cells – Lightsources.org

Understanding how motor proteins shape our cells

2022/08/092022/08/11

Understanding the busy networks inside our cells can help researchers develop new cancer treatments and prevent dangerous fungal infections.

With the help of the Canadian Light Source (CLS) at the University of Saskatchewan, a research team led by John Allingham from Queen’s University and Hernando Sosa from the Albert Einstein College of Medicine has shed light on a protein that regulates the intricate microscopic networks that give cells their shape and helps ship important molecules to diverse locations.

Using the CMCF beamline at the CLS and the cryo-EM facility at the Simons Electron Microscopy Center (SEMC) at the New York Structural Biology Center, the team found the missing pieces of an important puzzle.

In their published work, they are the first group to clearly describe the mechanism of action of a tiny motor protein called Kinesin-8 that enables it to control the structures of microtubule fiber networks inside the cell.

Read more on the CLS website

Image: Cells, Canadian Light Source.

Safely studying dangerous infections just got a lot easier

2022/02/252022/03/17

An extremely fast new 3D imaging method can show how cells respond to infection and to possible treatments

To combat a pandemic, science needs to move quickly. With safe and effective vaccines now widely available and a handful of promising COVID-19 treatments coming soon, there’s no doubt that many aspects of biological research have been successfully accelerated in the past two years.

Now, researchers from Lawrence Berkeley National Laboratory (Berkeley Lab) and Heidelberg University in Germany have cranked up the speed of imaging infected cells using soft X-ray tomography, a microscopic imaging technique that can generate incredibly detailed, three-dimensional scans.

Their approach takes mere minutes to gather data that would require weeks of prep and analysis with other methods, giving scientists an easy way to quickly examine how our cells’ internal machinery responds to SARS-CoV-2, or other pathogens, as well as how the cells respond to drugs designed to treat the infection.

“Prior to our imaging technique, if one wanted to know what was going on inside a cell, and to learn what changes had occurred upon an infection, they’d have to go through the process of fixing, slicing, and staining the cells in order to analyze them by electron microscopy. With all the steps involved, it would take weeks to get the answer. We can do it in a day,” said project co-lead Carolyn Larabell, a Berkeley Lab faculty scientist in the Biosciences Area. “So, it really speeds up the process of examining cells, the consequences to infection, and the consequences of treating a patient with a drug that may or may not cure or prevent the disease.”

Taking cellular freeze frames

Larabell is a professor of anatomy at UC San Francisco and director of the National Center for X-Ray Tomography, a facility based at Berkeley Lab’s Advanced Light Source (ALS). The facility’s staff developed soft X-ray tomography (SXT) in the early 2000s to fill in the gaps left by other cellular imaging techniques. They currently offer the SXT to investigators worldwide and continue to refine the approach. As part of a study published in Cell Reports Methods late last year, she and three colleagues performed SXT on human lung cell samples prepared by their colleagues at Heidelberg University and the German Center for Infection Research.

Read more on the Berkeley Lab website

Image: Digital images of cells infected with SARS-CoV-2, created from soft X-ray tomography taken of chemically fixed cells at the Advanced Light Source

Credit: Loconte et al./Berkeley Lab

How deadly parasites ‘glide’ into human cells

2020/10/262020/10/29

X-ray analysis reveals structure of molecular machinery of malaria and toxoplasmosis pathogens

An investigation at DESY’s X-ray source PETRA III provides new insights into the molecular machinery by which certain parasites travel through the human organism. The study, led by Christian Löw from the Hamburg branch of the European Molecular Biology Laboratory EMBL, analyzed the so-called gliding movement of the malaria and toxoplasmosis parasites. The results, which the interdisciplinary team presents in the journal Communications Biology, can aid the search for new drugs against the pathogens.

In biological terms, gliding refers to the type of movement during which a cell moves along a surface without changing its shape. This form of movement is unique to parasites from the phylum Apicomplexa, such as Plasmodium and Toxoplasma. Both parasites, which are transmitted by mosquitoes and cats, have an enormous impact on global heath. Plasmodium causes 228 million malaria infections and around 400 000 deaths per year. Toxoplasma, which infects even one third of the human population, can cause severe symptoms in some people, and is particularly dangerous during pregnancy.

