The cell: dissecting the new anatomy
Understanding how the body’s cells work and communicate is central to our understanding of disease and has been a major focus of research at King’s for several decades. In 1946 John Randall established the Biophysics Unit, the venue for the seminal works of Jean Hanson and Maurice Wilkins.
From the outset, scientists working in the unit sought and developed technologies based on physics to study biological problems; an aim that continues today. Jean Hanson (1919-1973) was one of the unit’s first pioneers. She used the newly developed electron microscope to describe the sliding motion of muscle filaments, and later became King’s first female Fellow of the Royal Society. Since then, King’s has maintained its position at the forefront of cell biology research, applying a range of advanced techniques to study complex cellular functions and to shed light on disease mechanisms.
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Randall and the Biophysics Unit at King’s
Biophysics began at King’s in 1946 when Sir John Randall became Wheatstone Professor of Physics and founding director of the newly established MRC Biophysics Research Unit. He developed a wide-ranging research programme that involved physicists, biologists and biochemists and employed state-of-the-art equipment, such as the electron microscope. New types of light microscopes were being employed to study muscle contraction and at the same time X-ray diffraction studies were developed to study the 3D structure of molecules including the double helix model of DNA.
Before Randall’s move to King’s he worked at St Andrew’s University with the physicist Maurice Wilkins (1916-2004), and when Randall started the Biophysics Research Unit, Wilkins joined him. Initially, Wilkins developed reflecting microscopes to study DNA within cells and viruses and measured the dry mass of cells using interference microscopes. He then went on to his X-ray diffraction studies of DNA and sperm heads for which he received a Nobel prize in 1962.
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King’s strength in muscle
Jean Hanson joined the Biophysics Research Unit in 1948 to study the structure of muscle and how it contracts. She was the first person to isolate myofibrils from rabbit skeletal muscle and make them contract in vitro in physiological salt solutions. This development marked the beginning of a new era of studies that allowed scientists to investigate the structural and biochemical properties of assemblies of molecules under controllable conditions.
In the early 1950s, Hanson went to the Massachusetts Institute of Technology, USA, to learn how to use an electron microscope and it was there that, with HE Huxley, she made the seminal description of the sliding filament mechanism of muscle contraction. Hanson and Huxley had realized that it was overlapping arrays of actin and myosin filaments which were responsible for the characteristic appearance of striated muscle and which slide past one another during contraction. When Hanson returned to the Biophysics Unit she went on to demonstrate the existence of the double filament mechanism in a wide variety of muscle types and molecular aspects of contraction. Hanson became Professor of Biology in 1966 and the first woman from the College to become a Fellow of the Royal Society.
On Randall’s retirement, Wilkins became Professor of Biophysics. The position was later held by Robert Simmons (b 1938) who, like Hanson, has contributed significantly to our understanding of muscle contraction. With AF Huxley he developed methods to measure muscle mechanics. During the 1990s, whilst on sabbatical at Stanford University, USA, he developed a laser apparatus that allowed activity of the muscle protein myosin to be measured accurately.
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Early models of cell membranes
During the time that Randall’s unit was breaking new ground in understanding muscle contraction and the cell’s inner workings, James Frederic Danielli (1911-1984) became Professor of Zoology at King’s. He had begun working on the structure of the cell membrane in the early 1930s and developed new methods for measuring the cell’s chemical components. Together with Hugh Davson, Danielli proposed the first generally accepted model of the cell membrane in 1935. They described cellular membranes as a sandwich of lipids covered on both sides with proteins. They later realised there were ‘active patches’ and protein-lined pores within the membranes and these could help move certain chemicals across the membrane. Danielli’s membrane model was later modified to the ‘fluid-mosaic’ model that proposes that the proteins are globular in arrangement and float within the lipid bilayer.
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On the move – cell shape and motility
There are many protein receptors integrated into the cell’s outer membrane that allow it to detect chemoattractant molecules around them and facilitate migration. Cell migration is a vital process in normal human development, wound healing and inflammatory immune responses. When the cell receives an attractive signal, receptors and energy releasing proteins become activated, and actin filaments are polymerized (a process regulated by a series of proteins) and this provides the force for the plasma membrane to change shape and for the cell to move.
Understanding this complex series of events will give insight into cancer metastasis, developmental defects, and defective healing. An example of this area of research at King’s is the finding that the major actin-nucleating protein in white blood cells is absolutely required for movement of macrophages and dendritic cells. Lack of this protein causes the immune deficiency disease Wiskott-Aldrich syndrome. Because their leukocytes cannot respond normally to chemoattractants, a normal defence mechanism, patients suffer repeated severe infections.
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The cytoskeleton – the cell’s scaffolding
The cytoskeleton is a protein network that provides strength and resilience for cell membranes and plays an active role in the passage of protein complexes to reach the surface of the cell. Scientists at King’s are studying the role of the cytoskeleton in cell movement and function. They use cutting edge imaging technology to study the locomotion of living cells and whilst studying the movement of malignant cells have found that signals passing between cells are critical for their movement.
