Sticky Cells: Cyclic Mechanical Reinforcement Extends Longevity of Bonds Between Cells

Research carried out by scientists at the Georgia Institute of Technology and The University of Manchester has revealed new insights into how cells stick to each other and to other bodily structures, an essential function in the formation of tissue structures and organs. It’s thought that abnormalities in their ability to do so play an important role in a broad range of disorders, including cardiovascular disease and cancer.

The study’s findings are outlined in the journal Molecular Cell and describe a surprising new aspect of cell adhesion involving the family of cell adhesion molecules known as integrins, which are found on the surfaces of most cells. The research uncovered a phenomenon termed “cyclic mechanical reinforcement,” in which the length of time during which bonds exist is extended with repeated pulling and release between the integrins and ligands that are part of the extracellular matrix to which the cells attach.

Professor Martin Humphries, dean of the faculty of life sciences at the University of Manchester and one of the paper’s co-authors, says the study suggests some new capabilities for cells: “This paper identifies a new kind of bond that is strengthened by cyclical applications of force, and which appears to be mediated by complex shape changes in integrin receptors. The findings also shed light on a possible mechanism used by cells to sense extracellular topography and to aggregate information through ‘remembering’ multiple interaction events.”

The cyclic mechanical reinforcement allows force to prolong the lifetimes of bonds, demonstrating a mechanical regulation of receptor-ligand interactions and identifying a molecular mechanism for strengthening cell adhesion through cyclical forces.

“Many cell functions such as differentiation, growth and the expression of particular genes depend on cell interaction with the ligands of the intracellular matrix,” said Cheng Zhu, a professor in the Coulter Department of Biomedical Engineering at Georgia Tech and Emory University and the study’s corresponding author. “The cells respond to their environment, which includes many mechanical aspects. This study has extended our understanding of how connections are made and how mechanical forces regulate interactions.”

The research was published online by the journal on February 14th. The work was supported by the National Institutes of Health (NIH) and the Wellcome Trust.

Peering into living cells — without dye nor fluophore

In the world of microscopy, this advance is almost comparable to the leap from photography to live television. Two young EPFL researchers, Yann Cotte and Fatih Toy, have designed a device that combines holographic microscopy and computational image processing to observe living biological tissues at the nanoscale. Their research is being done under the supervision of Christian Depeursinge, head of the Microvision and Microdiagnostics Group in EPFL’s School of Engineering.

Using their setup, three-dimensional images of living cells can be obtained in just a few minutes – instantaneous operation is still in the works – at an incredibly precise resolution of less than 100 nanometers, 1000 times smaller than the diameter of a human hair. And because they’re able to do this without using contrast dyes or fluorescents, the experimental results don’t run the risk of being distorted by the presence of foreign substances.

Being able to capture a living cell from every angle like this lays the groundwork for a whole new field of investigation. “We can observe in real time the reaction of a cell that is subjected to any kind of stimulus,” explains Cotte. “This opens up all kinds of new opportunities, such as studying the effects of pharmaceutical substances at the scale of the individual cell, for example.”

Watching a neuron grow

This month in Nature Photonics the researchers demonstrate the potential of their method by developing, image by image, the film of a growing neuron and the birth of a synapse, caught over the course of an hour at a rate of one image per minute. This work, which was carried out in collaboration with the Neuroenergetics and cellular dynamics laboratory in EPFL’s Brain Mind Institute, directed by Pierre Magistretti, earned them an editorial in the prestigious journal. “Because we used a low-intensity laser, the influence of the light or heat on the cell is minimal,” continues Cotte. “Our technique thus allows us to observe a cell while still keeping it alive for a long period of time.”

As the laser scans the sample, numerous images extracted by holography are captured by a digital camera, assembled by a computer and “deconvoluted” in order to eliminate noise. To develop their algorithm, the young scientists designed and built a “calibration” system in the school’s clean rooms (CMI) using a thin layer of aluminum that they pierced with 70nm-diameter “nanoholes” spaced 70nm apart.

