (3 ROI total, 2 from ARF6-CFP – vehicle, 1 from ARF6 Q67L-CFP – vehicle). Comparisons were made between images acquired under identical conditions. Representative confocal micrographs were adjusted for contrast uniformly using Adobe Photoshop (CC 2015.1.2) for display purposes only; no contrast adjustments were made prior to analysis. Confocal images (Leica TCS SP8) of fixed cells were acquired using a 40X (1.3 NA) oil immersion objective at 3X optical zoom in 1296 x 1296 format with a pixel area of 71 nm 2 as confocal z-stacks. Quantification of PANX1-EGFP/PANX1-RFP fluorescence intensity to describe ‘intracellular PANX1’ was performed at time zero and at 30 min post stimulation, as follows: a polygonal trace was drawn 1 μm inside the cell periphery and the encapsulated average PANX1 fluorescence intensity per pixel was computed. Quantification of cross-sectional cellular area was determined on same z-plane as intracellular area. Cross-sectional area per cell was the area encapsulated by the region of interest (ROI) traced along the cell periphery, as described above. Intracellular structure analysis To measure the size of potential macropinosomes, PANX1-EGFP signals were acquired with confocal microscopy using a 100X (1.3 NA) oil immersion objective in a 6008 x 6008 format, yielding a pixel size of 19 nm, and the pinhole size was set to 0.65 AU. Inclusion criteria for macropinosomes were round structures with a diameter of 0.2–5 μm that were positive for both PANX1-EGFP and/or TRITC-dextran. The diameters of the selected macropinosomes were measured using ImageJ (Version 2.14.0). Co-distribution analysis For co-distribution analysis of PANX1 and 70kDa Dextran, confocal images of fixed PANX1-EGFP N2a cells treated with 70 kDa Dextran-TRITC and ATP (500 μM, 30 min) were obtained using the Leica TCS SP8 with a 40X (1.3 NA) oil immersion objective at 2X optical zoom in a 1904 x 1904 format, resulting in a pixel area of 76.22 μm 2 as confocal z-stacks. A z-stack of three planes (z-step size set to 0.1 μm) was captured over the plane with the largest nucleus in the field of view. The z-plane displaying the largest plane of the nucleus among the three planes was selected for region of interest analysis. The selected plane contained at least 15 PANX1-EGFP positive cells. To quantify co-localization using Leica Application Suite, the threshold for both the intracellular PANX1 and Dextran intensity was set to 30%. Co-localization rate by computing the fluorescence intensity per pixel. Membrane Lipid Strip Interaction Assays Membrane Lipid Strips (Echelon Biosciences; hydrophobic membrane spotted with 15 different membrane lipids [GT – glyceryl tripalmitate, DAG – diacylglycerol, PA – phosphatidic acid, PS – phosphatidylserine, PE – phosphatidylethanolamine, PC – phosphatidylcholine, PG – phosphatidylglycerol, CL – cardiolipin, PI – phosphatidylinositol, PI(4)P – phosphatidylinositol 4-phosphate, PI(4,5)P 2 – phosphatidylinositol 4,5-bisphosphate, PI(3,4,5)P 3 – phosphatidylinositol 3,4,5-triphosphate, CHOL – cholesterol, SM – sphingomyelin, SULF – 3-sulfogalactosylceramide] and a blank [xylene cyanol FF]) were blocked for 1 h in blocking buffer (3% BSA in PBS-T). Strips were then transferred to blocking buffer containing purified protein of interest: PI(4,5)P 2 Grip (1 μg/mL, positive control from Echelon Biosciences), GST (5 μg/mL), or GST-fused PANX1 C-terminus (GST-PANX1 CT , 5 μg/mL) for 1 h, then washed three times in PBS-T and incubated for an additional hour in blocking buffer with primary antibody. Strips were again washed in PBS-T three times then incubated in corresponding HRP-conjugated secondary for 1 h. All incubations were performed at room temperature with gentle agitation. Statistical analysis The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. A biological replicate was defined as a coverslip obtained from an independent cell passage, where a technical replicate is each single cell on a given coverslip. Results were analysed using two-way ANOVA, one-way ANOVA or unpaired t tests, where applicable. Data are presented as mean ± S.E.M. Detailed information about statistical tests is available in the figure legends. RESULTS Elevated extracellular ATP increases N2a cell size through a putative PANX1- and macropinocytosis-dependent mechanism Growth factor-stimulated macropinocytosis is known to increase cell size (i.e. nerve growth factor 73 , insulin-like growth factor 74 , for review see Lloyd et al. 66 ). In our previous study, we identified that ATP-evoked PANX1 internalization could be disrupted by alanine substitution of the extracellular residue W74A. Here, we asked whether PANX1 internalization by ATP stimulation was accompanied by a change in cell size. Our model involves stable expression of PANX1-EGFP in murine neuroblastoma N2a cells, this clonal colony was selected for low level expression that distributes both to the cell surface and intracellular compartments 17 . EGFP-tagged PANX1 has a similar subcellular distribution to endogenous PANX1 (ie. cell surface with some intracellular localization) 75 . Here, to visualize internalization (using a protocol established in our previous studies 16 , 17 ), we bath applied ATP onto cultured N2a cells in the presence of transient protein-synthesis inhibition allows us to visualize steady state changes in PANX1 localization in cells devoid of de novo synthesized protein in the secretory pathway. To quantify changes in cell size, we acquired