You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Molecular determinants of large cargo transport into the nucleus}
Article:
Abstract:
Nucleocytoplasmic transport is tightly regulated by the nuclear pore complex( NPC). Among the thousands of molecules that cross the NPC, even very large( >15 nm) cargoes such as pathogens, mRNAs and pre-ribosomes can pass the NPC intact. For these cargoes, there is little quantitative understanding of the requirements for their nuclear import, especially the role of multivalent binding to transport receptors via nuclear localisation sequences( NLSs) and the effect of size on import efficiency. Here, we assayed nuclear import kinetics of 30 large cargo models based on four capsid-like particles in the size range of 17–36 nm, with tuneable numbers of up to 240 NLSs. We show that the requirements for nuclear transport can be recapitulated by a simple two-parameter biophysical model that correlates the import flux with the energetics of large cargo transport through the NPC. Together, our results reveal key molecular determinants of large cargo import in cells.
Introduction:
Cargo transport across the nuclear envelope is a hallmark of eukaryotic cells and is central to cellular viability( Knockenhauer and Schwartz, 2016; Jamali et al., 2011; Fahrenkrog and Aebi, 2003). In a typical HeLa cell, more than 2000 nuclear pore complexes( NPCs) span the nuclear envelope( Ribbeck and Görlich, 2001; Maul et al., 1972). With ≈120 MDa in metazoans( Reichelt et al., 1990) and roughly half that weight in yeast( Rout and Blobel, 1993; Yang et al., 1998), the NPC is among the largest macromolecular complexes found inside the cell. NPCs are the gatekeepers of nucleocytoplasmic transport and restrict access of cargoes larger than the typically reported threshold of 40 kDa( Paine et al., 1975; Keminer and Peters, 1999; Mohr et al., 2009), albeit recent work points to a rather ‘soft’ barrier model and a gradual decrease of passive transport rates with size( Timney et al., 2016). However, cargoes that present a nuclear localisation sequence( NLS) and bind nuclear transport receptors( NTRs) can rapidly enter into the nucleus. Several studies have characterized the NTR-mediated transport process, typically focusing on cargoes with one to five NLSs, and their nuclear import kinetics have been shown to follow a mono-exponential behaviour( Ribbeck and Görlich, 2001; Kopito and Elbaum, 2007; Timney et al., 2006). NPCs are remarkable in the diversity of sizes of cargoes they can transport, ranging from import of nuclear proteins( including histones and transcription factors), to viral import and nuclear export of pre-ribosomal subunits and mRNA complexes( Panté and Kann, 2002; Grünwald and Singer, 2010; Grünwald et al., 2011; Babcock et al., 2004; Mor et al., 2010; Au and Panté, 2012). How very large cargoes( >15 nm) can be efficiently transported is still an enigma, especially considering the dimensions and structure of the transport conduit itself. The NPC is formed by multiple copies of about 30 proteins, two thirds of which are folded proteins that assemble the NPC scaffold. The recent improvements in electron tomography( ET), paired with X-ray crystallography, have greatly expanded our knowledge on the organisation of these folded components of the NPC( von Appen et al., 2015; Szymborska et al., 2013; Lin et al., 2016; Kosinski et al., 2016). This pore-like scaffold is filled with multiple copies of ≈10 different intrinsically disordered proteins, known as FG nucleoporins( FG Nups), which form the NPC permeability barrier. FG repeats have been estimated to be at concentrations in the mM range inside the NPC( Aramburu and Lemke, 2017; Frey and Görlich, 2007). Our structural knowledge about the actual transport conduit compared to the scaffold is much lower, as its dynamic nature leads to a loss of electron density in the averaging process inherent to ET, leaving a ≈40 nm wide ‘hole’ inside the structural map of the NPC tomogram. As the transport of many large cargoes is believed not to irreversibly alter the structure of the NPC, substantial amounts of FG Nups mass must be displaced in order to facilitate such transport events. In addition to dynamics in the permeability barrier, dilation mechanisms in the scaffold structure itself have also been suggested( Beck and Hurt, 2017). Despite its high biological relevance, nuclear transport of large cargoes is still poorly understood. In order to address this gap, we designed a set of large model cargoes based on capsid-derived structures. In contrast to using fully physiological large cargoes, such as complete viruses, this strategy enabled us to titrate key features such as size, number of binding sites and surface properties. This reductionist approach opened the possibility to experimentally measure a rigorous set of biophysical parameters. We used a combination of spectroscopy and semi-automated microscopy assays to investigate the kinetics of nuclear import of cargoes ranging from 17 to 36 nm in diameter and with a number of NLSs between 0 and 240 in permeabilised cells. Our results uncovered the quantitative dependence of cargo size and NLS number in an understudied size range. The results are rationalized using a minimal physical model of nuclear transport that takes into account the energy gain from NTR binding to FG motifs, and the free energy cost needed for the insertion of a large particle into a densely filled channel.
