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.
Keywords: E. coli; NLS; capsid; cell biology; human; import kinetics; large cargo; molecular biophysics; nuclear transport; permeabilized cells; structural biology.
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.
© 2020, Paci et al.