Tag Archives: #cells

How Cells Control Mitochondria? (Biology)

Freiburg researchers discover a signaling protein that controls the assembly of human cellular “power plants”

Errors in the metabolic processes of mitochondria are responsible for a variety of diseases such as Parkinson’s and Alzheimer’s. Scientists needed to find out just how the necessary building blocks are imported into the complex biochemical apparatus of these cell areas. The TOM complex (translocase of the outer mitochondrial membrane) is considered the gateway to the mitochondrion, the proverbial powerhouse of the cell. The working group headed by Professor Chris Meisinger at the Institute of Biochemistry and Molecular Biology at the University of Freiburg has now demonstrated – in human cells – how signaling molecules control this gate. A signaling protein called DYRK1A modifies the molecular machinery of TOM and makes it more permeable for enzymes that are important for the cell metabolism. The group has thus discovered the first signaling protein that directly influences this import process in humans. Their work has been published in the journal Nature Communications.

Developmental disorders in a new light

In neurodevelopmental disorders such as autism, microcephaly and Down’s syndrome, DYRK1A is defective. “The connection with mitochondria is new. These results allow us to better understand these disorders and develop treatment strategies,” says Dr. Adinarayana Marada, a member of Meisinger’s team.

“For a long time, researchers thought that the TOM complex was a rigid structure in the mitochondrial membrane whose doors were always open,” Meisinger explains. His team recently demonstrated signaling mechanisms in baker’s yeast that alter the subunits of the TOM complex depending on the metabolic state of the cell, or in response to sudden stress. In this way, the cell can specifically control the influx of precursor proteins for building elements of the metabolism, and it can adapt the function of the mitochondria to an altered cellular state. Whether such mechanisms also exist in humans was previously unknown.

DYRK1A acts upon the TOM complex

The first authors of the study, Dr. Corvin Walter and Dr. Adinarayana Marada of Meisinger’s research group, developed a systematic approach to track down signaling mechanisms such as those triggered by protein kinases, in humans. Over several years, they tested candidates using cell biological and bioinformatic methods and found what they were looking for – DYRK1A, one such protein kinase, acts on the TOM complex. “With this, we actually found the needle in the haystack,” says Walter.

The work was done in collaboration with Professor Nora VögtleProfessor Tilman Brummer and Professor Claudine Kraft from the University of Freiburg as well as researchers at the Universities of Göttingen, Dortmund and Fribourg, Switzerland. Meisinger is speaker of the Collaborative Research Center “Dynamic Organization of Cellular Protein Mechanisms” at the University of Freiburg. In addition, he is a member of the Freiburg Cluster of Excellence CIBSS – Centre for Integrative Biological Signalling Studies, the research training group 2202 “Transport across and into Membranes”, and 2606 “ProtPath”.

Publication:
Walter, C., Marada, A., Suhm, T., Ernsberger, R., Muders, V., Kücükköse, C., Sánchez-Martín, P.,Hu,Z.,Aich, A., Loroch, S., Solari, F.A., Poveda-Huertes, D., Schwierzok, A., Pommerening, H., Matic, S., Brix, J., Sickmann, A., Kraft, C., Dengjel, J., Dennerlein, S., Brummer, T., Vögtle, F.N., and Meisinger, C. (2021): Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery. In: Nat. Commun. 12:4284. DOI: 10.1038/s41467-021-24426-9

Featured image: The network of mitochondria runs like a thread through the entire cell (marked in green and red with fluorescent proteins). The cell nucleus is stained in blue Photo: Pablo Sánchez-Martín/University of Freiburg


Provided by University of Freiburg

How Cells Measure Themselves? (Biology)

Ever since scientists discovered cells under the microscope more than 350 years ago, they have noted that each type of cell has a characteristic size. From tiny bacteria to inches-long neurons, size matters for how cells work. The question of how these building blocks of life regulate their own size, however, has remained a mystery.

Now we have an explanation for this long-standing biological question. In a study focusing on the growing tip of plants, researchers show that cells use their DNA content as an internal gauge to assess and adjust their size.

Professor Robert Sablowski, a group leader at the John Innes Centre and corresponding author of the study said: “It has been suggested for a long time that DNA could be used as a scale for cell size, but it was unclear how cells could read the scale and use the information. The key is to use the DNA as a template to accumulate the right amount of a protein, which then needs to be diluted before the cell divides. It’s exciting to come across such a simple solution to a long-standing problem.”