Muscle cells of the heart are subjected to continuous mechanical stress. Their activity is controlled by calcium fluxes at the cell surface via protein complexes and as the cell beats these complexes must withstand the movement. The cytoskeleton plays a central role in this, the cytoskeletal proteins linking to the calcium handling apparatus as well as the contractile elements of the cell. Several proteins are involved and it is believed that defective forms of these proteins can lead to cardiomyopathy.
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Unravelling the complexities of muscle
Muscle confers on animals one of their prime characteristics – mobility. Understanding the basis of muscle contraction and development is not only a challenge to physiologists, biochemists, physicists and geneticists but will also throw light on muscle diseases such as muscular dystrophy and cardiomyopathy. King’s continues to use biophysical techniques to investigate mechanisms of muscle contraction and its regulation. The motor protein myosin that drives muscle contraction is studied at both the isolated cell and single molecule levels. Optical tweezers are employed to observe directly the interaction of a single molecule of myosin with actin, showing that myosin ‘walks’ with two molecular heads along the actin monomer.
In collaboration with the National Institute for Medical Research, researchers have developed a method for real-time measurements of the orientation of protein domains in cells. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is being used to study the bending of the myosin molecule during contraction.
Another approach to understanding muscle is to study its embryonic development, and its abilities for growth and repair. In the mouse, two genes which regulate muscle and cartilage development (Meox1 and Meox2) have been identified. The next stage will be to identify the signals regulating these genes. Using the zebrafish as a model, work is in progress to understand how the basic programme for muscle development is modified to make individual muscles differ in size and contraction rate, and also how muscle repairs itself. With this knowledge the model can be used to test interventions to modify these functions and lead on to possible treatments for muscle disease.
An important area of research at King’s is the mechanisms that organise the smallest contractile unit of striated muscle, the sarcomere, and how these crosstalk to mechanisms controlling muscle growth. Scientists at King’s, in collaboration with the European Molecular Biology Group, have recently worked out how two muscle proteins – titin and telethonin – bond together. Titin is the largest of all human proteins and functions as a signalling molecule for the sarcomere. It bounces around rather like a bungee rope with hooks, making specific attachments to other proteins, including telethonin. Mutations in the titin gene can cause weakness of skeletal or cardiac muscle with serious consequences such as respiratory failure or heart failure.
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Taking an imaging approach
Visualizing biological processes such as protein interactions between live cells in disease may give new ways of monitoring and interfering with these biological processes. Interacting molecules can exchange energy and this can be visualized using fluorescence and microscopy. New techniques for deep tissue imaging are being developed to provide 3D images of molecular events in thick biological tissue samples and small organisms.
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Embryonic development and stem cells
When an egg is fertilized, it is programmed to divide and the cells begin to differentiate into different cell types – a process called pattern formation. This is a closely regulated process that takes place in all animal embryos and is being studied by scientists at King’s using the amphibian Xenopus.
Early stage embryos are also the source of embryonic stem cells and these have great therapeutic potential. The Stem Cell Laboratory at King’s wasthe first in the UK to successfully derive human embryonic stem cells (hES) and to deposit them in the UK stem cell bank. One cell line has been derived from an embryo screened for a genetic mutation that accounts for 70 per cent of cystic fibrosis cases. This will be an important tool for studying the pathophysiology of this disease. The group also aims to produce therapeutically important somatic stem cell populations from hES cells, and is exploring the possibility of stem cell therapies for a range of disorders including Parkinson’s, diabetes, and spinal cord damage.
The therapeutic potential of fetal and adult stem cells is also being studied. Scientists at King’s have cultured fetal neural stem cells in large numbers in the laboratory and transferred them into the brains of mice who have suffered stroke. The cells allowed the mice to recover completely. Researchers are also using imaging technologies to monitor the passage of the injected cells within the body.
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Teasing out the detail of muscle contraction
King’s scientists are using an apparatus with laser beams known as ‘optical tweezers’ to measure the activity of the muscle protein myosin. The apparatus was developed by Robert Simmons and Steve Chu, one of the inventors of the tweezers, whilst Simmons was on sabbatical from King’s at Stanford University in the US during the 1990s.
The tweezers work by moving a focused laser beam. If the object under study, such as a bead, moves away, the laser light is deflected and the particle is pushed in the opposite direction, back towards the focus. If the laser is moved the object moves, as with conventional tweezers. The tweezers can be set to alter their ‘stiffness’ so that the amount of force required to restore the object to the focus of the beam can be measured. The forces are similar to those exerted by the molecular motors that power muscle and actively transport material within cells.
Simmons studied movement of actin-coated beads on myosin-coated slides using the optical tweezers and found that actin-coated beads move in iscrete steps of about 10nm. The technology is now being used to understand further the intricacies of the interactions between actin and myosin during muscle contraction.
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