confocal Z-stacks and measured the cross-sectional area of vehicle and ATP-stimulated PANX1-EGFP or PANX1-W74A-EGFP expressing N2a cells at the plane where the nucleus was centered. Here, a transmitted light image was acquired to identify PANX1-EGFP at the cell periphery ( Figure 1A ). The cell periphery was traced and the cross-sectional area was quantified. Elevated extracellular ATP expanded cross-sectional cell area ( Figure 1B ), while no change was observed in response to elevated extracellular ATP with PANX1 W74A expression ( Figure 1B ), suggesting that PANX1 itself might regulate macropinocytosis. Download figure Open in new tab Figure 1. Elevated extracellular ATP increases N2a cell size through a putative PANX1- and macropinocytosis-dependent mechanism ( A ) Representative confocal micrographs of PANX1-EGFP (green) N2a cells overlaid with brightfield imaging following incubation with vehicle (water) or ATP (500 μM; 30 min). ( B ) Quantification of cross-sectional cellular area in PANX1-EGFP (representative outlined traces) or PANX1 W74A-EGFP N2a cells following treatment with vehicle (water) or ATP (500 μM). One-way ANOVA with Dunnett’s post hoc ( N = 3; *p<0.05, **p<0.01). ( C ) Representative confocal micrographs of PANX1-EGFP (green) N2a cells pre-treated with vehicle (DMSO), or amiloride (300 μM) for 1 h prior to vehicle (water) or ATP (500 μM; 30 min). ( D ) Cross-sectional cellular area and ( E ) intracellular PANX1 was quantified relative to vehicle control. N = 4, two-way ANOVA with Dunnett’s post hoc (interaction: F[1,12]=19.53, p=0.0008; inhibitor: F[1,12]=5.825, p=0.0327; ATP: F[1,12]=22.22, p=0.0005). Hoechst (cyan) was used as a nuclear counterstain. ( F ) Representative confocal micrographs of CHX-treated PANX1-RFP (green) N2a cells transiently-transfected with ARF6-CFP or ARF6-Q67L-CFP (magenta, 48 h) prior to vehicle (water) or ATP (500 μM; 30 min), indicating accumulation of intracellular PANX1 with co-expression of constitutively active ARF6 mutant. White arrows indicate overlap of internalized vesicles. ( G ) Quantification of cross-sectional cellular area and ( H ) intracellular PANX1 was quantified for each ARF6 construct relative to vehicle treatment. N = 3, (unpaired t-tests, WT: p=0.026, Q67L: p=0.96). ( I ) Representative confocal micrographs demonstrating accumulation of PANX1RFP (green) and ARF6-Q67L-CFP (magenta) in early endosomes (EEA1, cyan). N refers to the number of coverslips from independent passages (≥ 30 cells analyzed per coverslip). Given the effect of PANX1 mutation on cell size, we next addressed whether this ATP-evoked increase in cell size was triggered by macropinocytosis and whether PANX1 was internalized in the process. Macropinocytosis involves the coordinated recruitment and activation of several pH-sensitive GTPases in membrane ruffles 25 , 26 . Amiloride inhibits macropinocytosis by blocking the Na + /H + exchanger resulting in acidification of the submembranous space and inhibition of requisite small GTPases 31 . We first tested the impact of pre-treatment with amiloride (1 h; 300 μM) or vehicle (DMSO) on ATP-induced internalization of PANX1-EGFP constitutively expressed in murine neuroblastoma N2a cells using an analysis paradigm established in our previous studies 16 , 17 . Following amiloride pre-treatment, there was no change in intracellular PANX1 or cross-sectional area 30 min post-stimulation with 500 μM ATP ( Figure 1C-E ). This suggested that amiloride-sensitive macropinocytic GTPases were required for ATP-induced increase in cell size and PANX1 internalization. ARF6 regulates endocytic cargo transit between the plasma membrane and a clathrin-independent endosomal compartment that can mature into a macropinosome 23 , 24 . To further validate that the ATP-evoked increase in cell size was a macropinocytic mechanism, we used cyan fluorescent protein (CFP)-tagged wildtype ARF6 or GTP-hydrolysis resistant ( i . e ., constitutively active) ARF6 Q67L that would constitutively trigger internalization and thereby create a ceiling for increased cell size. We found that cross-sectional cellular area was increased in cells expressing constitutively active ARF6 Q67L, independent of ATP treatment, supporting our finding that ATP-mediated macropinocytosis increases N2a cell area ( Figure 1F,G ). We next assessed the role of ARF6 in ATP-dependent PANX1 internalization by co-expressing ARF6-CFP or ARF6 Q67L-CFP with PANX1-RFP in N2a cells. As expected, ARF6 Q67L expression triggered the formation and retention of large intracellular PANX1-positive/EEA1-positive vesicles independent of ATP stimulation ( Figure 1F-H ) while ATP-induced PANX1 internalization was not affected by co-expression of the wildtype ARF6 ( Figure 1H ). PANX1 internalizes to macropinosomes following ATP stimulation Given that amiloride and expression of constitutively active ARF6 mutant impacted internalization, we sought to further validate our hypothesis that PANX1 was internalized via macropinocytosis. Canonical endosomes ( i . e ., caveolae and clathrin-coated pits) are small ( 0.2 μm diameter) 1 . Using the fluorophore tetramethylrhodamine isothiocyanate (TRITC)-conjugated to a 70 kDa dextran (70 kDa-dextran-TRITC) can reliably label putative macropinosomes 30 , as it is excluded based on size from vesicles endocytosed through canonical endocytic pathways ( i . e ., clathrin or caveolin-mediated internalization). Cells were fixed after 30 min incubation and imaged using confocal