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Biomimetic nanopore for a mechanistic study of the nuclear pore complex

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Border control at the nuclear envelope

Scientists have created an unprecedented in vitro paradigm to study the mechanisms of single-molecule transport in and out of the nucleus. Based on excellent knowledge of structures, researchers can now begin assigning specific functions.

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Eukaryotic cells have membrane-bound nuclei that compartmentalise and protect the genome. However, transport of molecules including proteins and RNA across the nuclear envelope is necessary for most cellular functions. This communication is regulated by large nuclear pore complexes (NPCs) of about 30 distinct protein sub-units called nucleoporins, or Nups. The pores allow free passage to small molecules, water and ions, but control movement of other larger molecules via nuclear transport receptors. Although the structure and biochemistry of the NPCs is relatively well-established, the mechanisms of selective transport are not. Until now, in vitro reconstruction of the pore was impossible, hindering detailed functional studies and measurements. EU-funded scientists changed all that with pioneering work on the project 'Biomimetic nanopore for a mechanistic study of the nuclear pore complex' (BIONANOPORE). Researchers created a solid-state NPC to which it is possible to covalently bond various Nups, test their functions and compare differences in transport of single molecules. Importin beta (Imp beta) is a known nuclear import receptor. Scientists developed an experimental paradigm that enabled electrical monitoring of the translocation of Imp beta with high temporal resolution. The sensitivity of the protocol allowed investigators to quantify subtle differences among Nups. BIONANOPORE's biomimetic NPC selectively transported Imp beta, but blocked transport of the irrelevant protein bovine serum albumin from cows commonly used in cell culture protocols. In addition, the paradigm revealed subtle differences between two Nups. Although both selectively transported Imp beta on similar timescales, one was more selective for the Imp beta than the other. This observation can be explained by the group's previous finding that the apparently less selective one creates a larger open channel. This could allow other similar molecules into the pore to bind and be transported. The groundbreaking experimental technique established by BIONANOPORE researchers opens the door to detailed mechanistic studies of NPCs and their many protein sub-units. Monitoring of single-molecule transport and differences in that due to covalently bonding different Nups to the biomimetic nanopore will spur rapid discovery. Understanding how these NPCs work will help identify their potential roles in disease processes and could point the way to novel targeted therapies.

Keywords

Nuclear envelope, in vitro, single-molecule transport, nuclear pore complexes, nucleoporins, biomimetic, biomimetic nanopore, mechanistic study, solid-state, Imp beta

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