Read more on the DESY PETRA III website

Image: Molecular structure of essential light chain (ELC) protein in Plasmodium glideosome. Blue represents the electron density of the protein, with bonds between atoms indicated in yellow and water molecules indicated in red. The crystal structure at a resolution of 1.5 Ångström (0.15 millionths of a millimetre) was obtained at the EMBL beamlines at DESY’S X-ray source PETRA III. Credit: EMBL, Samuel Pazicky

X-ray microscopy at BESSY II: Nanoparticles can change cells

2020/02/132020/02/13

Nanoparticles easily enter into cells. New insights about how they are distributed and what they do there are shown for the first time by high-resolution 3D microscopy images from BESSY II.

For example, certain nanoparticles accumulate preferentially in certain organelles of the cell. This can increase the energy costs in the cell. “The cell looks like it has just run a marathon, apparently, the cell requires energy to absorb such nanoparticles” says lead author James McNally.
Today, nanoparticles are not only in cosmetic products, but everywhere, in the air, in water, in the soil and in food. Because they are so tiny, they easily enter into the cells in our body. This is also of interest for medical applications: Nanoparticles coated with active ingredients could be specifically introduced into cells, for example to destroy cancer cells. However, there is still much to be learned about how nanoparticles are distributed in the cells, what they do there, and how these effects depend on their size and coating.

Image: 3D architecture of the cell with different organelles: mitochondria (green), lysosomes (purple), multivesicular bodies (red), endoplasmic reticulum (cream).
Credit: Burcu Kepsutlu/HZB

Structure and functional binding epitopes of VISTA

2019/09/302019/10/10

V-domain Ig Suppressor of T-cell Activation (VISTA) is an immune checkpoint protein involved in the regulation of T cell activity. Checkpoint proteins are overexpressed by cancer cells or surrounding immune cells and prevent anti-tumor activity by co-opting natural regulation mechanisms to escape immune clearance. Compared to healthy tissues, VISTA is upregulated on tumor infiltrating leukocytes, including high expression on myeloid-derived suppressor cells (MDSCs). Through VISTA signaling, these inhibitory immune cells prevent effective antigen presentation and indirectly promote tumor growth. VISTA is implicated in a number of human cancers including skin (melanoma), prostate, colon, pancreatic, ovarian, endometrial, and non-small cell lung. VISTA is a known member of the B7 protein family but the mechanism of action is still unclear as VISTA has been shown to function as both a ligand^1,2 and a receptor³. In the model of VISTA as a receptor, the proposed ligand of interaction is V-set and immunoglobulin domain containing 3 (VSIG3)^4,5.

>Read more on the SSRL website

Image: Structure of human VISTA with extended C-C’ loop (blue), mapped VSTB/VSIG3 binding epitope (red), and disulfide bonds (yellow).

ALBA collaborates in the discovery of a new muscular disease: myoglobinopathy

2019/03/272019/04/17

An international collaboration led by IDIBELL identifies the first disease caused by a mutation in myoglobin.

At the MIRAS beamline of the ALBA Synchrotron they could demonstrate the presence of oxidized lipids in the damaged muscle cells.
Researchers of the Bellvitge Biomedical Research Institute (IDIBELL) led by Dr. Montse Olivé have described in Nature Communications a new muscular disease caused by a mutation in the myoglobin gene. The study has been possible thanks to a collaboration with a group of geneticists from the University of Western Australia (UWA), led by Prof. Nigel Laing, and researchers from the Karolinska Institute (Stockholm, Sweden).

Myoglobin, the protein that gives muscles their red colour, has as its main function the transportation and intracellular storage of oxygen, acting as an oxygen reservoir when there are low levels (hypoxia) or a total lack thereof (anoxia). It also acts as scavenger of free radicals and other reactive oxygen species, avoiding cell damage due to oxidative stress.

>Read more on the ALBA website

Image: Left, Typical μFTIR spectra and their second derivative of the muscle tissue where the lipid region has been highlighted in orange and the protein region in blue; the inset shows the lipid/protein ratio (calculated from the Infrared spectra) on an optical image of a tissue section with sarcoplasmic bodies. The color bar represents intensity of the ratio: blue and red mean low and high lipid content, respectively. The scale bar is 4 microns. Right, Infrared second derivative spectrum of the amide region of one sarcoplasmic body (green) showing an increase of β-sheet structures indicating protein aggregation. Second derivative of the amide region corresponding to the tissue surrounding the sarcoplasmic bodies (black).