AThe average cell size results from a balance between how much cells grow and how often they split in two. It has long been clear that cells grow to a certain size before they divide. But how can a cell know how much it has grown?

A good place to investigate this question is in the shoot meristem, the growing tip of the plant, which supplies new cells to make leaves, flowers and stems. Meristem cells constantly grow and divide. Their divisions are often not equal, producing cells of different sizes. Over time, these differences should build up, but the meristem cells stay within a narrow range of sizes over long periods.

In this study, which appears in Science, John Innes Centre researchers carefully followed the growth and division of meristem cells over time. They found that although cells can start their life with variable sizes, by the time the cells are ready to replicate their DNA (a necessary step before cell division, as each new cell needs its own copy of the DNA), most of the initial variability in cell sizes has been corrected.

They then monitored a protein called KRP4, whose role is to delay the start of DNA replication, and found that, regardless of their initial size, cells were always born with the same amount of KRP4. This means that when a cell is born too small, it receives a higher concentration of KRP4, which delays its progression to DNA replication, allowing time for the cell to catch up to the same size of the other cells. Conversely, if a cell is born too big, KRP4 is diluted so it can move quickly onto the next stage without growing further. Over time this keeps meristem cells within a narrow size range.

But what ensures that cells start off with the same amont of KRP4? It turned out that when cells divide, KRP4 “takes a ride” on the DNA, which is given in identical copies to each newborn cell. In this way, the initial amount of KRP4 becomes proportional to the cell’s DNA content. To make sure that KRP4 accumulates in the mother cell in proportion to the DNA content, any excess KRP4 not bound to the DNA is destroyed before cell division by another protein called FBL17. Mathematical models and using gene-edited mutants with varying quantities of these genetic components confirmed the mechanism.

Professor Robert Sablowski, explains this process, “One riddle we had to solve is how a cell can know how much it has grown when most of the components of a cell increase together in number and size so they cannot be used as a fixed ruler to measure size. One exception is DNA which exists in the cell in a discrete amount – its amount precisely doubles before cell division, but it does not vary with cell growth.”

Future experiments will seek to explain exactly how the regulatory protein KRP4 associates, then dissociates from chromosomes during cell division. The researchers also want to understand whether the mechanism is modulated in different cell types to produce different average sizes.

The findings may explain the relation between genome size and cell size – species with large genomes and, therefore a lot of DNA in their cells, tend to have larger cells. This is particularly important in crop plants, many of which have been selected to contain multiple copies of the genomes present in their wild ancestors, leading to larger cells and often larger fruits and seeds.

Components of the genetic mechanism that includes KRP4 are present in many organisms, and it has been suggested that these components are important to regulate cell size in human cells. Thus the mechanism uncovered in the study may also be relevant across biological Kingdoms, with implications for animal and human cell biology.

The study, “Cell size controlled in plants using DNA content as an internal scale” appears in Science.

Featured image: The image shows a shoot apical meristem (at the centre) with floral buds emerging on its flanks. Cells marked in green are about to enter DNA replication, whilst the magenta marker shows accumulation of KRP4, which is part of the mechanism that regulates cell size. © John Innes Centre


Provided by John Innes Center

Unparallelled Insights Into How Our Bodies Develop From A Single Cell (Biology)

Knowing what happens during normal development could help us to understand genetic diseases that arise during pregnancy

New insights into how our bodies come into being from a single cell have been generated by researchers at the Wellcome Sanger Institute, the Wellcome-MRC Cambridge Stem Cell Institute and the University of Cambridge. It is the first study of its kind to describe fetal development in humans by retracing how and when mutations are acquired during pregnancy. The team found higher rates of mutation in early cell divisions, with the ‘decision’ for whether cells become the fetus or become protective tissues like the placenta occurring much earlier than previously thought.

The study, published in Nature, highlights subtle differences between human biology and that of mice, which have previously been relied upon as models for such research. It provides an important reference of mutation under normal conditions for researchers seeking to understand the causes of diseases such as childhood cancers and rare developmental disorders, which often begin in utero.

Studying development is motivated in large part by the desire to understand how our bodies, with their incredible complexity, come into being from a single cell. Understanding how this is coordinated and which cells give rise to others under normal circumstances may help us to identify how and why development can sometimes go wrong.

