Nuclear Pore Transport: Toward an Integrated Perspective

Nuclear Pore Transport: Toward an Integrated Perspective | 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 Nuclear Pore Transport: Toward an Integrated Perspective Luca Lanzano , Francesco Cardarelli doi: https://doi.org/10.1101/2025.09.18.677018 Luca Lanzano a Department of Physics and Astronomy “Ettore Majorana”, University of Catania , Catania, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Francesco Cardarelli b NEST Laboratory - Scuola Normale Superiore , Piazza San Silvestro 12, Pisa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: francesco.cardarelli{at}sns.it Abstract Full Text Info/History Metrics Preview PDF Abstract Nuclear pore complexes (NPCs) mediate the selective exchange of proteins and RNAs between nucleus and cytoplasm. Despite decades of study, the molecular mechanism of transport remains debated, particularly the role of FG-nucleoporin dynamics in facilitating translocation. Here, we compare high-resolution single-molecule trajectories obtained with MINFLUX microscopy with earlier correlation-based measurements, revealing that actual pore-crossing events are typically completed within 2-4 localization steps, corresponding to 2-6 ms depending on localization rates. These values closely match transit times obtained from fluorescence fluctuation analysis and align with models in which cargo binding induces transient FG-nucleoporin collapse. Together with recent independent observations, this synthesis supports a converging view of NPC transport as a fast, directional, FG-assisted process. We propose that integrating fluctuation-based and trajectory-based approaches provides a robust framework for reconciling mechanistic hypotheses and refining our understanding of selective gating. Introduction: the unresolved question of nuclear transport dynamics Nucleocytoplasmic transport is essential for eukaryotic life, mediating the bidirectional exchange of proteins and RNA through nuclear pore complexes (NPCs)—massive assemblies embedded in the nuclear envelope that maintain the selectivity barrier between nucleus and cytoplasm 1 , 2 . Despite decades of research, the mechanism enabling fast and selective transport of cargoes remains largely unresolved. The debate centers on a single overarching question concerning the physical nature of the permeability barrier—do translocating molecules cross the NPC purely by diffusion, or rather via transient, directional interactions with FG-nucleoporins (FG-Nups)? These two translocation mechanisms correspond to two major experimental models that propose distinct structural organizations of FG-Nups within the NPC. The first envisions permeation as a diffusive process: FG-repeat domains assemble into a cohesive, gel-like mesh that acts as an entropic sieve, allowing receptor-bound cargos to pass while excluding inert macromolecules 3 . This idea was refined in the selective phase model, which proposed that FG domains organize into a polymeric network forming a thermodynamic barrier against unspecific passage, while still enabling passive, diffusion-driven translocation of cargos 4 , 5 . By contrast, a second model posits a directed mode of transport. In this framework, the FG-Nups within the permeability barrier undergo local rearrangements upon receptor binding and transiently collapse to create directional conduits that guide cargos across the pore 6 , 7 . Experimental support for this mechanism comes from in vitro observations—including AFM and EM evidence of receptor-induced FG condensation 6 , 7 —as well as from live-cell fluorescence correlation spectroscopy (FCS), which detected rapid and directional transport signatures at the single-pore level 8 , 9 . Over the past two decades, single-particle tracking (SPT) has provided foundational insights into nucleocytoplasmic transport. Early live-cell SPT studies, including those by Grünwald, Musser, and colleagues, demonstrated that individual cargos could be followed as they approached and entered the NPC, providing dwell time distributions and revealing heterogeneous transport behavior 10 - 15 . These experiments firmly established that transport could be studied at the single-molecule level in intact cells, even if spatial precision and temporal resolution were not yet sufficient to unambiguously resolve the millisecond dynamics of barrier crossing. The challenge, however, was not methodological but intrinsic: capturing the fast, confined motion within the ~50-nm central channel. Recent breakthroughs in single-molecule imaging now allow the field to revisit this question. Most notably, MINFLUX nanoscopy now enables the