microscopy and vehicle was compared to inhibitor pre-treatment in ATP-stimulated cells. Here, there was clear evidence of dextran engulfed by PANX1-positive membranous structures ( Figure 2A,B ). The distribution of diameters of these PANX1(+), dextran(+), and PANX1-dextran co-(+) structures were similarly distributed and primarily between 0.8-1.8 μm ( Figure 2C ), supporting that these were macropinosomes. Next, we applied several inhibitors or vehicle 1 h prior to initiating PANX1 internalization via infusion of ATP (500 μM, 30 min) along with the macropinosome cargo 70 kDa-dextran-TRITC (100 μg/mL) to the culture medium, then quantifying both intracellular PANX1, as well as its co-distribution with dextran-TRITC. Ethylisopropyl amiloride (EIPA), an amiloride derivative, inhibits Na + /H + exchangers and TRPP3 channels and is a more potent inhibitor of macropinocytosis than amiloride 31 , 76 . Relative to vehicle, cells treated with EIPA (25 μM) had less internalized PANX1 30 min after ATP stimulation ( Figure 2D,E ). Next, we bathed N2a cells in medium containing LY294002 (25 μM), a selective phosphoinositide-3 kinase (PI3K) inhibitor, which disrupts the closure phase of macropinosomes during internalization 77 . LY294002 also reduced PANX1 internalization following ATP stimulation ( Figure 2D,E ). Macropinocytosis is preceded by local actin-dependent filopodial dynamics prior to internalization and actin also plays an important role in closing the macropinosome following internalization 32 . Thus, to further support our hypothesis that PANX1 is internalized via macropinocytosis, we disrupted actin polymerization using latrunculin A (15 μM, LatA). As with EIPA and LY294002, ATP-induced PANX1 internalization was significantly reduced following LatA pre-treatment ( Figure 2D,E ). In support of our findings above, PANX1 distribution to 70 kDa dextran(+) structures was reduced in the presence of all macropinocytosis-targeting inhibitors (EIPA, LY294002, and LatA, Figure 2F ). Download figure Open in new tab Figure 2. Intracellular PANX1 distributes to macropinosomes. ( A ) Representative confocal micrographs with line scan over ROI demonstrate PANX1 localization in membrane-like structures surrounding macropinosome cargo 70 kDa dextran-TRITC. Inset highlights putative PANX1-positive macropinosome. ( B ) Line scan (10 μm) of putative macropinosome with PANX1-encompassing dextran-TRITC cargo. ( C ) Diameter of intracellular PANX1, TRITC-Dextran, and co-distributed structures show similar size distribution pattern. ( D ) Representative confocal micrographs of PANX1-EGFP (green) N2a cells pre-treated with vehicle or EIPA (25 μM), LY-294002 (25 μM), or latrunculin-A (15 μM) then incubated with 70 kDa-dextran-TRITC (100 μg/mL, magenta) and ATP (500 μM, 30 min) prior to fixation. ( E ) Intracellular PANX1 following ATP stimulation (30 min) was quantified relative to vehicle control. N = 14-15 per treatment group, one-way ANOVA with Dunnett’s post hoc (interaction: F[3,55]=9.124, p<0.0001). ( F ) Co-distribution rate of intracellular PANX1 with TRITC-dextran following ATP stimulation for each treatment ( N = 5 per treatment group, one-way ANOVA with Dunnett’s post hoc (interaction: F(3,16)=164, p<0.0001). Scale bar, 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. PANX1 C-terminus interacts with lipids involved in macropinocytosis The carboxy-tail of PANX1 (PANX1 CT ) is the location of PANX1-actin protein-protein interactions 18 , 65 , regulates internalization and cell surface trafficking 64 , 65 , and contains a highly-disordered putative membrane-associated region 78 ( Figure 3A ) that was unresolved in cryo-EM structures. Recent atomic resolution structures identified PANX1-lipid interactions 67 , 68 with functional studies suggesting that lipid subtypes might permeate PANX1 69 , 70 and regulate channel gating 68 , 71 . As PANX1 internalization was cholesterol-dependent 16 , 17 , we hypothesized that the PANX1 CT interacts with cholesterol or other lipids commonly co-distributed with cholesterol or known to be involved in cholesterol-dependent processes. To test this prediction, we incubated purified GST-tagged PANX1 CT or GST alone with a hydrophobic membrane spotted with 15 different membrane lipids. After washing off excess unbound PANX1 CT , we probed the membrane using an anti-GST ( Figure 3B ) or anti-Panx1 CT antibody ( Figure 3C ). We did not detect an enriched interaction between PANX1 CT and cholesterol; however, we found that PANX1 CT interacted with PA, PI(4)P, and PI(4,5)P 2 , known regulators of macropinocytosis 23,24 . Download figure Open in new tab Figure 3. The PANX1 C-terminus interacts with phospholipids involved in macropinocytosis. (A) Schematic of ( i ) full-length PANX1 as well as ( ii ) GST and GST-fused PANX1 C-terminus (GST-PANX1 CT ) indicating location of putative lipid interaction domain (blue rectangle) relative to the caspase cleavage site (red circle). Western blot (B) of Membrane Lipid Interaction Strip (hydrophobic membrane spotted with 15 different membrane lipids [GT – glyceryl tripalmitate, DAG – diacylglycerol, PA – phosphatidic acid, PS – phosphatidylserine, PE – phosphatidylethanolamine, PC – phosphatidylcholine, PG – phosphatidylglycerol, CL – cardiolipin, PI – phosphatidylinositol, PI(3)P – phosphatidylinositol 3-phosphate, PI(3,4)P2 – phosphatidylinositol 3,4-bisphosphate, PI(3,4,5)P3 – phosphatidylinositol 3,4,5-triphosphate, CHOL – cholesterol, SM – sphingomyelin, SULF – 3-sulfogalactosylceramide] and a blank [xylene cyanol FF]) incubated with purified peptides including positive control (PI(4,5)P2 Grip (1 μg/mL)), GST (5 μg/mL) or GST-PANX1 CT (5 μg/mL) and probed with GST antibody. (C) Western blot of Membrane Lipid Interaction Strip ( i ) that was pre-incubated with purified GST (5 μg/mL) or GST-PANX1 CT (5 μg/mL) and probed with PANX1CT antibody. (ii) Relative GST-PANX1 CT lipid interaction enrichment on Western blot, normalized to blank, was quantified using densitometry. N = 3, one-way ANOVA with Dunnett’s post-hoc (**p<0.01, ***p<0.001, ****p<0.0001). Download figure Open in new tab Figure 4. ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism. ATP evokes internalization of PANX1 via macropinocytosis with a PANX1-dependent increase in cell size. PANX1 internalization was disrupted through inhibition of actin polymerization, PI3K, and pH sensitive GTPases. The increase in cell size due to elevated extracellular ATP could arise from multiple, possibly convergent pathways, such as purinergic receptor signaling, and/or crosstalk between metabolic master regulators mTORC1 and AMPK. DISCUSSION Precise control of purinergic signalling via PANX1 is critical in many diverse cell types, including maintaining neural stem cell populations 18 , 19 , 79 , regulating the development of immature neurons 80 , 81 , tumorigenicity and metastasis of several cancers 82 – 85 . Macropinocytosis is also intimately involved in regulating these processes 10 , 11 . ATP-mediated macropinocytosis might not only change the receptor contribution but could also be exploited as an adaptive response for uptake of ATP and other nutrients in cells with elevated energy requirements ( i . e ., cancer 10 , 86 ). Macropinocytosis activity is elevated at the core of tumours where nutrients are least concentrated 87 . In several types of human lung cancer (as well as breast, liver, and pancreatic cancers), macropinocytosis is used as a mechanism of uptake for ATP, seen by colocalization of fluorescently tagged ATP and 70 kDa dextran 12 , 88 – 90 . In that context, internalized ATP is used as an energy source and promotes metastasis in nutrient-starved cancer cells 90 . Interestingly, PANX1 has also been implicated in the uptake of ATP in both human and yeast cells 91 , however, it was not determined whether this involved a channel- or internalization-based mechanism. Moreover, constitutive macropinocytosis promotes cell proliferation in many forms of cancer 92 . In previous work, we identified that PANX1 internalization occurs in response to elevated extracellular ATP in a dose-dependent manner via interaction with the ionotropic purinergic receptor P2X7R 16 , 17 . This interaction was disrupted by alanine-substitution of an extracellular tryptophan (W74A), which in turn inhibited PANX1 internalization. Here, we observe that elevated extracellular ATP also increase the size of mouse neuroblastoma N2a cells. PANX1 W74A, which disrupts the ATP-dependent interaction with P2X7R and downstream internalization 16 , 17 , eliminated the increase in mouse neuroblastoma N2a cell size triggered by extracellular ATP. Cryo-electron microscopy-based structures of PANX1 with atomic resolution demonstrated that W74 and its neighbouring residue R75 form the anionic selectivity filter for PANX1 45 – 47 , 49 . This filter is likely formed through the formation of a cation-π interaction that stabilizes both large and small anionic molecules as they move through the narrowest portion of the pore 49 . We and other have also demonstrated that mutation of this residue disrupts ATP-mediated PANX1 inhibition 63 and P2X7R-interaction dependent internalization 16 , 17 . ATP-induced increases in cell size, a common consequence of macropinocytosis 43 , 93 , were inhibited by amiloride and facilitated by a constitutively active mutant of the GTPase ARF6. ARF6 is a critical macropinocytosis effector and a driver of macropinosome trafficking following internalization 23 , 24 . Elevated ATP did not further increase intracellular PANX1 or cell size in the presence of ARF6 Q67L, suggesting that membrane internalization was saturated under these conditions. Here, each manipulation to disrupt ATP-dependent increases in cell size also disrupted PANX1 internalization, suggesting that ATP-induced PANX1 internalization occurs through macropinocytosis. In support of this, PANX1 internalization was sensitive to inhibition of amiloride-sensitive GTPases, PI3K, and actin, and internalized PANX1 had strong co-distribution with the macropinosome cargo 70 kDa dextran following ATP stimulation. Notably, novel PANX1 CT lipid interactors, PI(4,5)P 2 and PA, are produced in response to ARF6 activation and critical for multiple stages of macropinocytosis 23 , 24 . Given the role of ARF6 as a macropinsome effector and the sensitive of cell size perturbations to amiloride, these results support that ATP-evoked increases in cell size occur in a PANX1- and macropinocytosis-dependent mechanism. It is not yet clear whether PANX1 W74A disruption of ATP-induced cell area expansion implicates PANX1 directly (either through channel function, protein-protein interactions, or another mechanism) or indirectly (such as via the loss of an intracellular function) in the regulation of ATP-mediated macropinocytosis, but both may be possible. In addition to regulating purinergic signaling pathways, it is tempting to speculate that ATP release by PANX1 may, in