Finally, the assembled three-dimensional image of the cell, that looks as focused as a drawing in an encyclopedia, can be virtually “sliced” to expose its internal elements, such as the nucleus, genetic material and organelles.

Toy and Cotte, who have already obtained an EPFL Innogrant, have no intention of calling a halt to their research after such a promising beginning. In a company that’s in the process of being created and in collaboration with the startup Lyncée SA, they hope to develop a system that could deliver these kinds of observations in vivo, without the need for removing tissue, using portable devices. In parallel, they will continue to design laboratory material based on these principles. Even before its official launch, the start-up they’re creating has plenty of work to do - and plenty of ambition, as well.

'Zombie' replica cells may outperform live ones as catalysts and conductors

"Zombie" mammalian cells that may function better after they die have been created by researchers at Sandia National Laboratories and the University of New Mexico (UNM).

The simple technique coats a cell with a silica solution to form a near-perfect replica of its structure. The process may simplify a wide variety of commercial fabrication processes from the nano- to macroscale.

The work, reported in the Proceedings of the National Academy of Sciences (PNAS), uses the nanoscopic organelles and other tiny components of mammalian cells as fragile templates on which to deposit silica. The researchers then heat the cell to burn off its protein. The resultant hardened silica structures are faithful to the exterior and interior features of the formerly living cell, can survive greater pressures and temperatures than flesh ever could, and can perform some functions better than when they were alive, said lead researcher Bryan Kaehr, a Sandia materials scientist.

"It’s very challenging for researchers to build structures at the nanometer scale," said Kaehr. "We can make particles and wires, but 3-D arbitrary structures haven’t been achieved yet. With this technique, we don’t need to build those structures—nature does it for us. We only need to find cells that possess the machinery we want and copy it using our technique. And, using chemistry or surface patterning, we can program a group of cells to form whatever shape seems desirable."

UNM professor and Sandia Fellow Jeff Brinker added, “The process faithfully replicates features from the nanoscale to macroscale in a robust, three-dimensionally stable form that resists shrinkage even upon heating to over 500 degrees Centigrade [932 degrees Fahrenheit]. The refractoriness of these delicate structures is amazing.”

Listening to Cells: Scientists probe human cells with high-frequency sound

Sound waves are widely used in medical imaging, such as when doctors take an ultrasound of a developing fetus. Now scientists have developed a way to use sound to probe tissue on a much tinier scale. Researchers from the University of Bordeaux in France deployed high-frequency sound waves to test the stiffness and viscosity of the nuclei of individual human cells. The scientists predict that the probe could eventually help answer questions such as how cells adhere to medical implants and why healthy cells turn cancerous.

“We have developed a new non-contact, non-invasive tool to measure the mechanical properties of cells at the sub-cell scale,” says Bertrand Audoin, a professor in the mechanics laboratory at the University of Bordeaux. “This can be useful to follow cell activity or identify cell disease.” The work will be presented at the 57th Annual Meeting of the Biophysical Society (BPS), held Feb. 2-6, 2013, in Philadelphia, Pa.

The technique that the research team used, called picosecond ultrasonics, was initially applied in the electronics industry in the mid-1980s as a way to measure the thickness of semiconductor chip layers. Audoin and his colleagues, in collaboration with a research group in biomaterials led by Marie-Christine Durrieu from the Institute of Chemistry & Biology of Membranes & Nano-objects at Bordeaux University, adapted picosecond ultrasonics to study living cells. They grew cells on a metal plate and then flashed the cell-metal interface with an ultra-short laser pulse to generate high-frequency sound waves. Another laser measured how the sound pulse propagated through the cells, giving the scientists clues about the mechanical properties of the individual cell components.

“The higher the frequency of sound you create, the smaller the wavelength, which means the smaller the objects you can probe” says Audoin. “We use gigahertz waves, so we can probe objects on the order of a hundred nanometers.” For comparison, a cell’s nucleus is about 10,000 nanometers wide.