A step closer to early detection of multiple sclerosis

2019/03/082019/03/11

Synchrotron techniques identify the critical conditions that alter myelin structure

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease resulting in the destruction of myelin, a fatty substance that insulates nerves and increases the speed at which signals travel between nerve cells. MS affects more than 2.3 million people worldwide and has no cure. In work recently published in PNAS, a team of researchers from Tel Aviv University and the Technion-Israel Institute of Technology mapped, for the first time, the delicate and complicated force balance between the myelin sheath constituents, and their effect on the myelin structure. This new information will allow the identification of critical components involved in neurodegenerative diseases such as MS.

Structural insights into tiny bacterial harpoons

2019/02/282019/03/07

Bacteria produce complex nano-harpoons on their cell surface. One of their functions is to harpoon and inject toxins into cells that are close by. Producing such a complex weapon requires lots of different moving components that scientists are still trying to understand. Researchers from the University of Sheffield have been using some of Diamond’s crystallography beamlines to understand a particularly enigmatic piece of this tiny puzzle. The team led by David Rice and Mark Thomas worked on a protein component of the harpoon called TssA which they already knew was an integral piece of the machinery. However, unlike the other components of the harpoon, there are distinct variants of the TssA protein that contain radically different amino acid sequences at one end of the protein. The team showed that the structures of the variable region of two different TssA subunits were completely unrelated and they could assemble into distinctly different multisubunit complexes in terms of their size and geometry. This begged the question as to how different bacteria could use this protein with different structures to produce a harpoon with the same function across all species. They found that despite these differences, there was a very specific conserved region at the other end of the protein. They hypothesise that the conserved region is the part that does the work and helps the harpoon to function whereas the variable region acts as a scaffold. They used I02, I03 and I24 in their study and plan to do follow up work using X-ray crystallography and Cryo-EM such as those at the eBIC centre at Diamond. The research was published in Nature Communications.

Image: Macromolecular Crystallography (MX) at Diamond reveals the shape and arrangement of biological molecules at atomic resolution, knowledge of which provides a highly accurate insight into function.

A timely solution for the photosynthetic oxygen evolving clock

2019/01/252019/01/31

XFEL Hub collaboration reveals the intermediates of the photosynthetic water oxidation clock

A large international collaborative effort aided by the XFEL Hub at Diamond Light Source has generated the most detailed time-resolved studies to date of a key protein involved in photosynthesis. The pioneering work, recently published in Nature, shows how photosystem II harnesses light energy to produce oxygen – insights that could direct a next generation of photovoltaic cells.
>Read more on the Diamond Light Source website

Image: this figure is issued from a video you can watch here.

Know your ennemy

2018/12/212019/01/08

Light source identifies a key protein interaction during E. coli infection

Escherichia coli is a common source for contaminated water and food products, causing the condition known as gastroenteritis with symptoms that include diarrhea, vomiting, fever, loss of energy, and dehydration. In fact, for children or individuals with weakened immune systems, this bacterial infection in the gut can be life-threatening.

One of the microbes responsible for gastroenteritis, known formally as enteropathogenic E. coli (EPEC), causes infections by directing a pointed, needle-like projection into the human intestinal tract, releasing toxins that make people sick.

“Enteropathogenic E. coli can fire toxic proteins from inside the bacterium right into the cells of your gut lining,” says Dustin Little, a post-doctoral researcher in the Brian Coombes lab at McMaster University’s Department of Biochemistry and Biomedical Sciences.

Image: Dustin Little and Brian Coombes in the lab.
Credit: Dustin Little.

Mycoplasma genitalium’s cell adhesion mechanism revealed

2018/10/312018/11/08

Mycoplasma genitalium is a sexually transmitted bacterium responsible for several genitourinary disorders.

An estimated 1% of the adult population is infected with this bacterium. Using XALOC beamline at the ALBA Synchrotron it has been defined the structure of the protein involved in the pathogen’s adhesion process. The discovery opens the door to defining new therapeutic strategies to fight this pathogen which is becoming more and more resistant to antibiotics.