tracking of individual cargos traversing NPCs in intact cells with nanometer spatial precision and millisecond temporal resolution 16 , offering an unprecedented opportunity to directly confront existing models with observation. The renewed debate: insights from MINFLUX and complementary studies In a recent Nature study, Sau et al. applied 3D MINFLUX microscopy to visualize nuclear transport in living cells, achieving unmatched spatial and temporal accuracy 16 . Their trajectories reveal that import and export occur along overlapping annular pathways within the NPC and that cargos remain within a region defined as −25 to +25 nm from the midplane for an average of ~14 ms. These findings represent a technical milestone, adding valuable geometric detail to the long-standing question of transport routes. However, whether this “residence time” corresponds to the actual translocation step is uncertain. Examination of the authors’ published trajectories and videos suggests that the true crossing—from one side of the pore to the other across the central plane—is completed in far fewer steps than implied by the full ±25 nm range. Rather than a prolonged random walk, the transition appears as a short, directional burst embedded within a broader exploratory phase. Independent evidence for directed motion has recently emerged from cargo-centric tracking approaches. Li et al., in a 2024 Nature Physics article, reported that nuclear transport receptors exhibit anisotropic, non-random trajectories during NPC crossing 17 . Their results strongly support the concept of biased progression along defined paths rather than unrestricted Brownian diffusion. Taken together, these studies underscore that the NPC does not behave as a passive channel but as a dynamic structure that constrains and facilitates passage once productive interactions are established. Revisiting early evidence: fluctuation analysis of NPC transport More than a decade ago, we introduced a method that combined orbital tracking of individual NPCs with fluorescence fluctuation and cross-correlation analysis to probe molecular dynamics without reconstructing trajectories 8 , 9 . This approach exploited intensity fluctuations along a nanometric orbit encircling a single pore and identified directed transport statistically, using pair-correlation of signals recorded on opposite sides of the channel. This analysis yielded two key insights. First, transit times derived from multiple events showed sharply peaked distributions centered at 3–5 ms, implying a narrowly defined kinetic step for barrier crossing. Second, cross-correlation between cargo and Nup153 revealed synchronous fluctuations, suggesting transient co-movement, compatible with receptor-induced rearrangements of FG domains. These findings supported the mechanistic model proposed by Lim et al. 6 , 7 , in which binding of transport receptors induces FG domain collapse, generating a directional bias that facilitates selective transport. Although complementary to earlier SPT studies, our approach was distinctive in detecting transport signatures statistically—at 1-ms temporal resolution—without trajectory reconstruction. This latter difference also explains why some skepticism remained: correlation analysis, while powerful, does not provide direct visualization of individual trajectories. The advent of MINFLUX offers the opportunity to validate, at the single-trajectory level, the mechanistic insights previously inferred from fluctuation analysis. Re-analysis of MINFLUX trajectories: fast and directional crossing To test whether MINFLUX data align with correlation-based predictions, we re-examined representative import and export trajectories from Sau et al.’s supplementary movies. For each, we isolated the segment corresponding to true translocation—defined as the crossing of the 0-nm plane from one side of the NPC to the other—and mapped these localizations onto the observation geometry employed in our earlier orbital tracking experiments ( Figures 1 and 2 ). This alignment enables direct comparison between correlation-based inference and high-resolution imaging. Download figure Open in new tab Figure 1| Single import trajectory overlaid with orbital tracking geometry. Representative import trajectory extracted from Supplementary Movie 1 of Sau et al. (2025), displayed across selected frames (a–f). The nanometric observation geometry used in Cardarelli et al. (2012) is superimposed to illustrate the region probed by pair-correlation. Gray discs indicate point spread functions corresponding to correlation sites. White dashed lines mark ±25 nm boundaries from the pore center (0 nm, red). Four