The team faced challenges in applying picosecond ultrasonics to study biological systems. One challenge was the fluid-like material properties of the cell. “The light scattering process we use to detect the mechanical properties of the cell is much weaker than for solids,” says Audoin. “We had to improve the signal to noise ratio without using a high-powered laser that would damage the cell.” The team also faced the challenge of natural cell variation. “If you probe silicon, you do it once and it’s finished,” says Audoin. “If you probe the nucleus you have to do it hundreds of times and look at the statistics.”

The team developed methods to overcome these challenges by testing their techniques on polymer capsules and plant cells before moving on to human cells. In the coming years the team envisions studying cancer cells with sound. “A cancerous tissue is stiffer than a healthy tissue,” notes Audoin. “If you can measure the rigidity of the cells while you provide different drugs, you can test if you are able to stop the cancer at the cell scale.”

(Photo: Image courtesy of UCSD Jacobs)

NEUROSPHERE

Four differently stained images featuring a neurosphere and primary culture cells derived form glioblastoma – stained by MGMT/FITC, GFAP y3 and DAPT.

Image Courtesy: Marek Molcanyi, Marina Penner, Sandrine Pacchnini, Clemens Reinshagen, John Ivan Bianco, University of Cologne, Germany

(Source: leica-microsystems.com)

Mapping the living cell

To get a clear picture of what’s happening inside a cell, scientists need to know the locations of thousands of proteins and other molecules. MIT chemists have now developed a technique that can tag all of the proteins in a particular region of a cell, allowing them to more accurately map those proteins.

“That’s a holy grail for biology — to be able to get spatially and temporally resolved molecular maps of living cells,” says Alice Ting, the Ellen Swallow Richards Associate Professor of Chemistry at MIT. “We’re still really far from that goal, but the overarching motivation is to get closer to that goal.”

Ting’s new method, developed with researchers from the Broad Institute and Harvard Medical School, combines the strengths of two existing techniques — microscopic imaging and mass spectrometry — to tag proteins in a specific cell location and generate a comprehensive list of all the proteins in that area.

In a paper appearing in the Jan. 31 online edition of Science, Ting and colleagues used the new technique to identify nearly 500 proteins located in the mitochondrial matrix — the innermost compartment of the cellular organelle where energy is generated.

Using fluorescence or electron microscopy, scientists can determine protein locations with high resolution, but only a handful of a cell’s approximately 20,000 proteins can be imaged at once. “It’s a bandwidth problem,” Ting says. “You certainly couldn’t image all the proteins in the proteome at once in a single cell, because there’s no way to spectrally separate that many channels of information.”

With mass spectrometry, which uses ionization to detect the mass and chemical structure of a compound, scientists can analyze a cell’s entire complement of proteins in a single experiment. However, the process requires dissolving the cell membrane to release a cell’s contents, which jumbles all of the proteins together. By purifying the mixture and extracting specific organelles, it is then possible to figure out which proteins were in those organelles, but the process is messy and often unreliable.

The new MIT approach tags proteins within living cells before mass spectrometry is done, allowing spatial information to be captured before the cell is broken apart. This information is then reconstructed during analysis by noting which proteins carry the location tag.

Glial cells assist in the repair of injured nerves

Unlike the brain and spinal cord, the peripheral nervous system has an astonishing capacity for regeneration following injury. Researchers at the Max Planck Institute of Experimental Medicine in Göttingen have discovered that, following nerve damage, peripheral glial cells produce the growth factor neuregulin1, which makes an important contribution to the regeneration of damaged nerves.

From their cell bodies to their terminals in muscle or skin, neuronal extensions or axons in the peripheral nervous system are surrounded along their entire length by glial cells. These cells, which are known as Schwann cells, envelop the axons with an insulating sheath called myelin, which enables the rapid transmission of electrical impulses. Following injury to a peripheral nerve, the damaged axons degenerate. After a few weeks, however, they regenerate and are then recovered with myelin by the Schwann cells. For thus far unexplained reasons, however, the Schwann cells do not manage to regenerate the myelin sheaths completely. Thus the function of damaged nerves often remains permanently impaired and certain muscles remain paralysed in affected patients.

