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Body and mind

No more snapshots: now we can film cell processes

Before a sperm can fertilize an egg, a woman’s body needs to release a large volume of calcium all at once. This is just one of many cell processes in which transporting and regulating calcium are essential. Previously, researchers could only produce still images, but now they have released the first film of this vital calcium transport. This may improve the understanding and treatment of defects in the pump mechanism.

The last Nobel Prize a Danish researcher received was for a revolutionary discovery of how human cells create salt gradients across cell membranes. Jens Christian Skou was awarded the Prize in 1997 for discovering the enzyme that comprises the sodium-potassium pump in cell membranes. Although understanding of this vital pump mechanism has become more detailed, the final parts have remained unclear. A Danish research group has now published and shown the potential of a new imaging technique in studying these pumps in the prominent journal Nature.

“A huge number of molecules previously emitted signals, making it impossible to decipher the finer details of their functions, but we can now narrow the focus to one molecule and see it in action. This provides us with a video of the pumps in action with many fewer breaks in the filmstrip,” explains co-author Poul Nissen, Director of the PUMPkin Centre (Centre for Membrane Pumps in Cells and Disease) and the Danish Research Institute of Translational Neuroscience (DANDRITE).

A short video illustration of the pump’s cycle that shows how this transmits calcium ions out of the cell. The blue dots in the film represent calcium (Source: Nature Magazine)

No more pump fiction

The calcium pump is a close relative of the sodium-potassium pump, and the Danish research team succeeded in revealing how it functions. Each calcium pump is only a few nanometres – a few millionths of a millimetre – across, and they are in the membranes of all the cells in our bodies. The pump could only previously be illustrated in various stable individual states using X-ray crystallography.

“This is like stop-motion animation. Just for fun, we have called this ‘pump fiction’. Our spectroscopic technique [Förster resonance energy transfer] combines laser light and ultrasensitive cameras. This enables us to focus on an individual molecule and measure the tiny variations in fluorescent light and see what the molecules are doing here and now. We have therefore moved from ‘pump fiction’ to ‘pump live’.”

To film the calcium pump, the researchers positioned two dye molecules at two specific locations on the pump molecule, and the dye molecules move extensively in relation to each other when the pump functions. The researchers then illuminate one of the dye molecules, the donor, with laser light. The donor absorbs the light energy and emits it again as fluorescent light. Depending on the distance to the second dye molecule, some of the light energy absorbed is transferred to the second dye molecule, the acceptor, which then emits light in another colour. By measuring how much light the two dyes emit, the researchers can measure the distance between the donor and acceptor and therefore how the pump moves over time.

“The question we wanted to answer was how the pump managed to become unidirectional, solely pumping calcium out of the cell. We thought that this one-way pumping occurred when the energy-releasing adenosine triphosphate (ATP) molecule was split, but this proved not to be the case. Instead, we discovered a new state in the pump cycle, and the pump could only be in this state when a calcium ion came from within the cell. Once calcium escapes from this state, it is at the point of no return.”

The body’s engine room

Calcium pumps are responsible for actively transporting calcium out of cells. The concentration of calcium inside the cells is thus maintained at 10,000 times lower than the concentration outside. This massive difference means that the cells can rapidly increase their concentration of calcium by opening a channel to the surroundings.

“The pump is one reason why our muscles can contract and our nerve cells can send signals. If this tiny pump stopped functioning, cells would stop communicating and, without them, we would not be able to move or think. Cells therefore use a huge amount of energy to do this with the pumps using about one third of the body’s fuel, ATP.”

The calcium pump is therefore an essential molecular mechanism for humans, and this is the first time that researchers have been able to see inside this very fundamental engine room while it is functioning. Although the studies were carried out on Listeria monocytogenes bacteria, they add key knowledge to the understanding of the basic mechanisms of life processes in humans and the structural and mechanical principles of the calcium pump and can be used to combat disease.

“For example, if the pumps are not functioning, causing a small defect in brain cells, this can cause such diseases of the nervous system as migraine, one-sided paralysis and neurodegenerative disorders. Knowledge about ion pumps is therefore key to understanding the mechanisms of disease associated with defects in the pumps – especially to enable the long-term development of new drugs targeting the pumps.”

Dynamics of P-type ATPase transport revealed by single-molecule FRET” has been published in Nature. Poul Nissen, Professor, Department of Molecular Biology and Genetics, Aarhus University received the 2017 Novo Nordisk Prize. The Novo Nordisk Foundation awarded a grant in 2017 for his work on the structural determination of membrane proteins, ribosomes and enzyme complexes..

Poul Nissen
Professor
Poul Nissen has a background in crystallographic studies of translation factors and the ribosome. He became interested in P-type ATPases when he returned back to Denmark from his postdoctoral stay at Yale University where he worked on unraveling the structure and function of the ribosome in the laboratory of 2009 Nobel Prize laureate Thomas A. Steiz.