part, act in a homeostatic autocrine manner to normalize cell size. PANX1 demonstrates cell type-specific mechanosensitivity 52 , 58 , 60 , 94 , where changes in cell volume or mechanical stress activate PANX1 channels. In oocytes and HEK293T cells, osmotic shrinkage activates PANX1-mediated membrane currents (but no dye uptake) yet swelling does not impact PANX1 function 52 . While in airway epithelia, hypotonic challenge stimulated ATP release and dye-uptake 60 . Not only could PANX1 impact overall cell size in this manner, but there is also potential that, once internalized, PANX1 could regulate endosomal volume. Channel-dependent Cl - efflux from endosome compartments was recently demonstrated to regulate endosome volume 95 . The anion-selective PANX1 may be able to regulate endosome volume, if active, following internalization. Future investigations of the functional consequences of the relationship between PANX1 mechanosensation and macropinocytosis, and the function of endosome-resident PANX1 could resolve these outstanding questions. Taken together, our data suggest that elevated extracellular ATP drives a macropinocytosis- and PANX1-mediated increase in cell size. PANX1 internalization and cell size increases normally induced by elevated extracellular ATP are absent with PANX1 W74A suggesting that PANX1 may also directly or indirectly regulate ATP-evoked macropinocytosis and, consequently, cell size. The coordinated relationship between PANX1 and macropinocytosis has implications for many cellular behaviours where purinergic signalling is involved, particularly those, like cancer, where ATP can act as both a signalling molecule in the extracellular space and as a metabolite supporting enhanced cell growth when internalized. This highlights another possible mechanism for PANX1 to promote a survival advantage for cancer cells. AUTHOR CONTRIBUTIONS AKJB and LAS conceived of the studies. AKJB, HY, and LWS performed the experiments. AKJB, HY, and EVDS performed the analysis with input from JCSA and LAS. AKJB wrote the first draft and AKJB, LAS, and LWS revised the manuscript. DECLARATION OF INTERESTS The authors have no competing interests to declare. ACKNOWLEDGEMENTS The authors are grateful to Juan C. Sanchez-Arias and Emma van der Slagt for their initial input on study design and analysis not included in the updated version of the manuscript. This manuscript features data published in theses from AKJB 96 and HY 97 , featured in a bioRxiv preprint. This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-03889), from the Canadian Institutes of Health Research (MOP142215), and the University of Victoria-Division of Medical Sciences to LAS. LAS was also supported by a Michael Smith Foundation for Health Research and British Columbia Schizophrenia Society Foundation Scholar Award (5900). AKJB was supported by scholarships from NSERC (PGSD 459931-2014) and the University of Victoria (President’s Research Scholarship, Dr. Howard E. Petch and Dr. Julius F. Schleicher Memorial Scholarships) for this work and is currently supported in part by the National Institute of General Medical Sciences of the National Institute of Health under award P20GM109089. HY was supported by a NSERC CGS-M. LAS is also grateful for infrastructure support from the Canada Foundation for Innovation (29462) and the BC Knowledge Development Fund (804754) for the Leica SP8 microscope system. REFERENCES 1. ↵ Swanson , J. A. & King , J. S. The breadth of macropinocytosis research . Philosophical Transactions of the Royal Society B: Biological Sciences 374 , ( 2019 ). 2. ↵ Steinman , R. M. , Brodie , S. E. & Cohn , Z. A. Membrane flow during pinocytosis: A stereologic analysis . Journal of Cell Biology 68 , 665 – 687 ( 1976 ). OpenUrl Abstract / FREE Full Text 3. ↵ Roche , P. A. & Furuta , K. The ins and outs of MHC class II-mediated antigen processing and presentation . Nat Rev Immunol 15 , 203 – 216 ( 2015 ). OpenUrl CrossRef PubMed 4. ↵ Yoshida , S. , Pacitto , R. , Yao , Y. , Inoki , K. & Swanson , J. A. Growth factor signaling to mTORC1 by amino acid-laden macropinosomes . Journal of Cell Biology 211 , 159 – 172 ( 2015 ). OpenUrl Abstract / FREE Full Text 5. ↵ Wall , A. A. et al. Small GTPase Rab8a-recruited phosphatidylinositol 3-kinase γ regulates signaling and cytokine outputs from endosomal toll-like receptors . Journal of Biological Chemistry 292 , 4411 – 4422 ( 2017 ). OpenUrl Abstract / FREE Full Text 6. ↵ de Carvalho , T. M. U. , Barrias , E. & de Souza , W. Macropinocytosis: A pathway to protozoan infection . Front Physiol 6 , ( 2015 ). 7. Saeed , M. F. , Kolokoltsov , A. A. , Albrecht , T. & Davey , R. A. Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes . PLoS Pathog 6 , e1001110 ( 2010 ). OpenUrl CrossRef PubMed 8. Mercer , J. & Helenius , A. Gulping rather than sipping: Macropinocytosis as a way of virus entry . Curr Opin Microbiol 15 , 490 – 499 ( 2012 ). OpenUrl CrossRef PubMed 9. ↵ Francis , C. L. , Ryan , T. A. , Jones , B. D. , Smith , S. J. & Falkow , S. Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria . Nature 364 , 639 – 642 ( 1993 ). OpenUrl CrossRef PubMed Web of Science 10. ↵ Finicle , B. T. , Jayashankar , V. & Edinger , A. L. Nutrient scavenging in cancer . Nat Rev Cancer 18 , 619 – 633 ( 2018 ). OpenUrl CrossRef PubMed 11. ↵ Bloomfield , G. & Kay , R. R. Uses and abuses of macropinocytosis . J Cell Sci 129 , 2697 – 2705 ( 2016 ). OpenUrl Abstract / FREE Full Text 12. ↵ Wang , X. et al. Extracellular ATP, as an energy and phosphorylating molecule, induces different types of drug resistances in cancer cells through ATP internalization and intracellular ATP level increase . Oncotarget 8 , 87860 – 87877 ( 2017 ). OpenUrl CrossRef PubMed 13. ↵ Qian , Y. et al. Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells . Cancer Lett 351 , 242 – 251 ( 2014 ). OpenUrl CrossRef PubMed 14. ↵ Shukla , S. , Dalai , P. & Agrawal-Rajput , R. Metabolic crosstalk: Extracellular ATP and the tumor microenvironment in cancer progression and therapy . Cell Signal 121 , ( 2024 ). 15. ↵ Laird , D. & Penuela , S. Pannexin biology and emerging linkages to cancer . Trends in Cancer 7 , 1119 – 1131 ( 2021 ). OpenUrl PubMed 16. ↵ Boyce , A. K. J. & Swayne , L. A. P2X7 receptor cross-talk regulates ATP-induced pannexin 1 internalization . Biochemical Journal 474 , 2133 – 2144 ( 2017 ). OpenUrl Abstract / FREE Full Text 17. ↵ Boyce , A. K. J. , Kim , M. S. , Wicki-Stordeur , L. E. & Swayne , L. A. ATP stimulates pannexin 1 internalization to endosomal compartments . Biochemical Journal 470 , 319 – 330 ( 2015 ). OpenUrl Abstract / FREE Full Text 18. ↵ Wicki-Stordeur , L. E. & Swayne , L. A. Panx1 regulates neural stem and progenitor cell behaviours associated with cytoskeletal dynamics and interacts with multiple cytoskeletal elements . Cell Communication and Signaling 11 , ( 2013 ). 19. ↵ Wicki-Stordeur , L. E. , Dzugalo , A. D. , Swansburg , R. M. , Suits , J. M. & Swayne , L. A. Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation . Neural Dev 7 , 11 ( 2012 ). OpenUrl PubMed 20. ↵ Langlois , S. et al. Inhibition of PANX1 Channels Reduces the Malignant Properties of Human High-Risk Neuroblastoma . J Cancer 14 , 689 – 706 ( 2023 ). OpenUrl CrossRef PubMed 21. ↵ Grimmer , S. , van Deurs , B. & Sandvig , K. Membrane ruffling and macropinocytosis in A431 cells require cholesterol . J Cell Sci 115 , 2953 – 2962 ( 2002 ). OpenUrl Abstract / FREE Full Text 22. ↵ Naslavsky , N. , Weigert , R. & Donaldson , J. G. Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides . Mol Biol Cell 14 , 417 – 431 ( 2003 ). OpenUrl Abstract / FREE Full Text 23. ↵ Honda , A. et al. Phosphatidylinositol 4-phosphate 5-kinase α is a downstream effector of the small G protein ARF6 in membrane ruffle formation . Cell 99 , 521 – 532 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 24. ↵ Brown , F. D. , Rozelle , A. L. , Yin , H. L. , Balla , T. & Donaldson , J. G. Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic . Journal of Cell Biology 154 , 1007 – 1017 ( 2001 ). OpenUrl Abstract / FREE Full Text 25. ↵ Zhang , Q. , Calafat , J. , Janssen , H. & Greenberg , S. ARF6 Is Required for Growth Factor- and Rac-Mediated Membrane Ruffling in Macrophages at a Stage Distal to Rac Membrane Targeting . Mol Cell Biol 19 , 8158 – 68 ( 1999 ). OpenUrl Abstract / FREE Full Text 26. ↵ Daste , F. et al. Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature . Journal of Cell Biology 216 , 3745 – 3765 ( 2017 ). OpenUrl Abstract / FREE Full Text 27. ↵ Dharmawardhane , S. et al. Regulation of macropinocytosis by p21-activated kinase-1 . Mol Biol Cell 11 , 3341 – 3352 ( 2000 ). OpenUrl Abstract / FREE Full Text 28. ↵ Liberali , P. et al. The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS . EMBO Journal 27 , 970 – 981 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 29. ↵ Bohdanowicz , M. & Grinstein , S. Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis . Physiol Rev 93 , 69 – 106 ( 2013 ). OpenUrl CrossRef PubMed 30. ↵ Lin , X. P. , Mintern , J. D. & Gleeson , P. A. Macropinocytosis in Different Cell Types: Similarities and Differences . Membranes (Basel) 10 , 177 ( 2020 ). OpenUrl PubMed 31. ↵ Koivusalo , M. et al. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling . Journal of Cell Biology 188 , 547 – 563 ( 2010 ). OpenUrl Abstract / FREE Full Text 32. ↵ Swanson , J. A. Shaping cups into phagosomes and macropinosomes . Nat Rev Mol Cell Biol 9 , 639 – 649 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 33. ↵ Canton , J. Macropinocytosis: New insights into its underappreciated role in innate immune cell surveillance . Front Immunol 9 , ( 2018 ). 34. ↵ Amyere , M. et al. Constitutive macropinocytosis in oncogene-transformed fibroblasts depends on sequential permanent activation of phosphoinositide 3-kinase and phospholipase C . Mol Biol Cell 11 , 3453 – 3467 ( 2000 ). OpenUrl Abstract / FREE Full Text 35. ↵ Bryant , D. M. et al. EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin . J Cell Sci 120 , 1818 – 1828 ( 2007 ). OpenUrl Abstract / FREE Full Text 36. ↵ Racoosin , E. L. & Swanson , J. A. Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages . Journal of Experimental Medicine 170 , 1635 – 1648 ( 1989 ). OpenUrl Abstract / FREE Full Text 37. ↵ Tanaka , G. et al. CXCR4 stimulates macropinocytosis: Implications for cellular uptake of arginine-rich cell-penetrating peptides and HIV . Chem Biol 19 , 1437 – 1446 ( 2012 ). OpenUrl CrossRef PubMed 38. ↵ Burnstock , G. & Ulrich , H. Purinergic signaling in embryonic and stem cell development . Cellular and Molecular Life Sciences 68 , 1369 – 1394 ( 2011 ). OpenUrl CrossRef PubMed 39. Franke , H. & Illes , P. Involvement of P2 receptors in the growth and survival of neurons in the CNS . Pharmacol Ther 109 , 297 – 324 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 40. Zimmermann , H. Extracellular ATP and other nucleotides—ubiquitous triggers of intercellular messenger