Tracking development forward through time can be achieved through lineage tracing, which involves ‘marking’ cells in a way that this is passed on to the offspring of a cell. This allows you to map how cells are related to each other and create a ‘family tree’. This technique requires manipulation of the developing embryo, however, so is not ethical or feasible in humans. Study of human development has therefore been limited primarily to careful microscopy, with much of our knowledge of development based on model organisms such as zebra fish and mice.

For this study, researchers at the Wellcome Sanger Institute and Wellcome-MRC Cambridge Stem Cell Institute collected eight week and eighteen week-old haematopoietic stem and progenitor cells (HSPCs) from human fetal tissue1 and grew them into 511 single-cell-derived colonies.

DNA from hundreds of these colonies underwent whole genome sequencing to identify somatic mutations that could be used to trace the lineage of blood cells back to the first division of the embryo. Looking for these ‘marker’ mutations in tiny biopsies from other tissues then allowed the researchers to see when these tissues diverged from the blood cell population.

The team found that by week eight of development, cells had acquired 25 mutations and 42 by week 18, indicating a higher rate of mutation in early cell divisions. They also timed the ‘decision’ for which cells would become the fetus and which would become extra-embryonic tissue, which includes the placenta and yolk-sac, occurred between four and 16 cells.

“The findings of this study have challenged some of our previous understanding about how the fetus grows from one cell during the earliest stages of life, such as when the embryonic and extra-embryonic tissues diverge. This kind of resolution will be essential if we are to try to pinpoint the origin of diseases that have their roots in development.”

— Dr Anna Ranzoni,a first author of the study from the Wellcome-MRC Cambridge Stem Cell Institute and Department of Haematology at the University of Cambridge

There was also evidence that the extra-embryonic mesoderm and the blood cells that deliver oxygen to the fetus in the first trimester of pregnancy arise from the hypoblast, which is generally considered an extra-embryonic tissue – a clear difference between human and mouse biology.

“Mice have been an excellent model for studying human development, but there was always the question of whether mouse biology was the same as our biology or merely similar. We found evidence that primitive human blood cells arise from the hypoblast, which is different to mice, settling a question that has been debated for decades.”

Dr Michael Spencer Chapman,a first author of the study from the Wellcome Sanger Institute

These insights into the precise biological processes involved in human development provide an essential reference of developmental dynamics under normal circumstances for those studying childhood cancers, which often begin in utero, as well as rare developmental disorders.

“Our study provides important insights into the incredibly complex biological processes at work in the earliest weeks of life, which have simply not been possible until now. This resource will be an invaluable reference for what happens under normal circumstances, so that we can start to unravel what happens when development goes wrong.”

Dr Ana Cvejic,a senior author of the study from the Wellcome-MRC Cambridge Stem Cell Institute and Department of Haematology at the University of Cambridge

More information

Notes to Editors:

1 Human tissue used in the study was provided by the Human Developmental Biology Resource, in accordance with ethical approval by the NHS Health Research Authority (HRA).

Publication:

Michael Spencer Chapman, Anna Maria Ranzoni and Brynelle Myers et al. (2021). Lineage tracing of human development through somatic mutations. Nature. DOI: https://doi.org/10.1038/s41586-021-03548-6

Funding:

This work was supported by the European Research Council, EMBO, the Medical Research Council (MRC) and Wellcome.

Featured image credit: K Hardy_CC BY 4.0


Provided by Wellcome Sanger Institute

How Human Cells And Pathogenic Shigella Engage in Battle? (Biology)

One member of a large protein family that is known to stop the spread of bacterial infections by prompting infected human cells to self-destruct appears to kill the infectious bacteria instead, a new study led by UT Southwestern scientists shows. However, some bacteria have their own mechanism to thwart this attack, nullifying the deadly protein by tagging it for destruction.

The findings, published online today in Cell, could lead to new antibiotics to fight bacterial infections. And insight into this cellular conflict could shed light on a number of other conditions in which this protein is involved, including asthma, Type 1 diabetes, primary biliary cirrhosis, and Crohn’s disease.