consecutive localizations (green to red arrows) span the crossing event. Assuming localization intervals of 0.52–1.6 ms, this corresponds to a transit time of ~2–6 ms, matching correlation-based estimates. Download figure Open in new tab Figure 2| Single export trajectory overlaid with orbital tracking geometry. As in Figure 1 , but for an export event extracted from Supplementary Movie 2 of Sau et al. (2025). The translocation segment comprises only two consecutive steps, yielding an estimated crossing time of ~1–3 ms. These values reinforce the convergence between MINFLUX tracking and fluctuation-derived kinetics. The analysis highlights two consistent features. First, the active crossing phase typically involves only 2–4 consecutive localizations, far fewer than suggested by the broader ±25 nm interval. Second, applying Sau et al.’s reported localization intervals (0.52 ms minimum, 1.6 ms average) yields estimated crossing durations of ~2–6 ms for import and ~1–3 ms for export— values that fall squarely within the distributions we reported over a decade ago 8 , 9 . Figures 1 and 2 illustrate these findings, showing how the mechanistic signal extracted from fluctuation analysis corresponds to a brief, directional event now directly observable with MINFLUX. Toward a unified mechanistic framework The convergence of fluctuation spectroscopy, MINFLUX imaging, and cargo-centric analysis strengthens the case for a model in which FG-Nups act as dynamic, receptor-responsive polymers. This view is consistent with nanomechanical data showing receptor-induced FG condensation and with structural models proposing reversible collapse as a gating principle 6 , 7 , 11 . Directional bias may emerge from sequential stabilization of receptor– FG contacts, enabling “hand-over-hand” translocation that reconciles selectivity with high speed. These considerations help redefine the priorities for experimental design. Future studies should integrate high-speed, multi-color tracking with probes reporting FG-Nup conformation, alongside statistical approaches capable of resolving microsecond fluctuations. Such integrative strategies will be essential to capture the interplay of molecular recognition, polymer dynamics, and confinement that underpins the NPC’s extraordinary performance. More than a decade ago, correlation analysis suggested that NPC transport is fast, directional, and FG-mediated 8 , 9 . Today, MINFLUX and complementary approaches provide direct support for this interpretation 16 , 17 . Far from being contradictory, these methods converge on a coherent picture: the NPC is not a static filter but a responsive molecular machine. The challenge ahead lies in integrating these insights into a comprehensive, experimentally grounded model of selective gating. Conflict of interest The authors declare no competing interests Funding This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 866127, project CAPTUR3D). Acknowledgments We are deeply grateful to Enrico Gratton, whose mentorship and vision shaped the foundations of this work. Funder Information Declared European Research Council, https://ror.org/0472cxd90 , 866127 References 1. ↵ Weis K ( 2003 ). Regulating access to the genome: Nucleocytoplasmic transport throughout the cell cycle . Cell 112 : 441 – 451 . 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Yang W , Musser SM ( 2006 ). Nuclear import time and transport efficiency depend on importin β concentration . J Cell Biol 174 : 951 – 961 . OpenUrl Abstract / FREE Full Text 13. Dange T , Grunwald D , Grunwald A , Peters R , Kubitscheck U ( 2008 ). Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study . J Cell Biol 183 : 77 – 86 . OpenUrl Abstract / FREE Full Text 14. Grünwald D , Singer RH ( 2010 ). In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport . Nature 467 : 604 – 607 . OpenUrl CrossRef PubMed Web of Science 15. ↵ Ma J , et al. ( 2013 ). High-resolution three-dimensional mapping of mRNA export through the nuclear pore . Nat Commun 4 : 2414 . OpenUrl CrossRef PubMed 16. ↵ Sau A , et al. ( 2025 ). Overlapping nuclear import and export paths unveiled by two-colour MINFLUX . Nature 640 : 821 – 827 . OpenUrl PubMed 17. ↵ Panagaki F , Tapia-Rojo R , Zhu T , et al. ( 2024 ). Structural anisotropy results in mechano-directional transport of proteins across nuclear pores . Nat Phys 20 : 1180 – 1193 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 19, 2025. Download PDF 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 Nuclear Pore Transport: Toward an Integrated Perspective 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. 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