Researchers from the Molecular Biology Institute of Barcelona (IBMB-CSIC) and the Institute of Biotechnology and Biomedicine (IBB-UAB) have discovered the mechanism by which the bacterium Mycoplasma genitalium (Mgen) adheres to human cells. This adhesion is essential for the onset of bacterial infection and subsequent disease development.
Mgen is an emerging pathogen responsible for several infectious genitourinary disorders. In men, it is the most common cause of urethritis (15-20%) while in women, it has been associated with cervicitis, pelvic inflammatory disease, premature birth and spontaneous abortions. So far, it was known that adherence to the genitourinary tract was possible thanks to proteins known as adhesins, which recognise specific cell surface receptors.
In this study, IBMB-CSIC researchers determined the three-dimensional structure of the Mgen’s P110 adhesins interacting with these cell receptors using X-rays diffraction and protein crystallography at the XALOC beamline. “We made a protein crystal of the P110 adhesin bound to these receptors and diffracted with the synchrotron’s X-rays to determine the exact position of the atoms within the protein, and we were able to decipher the three-dimensional structure”, explains IBMB researcher David Aparicio.

>Read more on the ALBA website

Image: Overall structure of P110. Two views, 90° apart from each other, of the extracellular region of P110 that is formed by a large N-domain, with a seven blade β-propeller (green), the crown (brown), and the C-domain (orange). In the right side panel the view is along the central axis of the β-propeller. The situation of the seven blades in the propeller is explicitly indicated showing that the two terminal blades I and VII are close to the C-terminal domain and opposite to the crown.

X-rays reveal L-shape of scaffolding protein

2018/09/142018/09/20

Structural biologists discover unexpected results at PETRA III at DESY in Germany.

An investigation at DESY’s X-ray light source PETRA III has revealed a surprising shape of an important scaffolding protein for biological cells. The scaffolding protein PDZK1 is comprised of four so-called PDZ domains, three linkers and a C-terminal tail. While bioinformatics tools had suggested that PDZK1’s PDZ domains and linkers would behave like beads on a string moving around in a highly flexible manner, the X-ray experiments showed that PDZK1 has a relatively defined L-shaped conformation with only moderate flexibility. The team led by Christian Löw from the Centre for Structural Systems Biology CSSB at DESY and Dmitri Svergun from the Hamburg branch of the European Molecular Biology Laboratory EMBL report their results in the journal Structure.

Similar to metal scaffolding which provides construction workers with access points to a building, scaffolding proteins mediate interactions between proteins situated on the membrane of the human cell. While the molecular structure of each of PDZK1’s four individual PDZ domains has been solved using X-ray crystallography and NMR spectroscopy, the overall arrangement of the domains in the protein as well as their interactions was not yet understood.

Image: Artistic shape interpretation of the scaffolding protein PDZK1. (Credit: Manon Boschard)tistic shape interpretation of the scaffolding protein PDZK1.
Credit: Manon Boschard

Shining a new light on biological cells

2018/09/072018/09/12

Combined X-ray and fluorescence microscope reveals unseen molecular details

A research team from the University of Göttingen has commissioned at the X-ray source PETRA III at DESY a worldwide unique microscope combination to gain novel insights into biological cells. The team led by Tim Salditt and Sarah Köster describes the combined X-ray and optical fluorescence microscope in the journal Nature Communications. To test the performance of the device installed at DESY’s measuring station P10, the scientists investigated heart muscle cells with their new method.

Modern light microscopy provides with ever sharper images important new insights into the interior processes of biological cells, but highest resolution is obtained only for the fraction of biomolecules which emit fluorescence light. For this purpose, small fluorescent markers have to be first attached to the molecules of interest, for example proteins or DNA. The controlled switching of the fluorescent dye in the so-called STED (stimulated emission depletion) microscope then enables highest resolution down to a few billionth of a meter, according to principle of optical switching between on- and off-state introduced by Nobel prize winner Stefan Hell from Göttingen.

Image: STED image (left) and X-ray imaging (right) of the same cardiac tissue cell from a rat. For STED, the network of actin filaments in the cell, which is important for the cell’s mechanical properties, have been labeled with a fluorescent dye. Contrast in the X-ray image, on the other hand, is directly related to the total electron density, with contributions of labeled and unlabeled molecules. By having both contrasts at hand, the structure of the cell can be imaged in a more complete manner, with the two imaging modalities “informing each other”.
Credit: University of Göttingen, M. Bernhardt et al.

Just like lego – studying flexible protein for drug delivery

2018/08/302018/09/06

Researchers from the Sapienza University of Rome and its spin-off company MoLiRom (Italy) are spending the weekend at the ESRF to study a protein that could potentially transport anticancer drugs.