release . Purinergic Signal 12 , 25 – 57 ( 2016 ). OpenUrl CrossRef PubMed 41. ↵ Erlinge , D. Extracellular ATP: A growth factor for vascular smooth muscle cells . Gen Pharmacol 31 , 1 – 8 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 42. ↵ Yoshida , S. , Pacitto , R. , Yao , Y. , Inoki , K. & Swanson , J. A. Growth factor signaling to mTORC1 by amino acid-laden macropinosomes . Journal of Cell Biology 211 , 159 – 172 ( 2015 ). OpenUrl Abstract / FREE Full Text 43. ↵ Yoshida , S. , Pacitto , R. , Inoki , K. & Swanson , J. Macropinocytosis, mTORC1 and cellular growth control . Cellular and Molecular Life Sciences vol. 75 1227 – 1239 Preprint at doi: 10.1007/s00018-017-2710-y ( 2018 ). OpenUrl CrossRef 44. ↵ Lazarowski , E. R. Vesicular and conductive mechanisms of nucleotide release . Purinergic Signal 8 , 359 – 373 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 45. ↵ Ruan , Z. , Orozco , I. J. , Du , J. & Lü , W. Structures of human pannexin 1 reveal ion pathways and mechanism of gating . Nature 584 , 646 – 651 ( 2020 ). OpenUrl CrossRef PubMed 46. Deng , Z. et al. Cryo-EM structures of the ATP release channel pannexin 1 . Nat Struct Mol Biol 27 , 373 – 381 ( 2020 ). OpenUrl CrossRef PubMed 47. ↵ Mou , L. et al. Structural basis for gating mechanism of Pannexin 1 channel . Nature 30 , 452 – 454 ( 2020 ). OpenUrl 48. ↵ Chiu , Y. H. et al. A quantized mechanism for activation of pannexin channels . Nat Commun 8 , ( 2017 ). 49. ↵ Michalski , K. et al. The Cryo-EM structure of a pannexin 1 reveals unique motifs for ion selection and inhibition . Elife 12 , e54670 ( 2020 ). OpenUrl 50. ↵ Ma , W. , Hui , H. , Pelegrin , P. & Surprenant , A. Pharmacological characterization of pannexin-1 currents expressed in mammalian cells . Journal of Pharmacology and Experimental Therapeutics ( 2009 ) doi: 10.1124/jpet.108.146365 . OpenUrl Abstract / FREE Full Text 51. ↵ Ma , W. et al. Pannexin 1 forms an anion-selective channel . Pflugers Arch 463 , 585 – 592 ( 2012 ). OpenUrl CrossRef PubMed 52. ↵ Nielsen , B. S. et al. Pannexin 1 activation and inhibition is permeant-selective . Journal of Physiology ( 2020 ) doi: 10.1113/JP278759 . OpenUrl CrossRef 53. ↵ Nielsen , A. C. et al. Non-ionotropic NMDAR signalling activates Panx1 to induce P2X4R-dependent long-term depression in the hippocampus . J Physiol 1 – 15 ( 2024 ) doi: 10.1113/JP285193#support-information-section . OpenUrl CrossRef 54. ↵ Yang , Y. et al. Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium . The Journal of Immunology 204 , 2995 – 3007 ( 2020 ). OpenUrl PubMed 55. ↵ Medina , C. B. et al. Metabolites released from apoptotic cells act as tissue messengers . Nature 580 , 130 – 135 ( 2020 ). OpenUrl CrossRef PubMed 56. ↵ Sanchez Arias , J. C. et al. PANX1 in inflammation heats up: New mechanistic insights with implications for injury and infection . Cell Calcium 90 , ( 2020 ). 57. ↵ Sandilos , J. K. et al. Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region . Journal of Biological Chemistry ( 2012 ) doi: 10.1074/jbc.M111.323378 . OpenUrl Abstract / FREE Full Text 58. ↵ Bao , L. , Locovei , S. & Dahl , G. Pannexin membrane channels are mechanosensitive conduits for ATP . FEBS Lett ( 2004 ) doi: 10.1016/j.febslet.2004.07.009 . OpenUrl CrossRef PubMed Web of Science 59. Chekeni , F. B. et al. Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis . Nature ( 2010 ) doi: 10.1038/nature09413 . OpenUrl CrossRef PubMed Web of Science 60. ↵ Seminario-Vidal , L. et al. Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia . Journal of Biological Chemistry 286 , 26277 – 26286 ( 2011 ). OpenUrl Abstract / FREE Full Text 61. ↵ Giuliani , A. L. , Sarti , A. C. & Di Virgilio , F. Extracellular nucleotides and nucleosides as signalling molecules . Immunol Lett 205 , 16 – 24 ( 2019 ). OpenUrl CrossRef PubMed 62. ↵ Qiu , F. & Dahl , G. A permeant regulating its permeation pore: Inhibition of pannexin 1 channels by ATP . Am J Physiol Cell Physiol 296 , C250 – C255 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 63. ↵ Qiu , F. , Wang , J. & Dahl , G. Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity . Purinergic Signal 8 , 81 – 90 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 64. ↵ Gehi , R. , Shao , Q. & Laird , D. W. Pathways regulating the trafficking and turnover of pannexin1 protein and the role of the C-terminal domain . J Biol Chem 286 , 27639 – 53 ( 2011 ). OpenUrl Abstract / FREE Full Text 65. ↵ Bhalla-Gehi , R. , Penuela , S. , Churko , J. M. , Shao , Q. & Laird , D. W. Pannexin1 and pannexin3 delivery, cell surface dynamics, and cytoskeletal interactions . Journal of Biological Chemistry 285 , 9147 – 9160 ( 2010 ). OpenUrl Abstract / FREE Full Text 66. ↵ Lloyd , A. C. The regulation of cell size . Cell 154 , 1194 – 1205 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 67. ↵ Ruan , Z. , Orozco , I. J. , Du , J. & Lü , W. Structures of human pannexin 1 reveal ion pathways and mechanism of gating . Nature 584 , 646 – 651 ( 2020 ). OpenUrl CrossRef PubMed 68. ↵ Kuzuya M et al. 35133866 . doi: 10.1126/scisignal.abg6941 . OpenUrl CrossRef PubMed 69. ↵ Anderson , C. L. et al. The Pannexin-1 N-terminal Helix Gates a Switch between Ion Conductance and Anandamide Transport . Preprint at doi: 10.1101/2024.03.11.584466 ( 2024 ). OpenUrl Abstract / FREE Full Text 70. ↵ Bialecki , J. et al. Suppression of presynaptic glutamate release by postsynaptic metabotropic NMDA receptor signalling to pannexin-1 . Journal of Neuroscience 40 , 729 – 742 ( 2020 ). OpenUrl Abstract / FREE Full Text 71. ↵ Henze , E. et al. ATP-release pannexin channels are gated by lysophospholipids . Elife 14 , ( 2025 ). 