Discussion:
Our approach based on modified capsids with tuneable surface properties and quantitative imaging in permeabilised cells enabled us to arrive at a substantially enhanced quantitative understanding of large cargo transport through the NPC. Assaying nuclear import kinetics in an unprecedented cargo size and #NLSs range, we have shown that the requirements for transport scale non-linearly with size and can be recapitulated by a two-parameter biophysical model that correlates the import flux to the energetic requirements for nuclear transport. For small cargo transport, biochemical or physicochemical properties of the cargo surface have been shown to influence nuclear transport( Frey et al., 2018; Naim et al., 2009). While we do not claim that surface effects play no role in large cargo transport, based on the prediction from our experimental assay we would suggest that the binding of multiple Importin complexes seem to partially mask the cargo surface properties. Our work significantly expands the range of sizes and #NLSs for which nuclear import has been characterised: Tu et al., 2013 previously reported a single molecule study of a β-galactosidase cargo, which has four NLSs. This approximately cylindrical molecule is 18 nm at its longest axis, similar to MS2S37P, and 9 nm along its shorter axis. If the cargo crosses the NPC in a favourable orientation( through its narrow end), this would result in a lower cost of insertion and explain well why for this substrate 4 NLSs are sufficient for import( Tu et al., 2013). By comparison, our smallest cargo, MS2S37P, which is spherical with a 17 nm diameter, was not substantially imported below 10 NLSs within the timeframe of our assay. For the larger MS2, more than 30 NLSs were required to detect a clear signal. It is important to note that in addition to cargo shape( Mohr et al., 2009), its mechanical stability and rigidity are likely to play a role in nucleocytoplasmic transport: the import rate of proteins is inversely correlated with its mechanical stability( Infante et al., 2019), and flexibility is likely relevant for the transport of large deformable synthetic cargoes, such as polymer vesicles( Zelmer et al., 2020). While our simple biophysical model can explain the experimental data very well with only two fitting parameters per capsid( FR and ϵ) it also raises several interesting questions. The model provides quantitative estimates of the free energy cost of capsid insertion into the FG Nup assembly, as well as the effective binding energy needed to compensate for the insertion cost. Notably, despite the fact that a single MS2 capsid already occupies ≈1/3 of the estimated volume of the central NPC channel( Isgro and Schulten, 2005)( as illustrated in the cartoon in Figure 5) the free energy cost of insertion is relatively low( on the order of a few kBT’s), and is similar for the capsids of different sizes. This might indicate that further mechanisms facilitate large cargo transport, such as NPC scaffold dilation, a hypothesis supported also by multiple evidences for tentative hinge elements in the NPC scaffold structures( Bui et al., 2013; Kelley et al., 2015), or bistability in the FG density in the radial direction induced by such extremely large cargo( Osmanović et al., 2013). In terms of the effective interaction energy ϵ, the largest MS2 capsid required a fit with the lowest effective ϵ. This finding is surprising at first glance, because one would expect that the main difference between the capsids would stem from their size difference, while the interaction energy of an NTR with an FG motif stays relatively constant. One can think of several potential origins for this effect, among those are the lack of independence in the NTR binding of the FG repeats in case of large surface coverage, or the loss of accessibility of the FG motifs due to the high compression of the FG assembly by the largest capsid, which will be explored in future work. Nevertheless, we found that all these features can be explained in a minimal model that incorporates the potential heterogeneity of the FG Nup distribution along the NPC axis, whereby there are at least two different regions of different FG Nup densities, as has been previously suggested in a 'two gate' or 'vestibule' pictures of the NPC( Tu et al., 2013; Lowe et al., 2015; Yamada et al., 2010). In Figure 5—figure supplement 3, we show that such a spatially heterogeneous model would be consistent with the data