In a current research study, the scientists have succeeded in showing that the growth factor neuregulin1 supports nerve repair and the redevelopment of the myelin layer. This protein is usually produced by neurons and is localised on axons where it acts as an important signal for the maturation of Schwann cells and myelin formation. Because the axons rapidly degenerate after injury, the remaining Schwann cells lose their contact with the axons. They thus lack the neuregulin1 signal of the nervous fibres. “In the phase following nerve damage, in which the axons are missing, the Schwann cells must carry out many tasks without the help of axonal signals. If the Schwann cells cannot overcome this first major obstacle in the aftermath of nerve injury, the nerve cannot be adequately repaired,” explains Ruth Stassart, one of the study authors.

To prevent this, the Schwann cells themselves take over the production of the actual neuronal signal molecule. After nerve damage, they synthesise the neuregulin1 protein until the axons have grown again. With the help of genetically modified mice, the researchers working on this study were able to show that the neuregulin1 produced in Schwann cells is necessary for the new maturation of the Schwann cells and the regeneration of the myelin sheath after injury. “In mice that lack the neuregulin1 gene in their Schwann cells, the already incomplete nerve regeneration process is extensively impaired,” explains co-author Robert Fledrich.

The researchers would now like to examine in greater detail how the Schwann cells contribute to the complete repair of myelinated axons after nerve damage, so that this information can also be used for therapeutic purposes.

Cell biologists show molecular forces are key to proper cell division

Studies led by assistant professor of Biology Thomas Maresca are revealing new details about a molecular surveillance system that helps detect and correct errors in cell division that can lead to cell death or human diseases. Findings are reported in the current issue of the Journal of Cell Biology.

The purpose of cell division is to evenly distribute the genome between two daughter cells. To achieve this, every chromosome must properly interact with a football-shaped structure called the spindle. However, interaction errors between the chromosomes and spindle during division are amazingly common, occurring in 86 to 90 percent of chromosomes, says Maresca, an expert in mitosis.

“This is not quite so surprising when you realize that every single one of the 46 chromosomes has to get into perfect position every time a cell divides,” he notes. The key to flawless cell division is to correct dangerous interactions before the cell splits in two.

Working with fruit fly tissue culture cells, Maresca and graduate students Stuart Cane and Anna Ye have developed a way to watch and record images of the key players in cell division including microtubule filaments that form the mitotic spindle and sites called kinetochores that mediate chromosome-microtubule interactions. They also examined the contribution of a force generated by molecular engines called the polar ejection force (PEF), which is thought to help line up the chromosomes in the middle of the spindle for division. For the first time, they directly tested and quantified how PEF, in particular, influences tension at kinetochores and affects error correction in mitosis.

“We also now have a powerful new assay to get at how this tension regulates kinetochore-microtubule interactions,” Maresca adds. “We knew forces and tension regulated this process, but we didn’t understand exactly how. With the new technique, we can start to dissect out how tension modulates error correction to repair the many erroneous attachment intermediates that form during division.”

Four-stranded ‘quadruple helix’ DNA structure proven to exist in human cells

In 1953, Cambridge researchers Watson and Crick published a paper describing the interweaving ‘double helix’ DNA structure – the chemical code for all life.

Now, in the year of that scientific landmark’s 60^th Anniversary, Cambridge researchers have published a paper proving that four-stranded ‘quadruple helix’ DNA structures – known as G-quadruplexes – also exist within the human genome. They form in regions of DNA that are rich in the building block guanine, usually abbreviated to ‘G’.

The findings mark the culmination of over 10 years investigation by scientists to show these complex structures in vivo – in living human cells – working from the hypothetical, through computational modelling to synthetic lab experiments and finally the identification in human cancer cells using fluorescent biomarkers.

The research, published in Nature Chemistry and funded by Cancer Research UK, goes on to show clear links between concentrations of four-stranded quadruplexes and the process of DNA replication, which is pivotal to cell division and production.