Researchers at Mainz University have measured protein dynamics within the nanosized nuclear pore complex of a living cell / Publication in Nature

Our genetic material is hidden inside the nucleus of our cells, well protected by a double membrane. All molecules wishing to pass through must thread their way through one of several thousand nuclear pores. The scaffold structure of these pores is well known. However, exactly how transport takes place within them has been the subject of much debate. Researchers at Johannes Gutenberg University Mainz (JGU), the Max Planck Institute for Biophysics (MPI-BP) in Frankfurt am Main and Wuhan University in China have now shed light on the matter. “For the first time, we have been able to measure intrinsic protein dynamics within the nanocanal of a living cell and demonstrate that the interior of this canal is fluid-like,” said Professor Edward Lemke, Professor of Synthetic Biophysics at JGU, who led the current study together with Professor Gerhard Hummer from the MPI-BP. The researchers at the MPI-BP, in turn, have succeeded in creating the most comprehensive simulation model of a nuclear pore to date, based on the findings of Lemke and his team. In particular, this model answers the question of the transport pathway through the pore. “These complex calculations were made possible by JUPITER, Europe’s first exascale supercomputer at the Jülich Research Centre,” said Hummer. The researchers published their findings today in the journal Nature. “As the nuclear pore protects the genome and controls all exchange between the cell nucleus and the cytoplasm, it is one of the most important molecular machines in the human cell,” said Professor Miao Yu from Wuhan University, co-first author of the study. “And also one of the most complex,” added co-first author Maziar Heidari from the MPI-BP.

Fluorescent dyes as probes

Anyone travelling to a town in the Middle Ages first had to pass through one of the town gates – and might well have been turned away there. The situation at the membrane, which protectively surrounds the interior of human cell nuclei containing the valuable genetic information, is comparable in a certain sense. If signalling molecules or pathogens wish to pass through, the only route is via the nuclear pores, each of which controls tens of thousands of visitors per minute. Whilst certain molecules are specifically permitted to pass, others are held back. Essential to this selection process is a barrier of proteins that fills the opening – that much was already known. However, opinions differed widely regarding the exact nature of this barrier: studies on isolated proteins yielded varying results, ranging from stretched polymers to liquid-like condensates and even solid-like gels with a consistency similar to that of gummy bears. “Such studies did not, however, allow any direct conclusions to be drawn about how the barrier behaves within the living cell,” said Lemke. How dynamic are these protein chains anchored within the pore in their natural environment? Do they act more like a rigid comb, or are they soft and flexible like yoghurt or a liquid? And to what extent do their dynamics contribute to selective transport? Such questions have remained unanswered until now.

To find answers, Lemke and his team specifically coupled fluorescent dyes to the proteins in the nuclear pores as probes: tiny glowing dots whose mobility reveals a great deal about the dynamics of the proteins. “As the dyes move extremely quickly, the state inside the nuclear pores must be fluid-like,” said Lemke.

What keeps the barrier fluid

In living cells, the proteins remained highly mobile throughout the entire two-hour observation period. The team compared these results with an in vitro model, i.e. with isolated proteins in a test tube: There, however, after about two hours, the protein mass invariably turned into a rigid ‘gummy bear’ or even a solid resembling the deposits found in the brains of people with neurodegenerative diseases such as Alzheimer’s or Parkinson’s. “This was a crucial turning point: there had to be something keeping the barrier in the cells dynamic,” said Lemke. The solution to the puzzle: on the one hand, a transport receptor is required; on the other, the barrier proteins must be glycosylated – that is, modified with sugar groups – so that they do not stick together too tightly. “When we add these two components, we observe a similar mobility in the model system to that found in the nuclear pores of living cells,” said Lemke.

Transport primarily via the edge of the pore

Another unresolved question concerned the transport pathway. Whilst the flow velocity in pipes or rivers, for example, is higher in the center than at the edges, earlier studies suggested that the opposite is true in nuclear pores. The scientific community’s hypothesis was that there must be a biological reason for this – such as a plug in the centre of the pore. The researchers led by Hummer carried out simulations to investigate this. They based their work on the degree of cross-linking that the Mainz team had determined in their experiments. “The simulations confirm that the main transport route runs along the edge of the pore,” said Hummer. “Whilst we do not rule out a biological cause such as a plug, it is not necessary from a physicochemical point of view.” The findings are not only important for a fundamental understanding of how human cells function, but also help to shed light on neurodegenerative processes – after all, these also involve the hardening of previously flexible proteins.