across all three capsid data sets, without invoking different effective interaction energies for the different capsids. In our model, the energetic terms represent the binding between FG repeats and NTRs. The microscopic binding mechanism between NTRs and FG repeats during NPC passage is probably similar both for import and export, with a few exceptions- such as the binding of the export factor Crm1 to a specific stretch of Nup214,( which has likely a larger role in undocking than permeability barrier passage)( Port et al., 2015; Tan et al., 2018). We thus anticipate that basic principles of our work could also help in the future to better understand export of large cargoes, such as pre-ribosomal subunits and large RNA export complexes. The theoretical model used in this paper implicitly assumes that the capsids do not interact with each other during transport through the pore. We cannot exclude multiple capsids colliding with each other in a single pore with absolute certainty- and this has indeed been observed in EM of HBV capsids injected in Xenopus oocytes( Panté and Kann, 2002). However, the hallmark of jamming resulting from multi-particle occupancy is the non-monotonic dependence of the flux on the interaction strength and thus on the #NLS on the capsid( Zilman, 2009; Pagliara et al., 2013)- a trend that is currently not apparent in our data within the experimental accuracy, at least in the initial rate, which is the focus of our analysis. A more complete picture of nuclear transport and refined model building in the future would require taking into account additional features in more detail, such as docking and undocking from the barrier, more realistic modelling of the capsid cargo passage through the pore, and complex entropic effects of capsid-FG Nup interactions. Future studies using our cargo substrates and time resolved high-resolution measurements could provide further insights into the individual kinetic steps of NPC binding, barrier passage and undocking and how those link to FG Nup and potentially scaffold dynamics in the NPC.
Eukaryotes , such as animals , plants and fungi , store the genetic material within their cells inside a specific compartment called the nucleus . Surrounding the nucleus is a protective membrane which molecules must pass across in order to reach the cell’s DNA . Straddling the membrane are nuclear pore complexes , or NPCs for short , which act as the gatekeepers to the nucleus , shuttling thousands of different molecules back and forth whilst restricting access to others . Large cargoes need to have specific markers on their surface called nuclear localization signals in order to be transported by NPCs . Certain transporter proteins help the NPC carry large molecules across the membrane by binding to these signals . This generates the energy needed to overcome the barrier of transporting it across the membrane . Some viruses have nuclear localization signals of their own , which can exploit this transport system; these signals allow the virus to enter the nucleus and hijack the genetic machinery of the cell . It has been suggested that viruses have multiple copies of these surface signals to improve their chances of reaching the nucleus . However , it remained unclear how the number of nuclear localization signals affects the transport of large molecules into the nucleus . To answer this question , Paci et al . engineered a range of different sized particles derived from viral structures which had varying numbers of nuclear localization signals on their surface . These particles were inserted into human cell lines grown in the laboratory , and imaged to see how they were transported into the nucleus . The rate of nuclear transport was then measured for each particle , and this data was used to create a mathematical model . Paci et al . found that the larger the cargo , the more nuclear localization signals it needed to be efficiently transported across the membrane into the nucleus . This is because inserting big cargoes into the NPC requires more energy . Therefore , by increasing the number of surface signals transporter proteins can bind to , larger molecules are able to interact with the NPC and generate the energy required for crossing . These findings improve our current understanding of how nuclear transport could be hijacked by viruses . It could also help scientists who are developing targeted nanoparticles to deliver therapies for genetic conditions to the nucleus .