Neal M. Alto, Ph.D.
Neal M. Alto, Ph.D. © UT Southwestern Medical Center

“This is a wonderful example of an arms race between infectious bacteria and human cells,” says study leader Neal M. Alto, Ph.D., professor of microbiology at UTSW and a member of the Harold C. Simmons Comprehensive Cancer Center.

Previous research has shown that the protein, called gasdermin B (GSDMB), was different from other members of the mammalian gasdermin family. Related gasdermin proteins form pores in the membranes of infected cells, killing them while allowing inflammatory molecules to leak out and incite an immune response. However, GSDMB – found in humans but not in some other mammalian species, including rodents – doesn’t form pores in the membranes of cultured mammalian cells, leaving its target a mystery.

Using a novel screening technology, Alto and colleagues discovered that a protein toxin called IpaH7.8 from shigella flexneri, a bacterium that causes diarrheal disease, directly inhibits GSDMB. Biochemical experiments show that IpaH7.8 places a chemical tag on GSDMB that marks it for cellular destruction.

To understand why shigella flexneri rids human cells of GSDMB, the researchers placed GSDMB within synthetic mammalian and bacterial cell membranes. While GSDMB left the synthetic mammalian membranes unharmed, it poked holes in the bacterial membranes. Further investigation showed that immune cells called natural killer cells stimulate this process.

Alto notes that inhibiting the ability of shigella IpaH7.8 to counteract GSDMB could lead to new types of antibiotics. And because genetic variants of GSDMB have been linked to a variety of inflammatory diseases and cancer, better understanding this protein could lead to new treatments for these conditions too.

Other UTSW researchers who contributed to this study include Justin M. Hansen, Maarten F. de Jong, Qi Wu, Li-Shu Zhang, David B. Heisler, and Laura T. Alto.

This research was funded by grants from the National Institutes of Health (AI083359), The Welch Foundation (I-1704), the Burroughs Wellcome Fund, (1011019) and the Howard Hughes Medical Institute and Simons Foundation Faculty Scholars Program (55108499).

Featured image: A new study finds that gasdermin B (GSDMB) pokes holes in bacterial membranes containing cardiolipin as a novel immune defense strategy. Shown are pictures of GSDMB pores embedded in synthetic bacterial membranes. The inset image shows a purified GSDMB pore. Credit: Justin M. Hansen


Reference: Justin M. Hansen, Maarten F. de Jong et al., “Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions”, Cell, 2021. DOI: https://doi.org/10.1016/j.cell.2021.04.036


Provided by UT Southwestern Medical Center

Researchers Discover New Mechanism for Lipid Storage Inside Cell (Biology)

In a study published in Journal of Cell Biology, a research group led by Prof. HU Junjie from the Institute of Biophysics of the Chinese Academy of Sciences reported that endoplasmic reticulum (ER) tubule-forming proteins and septin cytoskeletons are involved in the regulation of lipid droplet (LD) biogenesis via their interactions with ER membrane protein FIT2.

LD is the major place for lipid storage, and it plays crucial roles in lipid metabolism and cellular stress responses. In eukaryocytes, ER accomadates neutral lipid synthesis and nascent LD biogenesis. The accumulation of neutral lipids between the leaflets of ER membranes leads to a growing lens-like structure, which eventually buds into the cytosol as nascent LD. However, the details mechanism and regulation of LD biogenesis is largely unclear.

The ER consists of two distinct morphological domains: tubules and sheets. The tubular ER network is generated by reticulons(Rtns)/REEPs, which stabilize membrane curvature, and subsequently connected by dynamin-like GTPase atlastin (ATL). Converging evidence suggests that ER tubules are mechanistically linked to LD formation.

Prof. HU’s team used C. elegans to screen new regulators of ER morphology, and found the deletion of FIT2 caused ER sheets expansion.

Biochemical analysis revealed that FIT2 physically interacted with ER tubule-forming proteins, including Rtn4 and REEP5, and septin cytoskeletons. Deletion or depletion of these proteins caused defective LD formation in mammalian cells and in worms. FIT2-interacting proteins were up regulated during adipocyte differentiation. Importantly, FIT2, ER tubule-forming proteins and septins were transiently enriched at nascent LD formation sites, as shown by super-resolution live cell imaging.

In summary, neutral lipids in the ER accumulate with FIT2, which in turn recruits ER tubule-forming proteins to stabilize the curvature at the growing oil lens and promote lipid preparation. The outward bulging of the oil lens is reinforced by FIT2-interacting septins, serving as a handrail.