Ferritin is a large spherical protein (20 times bigger than haemoglobin) that stores iron within its cavity in every organism. Just like a lego playset, Ferritin assembles and disassembles. It is also naturally targeted to cancer cells. These are the reasons why Ferritin is a great candidate as a drug-transport protein to fight cancer. An international team of scientists from “Sapienza” University of Rome and the SME MoLiRom (Italy) came to the ESRF to explore a special kind of ferritin that shows promising properties. “This is an archaebacterial ferritin that have transformed into a humanised ferritin to try to tackle cancer cells”, explains Matilde Trabuco, a scientist at the Italian SME MoLiRom.

The mechanism looks simple enough: “Ferritin has a natural attraction to cancer cells. If we encapsulate anti-cancer drugs inside it, it will act as a Trojan horse to go inside cells, then it will open up and deliver the drug”.

Ferritins have been widely used as scaffolds for drug-delivery and diagnostics due to their characteristic cage-like structure. Most ferritins are stable and disassemble only by a harsh pH jump that greatly limits the type of possible cargo. The humanised ferritin was engineered to combine assembly at milder conditions with specific targeting of human cancer cells.

Unprecedented 3D images of neurons in healthy and epileptic brains

2018/08/242018/10/08

Results open new perspectives for the study of neurodevelopment and neurodegenerative diseases.

A comprehensive understanding of the brain, its development, and eventual degeneration, depends on the assessment of neuronal number, spatial organization, and connectivity. However, the study of the brain architecture at the level of individual cells is still a major challenge in neuroscience.
In this context, Matheus de Castro Fonseca, from the Brazilian Biosciences National Laboratory (LNBio), and collaborators [1] used the facilities of the Brazilian Synchrotron Light Laboratory (LNLS) to obtain, for the first time, three-dimensional images in high resolution of part of the neuronal circuit, observed directly in the brain and with single cell resolution.

The researchers used the IMX X-Ray Microtomography beamline, in combination with the Golgi-Cox mercury-based impregnation protocol, which proved to be an efficient non-destructive tool for the study of the nervous system. The combination made it possible to observe the points of connectivity and the detailed morphology of a region of the brain, without the need for tissue slicing or clearing.
The mapping of neurons in healthy and unhealthy tissues should improve the research in neurodegenerative and neurodevelopmental diseases. As an example of this possibility, the work presents, for the first time in 3D, the neuronal death in an animal model of epilepsy.

The researchers are now working to extend the technique to animal models of Parkinson’s disease. The intention is to better understand the cellular mechanisms involved in the onset and progression of the disease. In the future, with the inauguration of the new Brazilian synchrotron light source, Sirius, the researchers believe that it will be possible to obtain images at the subcellular level, that is, images of the interior of the neurons.

Image: X-ray microtomography of the cerebral cortex showing the segmentation of individual neurons. Each color represents a single neuron or a group of neurons.

Infrared beams show cell types in a different light

2018/08/232018/08/30

Berkeley Lab scientists developing new system to identify cell differences.

By shining highly focused infrared light on living cells, scientists at the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) hope to unmask individual cell identities, and to diagnose whether the cells are diseased or healthy.
They will use their technique to produce detailed, color-based maps of individual cells and collections of cells – in microscopic and eventually nanoscale detail – that will be analyzed using machine-learning techniques to automatically sort out cell characteristics.

Using microscopic color maps to unlock cell identity

Their focus is on developing a rapid way to easily identify cell types, and features within cells, to aid in biological and medical research by providing a way to probe living cells in their native environment without harming the cells or requiring obtrusive cell-labeling techniques.
“This is totally noninvasive,” said Cynthia McMurray, a biochemist and senior scientist in Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging (MBIB) Division who is leading this new imaging effort with Michael Martin, a physicist and senior staff scientist at Berkeley Lab’s Advanced Light Source (ALS).
The ALS has dozens of beamlines that produce beams of intensely focused light, from infrared to X-rays, for a broad range of experiments.

Image: From left to right: Aris Polyzos, Edward Barnard, and Lila Lovergne, pictured here at Berkeley Lab’s Advanced Light Source, are part of a research team that is developing a cell-identification technique based on infrared imaging and machine learning.
Credit: Marilyn Chung/Berkeley Lab