72. ↵ Penuela , S. et al. Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins . J Cell Sci 120 , 3772 – 3783 ( 2007 ). OpenUrl Abstract / FREE Full Text 73. ↵ Purves , D. , Snider , W. D. & Voyvodic , J. T. Trophic regulation of nerve cell morphology and innervation in the autonomic nervous system . Nature 336 , 123 – 128 ( 1988 ). OpenUrl CrossRef PubMed Web of Science 74. ↵ Laplante , M. & Sabatini , D. M. MTOR signaling in growth control and disease . Cell 149 , 274 – 293 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 75. ↵ Penuela , S. , Celetti , S. J. , Bhalla , R. , Shao , Q. & Laird , D. W. Diverse subcellular distribution profiles of pannexin1 and pannexin3 . Cell Commun Adhes 15 , 133 – 142 ( 2008 ). OpenUrl CrossRef PubMed 76. ↵ Lin , H. P. et al. Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs . Br J Pharmacol 175 , 3640 – 3655 ( 2018 ). OpenUrl CrossRef PubMed 77. ↵ Araki , N. , Johnson , M. T. & Swanson , J. A. A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages . Journal of Cell Biology ( 1996 ) doi: 10.1083/jcb.135.5.1249 . OpenUrl Abstract / FREE Full Text 78. ↵ Spagnol , G. , Sorgen , P. L. & Spray , D. C. Structural order in Pannexin 1 cytoplasmic domains . Channels 8 , 157 – 166 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 79. ↵ Wicki-Stordeur , L. E. et al. Pannexin 1 differentially affects neural precursor cell maintenance in the ventricular zone and Peri-Infarct cortex . Journal of Neuroscience 36 , 1203 – 1210 ( 2016 ). OpenUrl Abstract / FREE Full Text 80. ↵ Sanchez-Arias , J. C. , Candlish , R. C. , van der Slagt , E. & Swayne , L. A. Pannexin 1 Regulates Dendritic Protrusion Dynamics in Immature Cortical Neurons . eNeuro 7 , ( 2020 ). 81. ↵ Sanchez-Arias , J. C. et al. Pannexin 1 regulates network ensembles and dendritic spine development in cortical neurons . eNeuro 6 , ( 2019 ). 82. ↵ Penuela , S. et al. Loss of pannexin 1 attenuates melanoma progression by reversion to a melanocytic phenotype . Journal of Biological Chemistry 287 , 29184 – 29193 ( 2012 ). OpenUrl Abstract / FREE Full Text 83. Freeman , T. J. et al. Inhibition of pannexin 1 reduces the tumorigenic properties of human melanoma cells . Cancers (Basel) 11 , 102 ( 2019 ). OpenUrl PubMed 84. Furlow , P. W. et al. Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival . Nat Cell Biol 17 , 943 – 952 ( 2015 ). OpenUrl CrossRef PubMed 85. ↵ Di Virgilio , F. , Sarti , A. C. , Falzoni , S. , De Marchi , E. & Adinolfi , E. Extracellular ATP and P2 purinergic signalling in the tumour microenvironment . Nat Rev Cancer 18 , 601 – 618 ( 2018 ). OpenUrl CrossRef PubMed 86. ↵ Commisso , C. et al. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells . Nature 497 , 633 – 637 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 87. ↵ Lee , S. W. et al. EGFR-Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis . Dev Cell 50 , 381 – 392 ( 2019 ). OpenUrl CrossRef PubMed 88. ↵ Recouvreux , M. V. & Commisso , C. Macropinocytosis: A metabolic adaptation to nutrient stress in cancer . Front Endocrinol (Lausanne) 8 , 261 ( 2017 ). OpenUrl PubMed 89. Qian , Y. , Wang , X. , Li , Y. , Cao , Y. & Chen , X. Extracellular ATP a new player in cancer metabolism: NSCLC cells internalize ATP in vitro and in vivo using multiple endocytic mechanisms . Molecular Cancer Research 14 , 1087 – 1096 ( 2016 ). OpenUrl Abstract / FREE Full Text 90. ↵ Cao , Y. et al. Extracellular and macropinocytosis internalized ATP work together to induce epithelial-mesenchymal transition and other early metastatic activities in lung cancer . Cancer Cell Int 19 , ( 2019 ). 91. ↵ Forte , G. M. et al. Import of extracellular ATP in yeast and man modulates AMPK and TORC1 signalling . J Cell Sci 132 , ( 2019 ). 92. ↵ Stow , J. L. , Hung , Y. & Wall , A. A. Macropinocytosis: Insights from immunology and cancer . Curr Opin Cell Biol 65 , 131 – 140 ( 2020 ). OpenUrl CrossRef PubMed 93. ↵ Xu , G. , Zhang , Q. , Cheng , R. , Qu , J. & Li , W. Survival strategies of cancer cells: the role of macropinocytosis in nutrient acquisition, metabolic reprogramming, and therapeutic targeting . Autophagy vol. 21 693 – 718 Preprint at doi: 10.1080/15548627.2025.2452149 ( 2025 ). OpenUrl CrossRef PubMed 94. ↵ Locovei , S. , Bao , L. & Dahl , G. Pannexin 1 in erythrocytes: Function without a gap . Proc Natl Acad Sci U S A 103 , 7655 – 7659 ( 2006 ). OpenUrl Abstract / FREE Full Text 95. ↵ Freeman , S. A. et al. Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance . Science (1979) 367 , 301 – 305 ( 2020 ). OpenUrl Abstract / FREE Full Text 96. ↵ Boyce , A. K. J. Regulation of pannexin 1 trafficking by adenosine triphosphate . ( University of Victoria , 2017 ). 97. ↵ You , H. Investigating macropinocytosis as the mechanism of ATP-induced PANX1 internalization in Neuro2a cells . ( University of Victoria, Victoria , 2024 ). View the discussion thread. Back to top Previous Next Posted September 12, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism Andrew K.J. Boyce , Haifei You , Leigh E. Wicki-Stordeur , Leigh Anne Swayne bioRxiv 2025.09.11.675618; doi: https://doi.org/10.1101/2025.09.11.675618 Share This Article: Copy Citation Tools ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism Andrew K.J. Boyce , Haifei You , Leigh E. 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