This study provides important insight into the early steps of LD formation.


Featured image: Fang Chen, Bing Yan, Jie Ren, Rui Lyu, Yanfang Wu, Yuting Guo, Dong Li, Hong Zhang, Junjie Hu; FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins. J Cell Biol 3 May 2021; 220 (5): e201907183. doi: https://doi.org/10.1083/jcb.201907183


Provided by Chinese Academy of Sciences

Force Transmission Between Cells Orchestrates Collective Cellular Motion (Biology)

How do the billions of cells communicate in order to perform tasks? The cells exert force on their environment through movement – and in doing so, they communicate. They work as a group in order to infiltrate their environment, perform wound healing and the like. They sense the stiffness or softness of their surroundings and this helps them connect and organize their collective effort. But when the connection between cells is distrubeddisturbed, a situation just like when cancer is initiated, can appear.

Assistant Professor Amin Doostmohammadi at the Niels Bohr Institute, University of Copenhagen has investigated the mechanics of cell movement and connection in an interdisciplinary project, collaborating with biophysicists in France, Australia, and Singapore, using both computer modelling and biological experiments. The result is now published in Nature Materials.

Amin Doostmohammadi explains: “We need to understand how cells translate this “knowledge from sensing” at the individual cell level and transform it into action on the collective level. This is still kind of a black box in biology – how do cell talk to their neighbors and act as a collective?”

The force of surrounding tissue dictates cell behavior

Individual cells have a contractile mode of motion: they pull on the surface they are located on to move themselves forward. However, cells lining up cavities and surfaces in our body, like the tubes of blood vessels or the cells at the surface of organs, are able to generate extensile forces. They do the opposite, they stretch instead of contract – and they form strong connections with their neighbors. Contractile cells are able to switch to becoming extensile cells, when coming into contact with their neighbors. If, for instance, when contractile cells sense a void or an empty space, like when a wound appears, they can loosen their cell – cell connection, become more individual, and when healing the wound, they form strong connections with their neighbors again, becoming extensile, closing the gap, so to speak.

Weakening cell connection can be the hallmark of cancer initiation

The cells connect to their neighbors by adherens junctions. They connect their internal cytoskeleton to one another and become able to transmit forces through the strong contacts. “So we asked ourselves what would happen if we prohibited the cells from making this strong connection – and it turned out that extensile, strongly connected cells turned into contractile cells with weaker connections. This is significant, because the loss of this contact is the hallmark of cancer initiation. The cells losing contact start behaving more as individuals and become able to infiltrate their surroundings. This process also happens when an embryo develops, but the key difference here is that when the healthy cells have achieved their goal, like forming an organ, they go back to their original form. Cancer cells do not. They are on a one way street”, Amin Doostmohammadi says.

The basic action and reaction of cells are determined by surroundings and communication

How cells “decide” when to go from one form to another is a complicated mix of reacting to their environment, changes in the chemical composition of it, the mechanical stiffness or softness of the tissue – and many proteins in the cells are involved in the process. The key finding of this study is that this reaction to surroundings is constantly shifting: There is a constant cross-talk between cell – surroundings and cell – cell, and this is what determines the actions and reactions of the cells.

Are treatments for cancer within the scope of this new understanding in cell mechanics?

“We must always be careful, when talking about a serious and very complex disease like cancer”, Amin Doostmohammadi says. “But what we can say is that this study brings us one step closer to understanding the basic mechanics of cell behavior, when the cells go from the normal behavior to the aggressive, cancer type cell behavior. So, one of the big questions this study raises is if we might be able to target the mechanics of the cells by some form of therapy or treatment, instead of targeting the DNA or chemical composition of the cells themselves? Could we target the environment instead of the cells? This is basic research, connecting physics and biology, into the mechanics of cell behavior, based on their sensing and responding to the surroundings and coordinating their effort – our improved understanding of this may well lead to new therapies, and there are trials going on at the moment at a preliminary stage”.

Link to the scientific article: https://www.nature.com/articles/s41563-021-00919-2

Featured image: Mixed cell populations autonomously sort themselves into separate domains: islands of extensile cells with normal cell-cell contacts (purple) surrounded by contractile cells that have weakened cell-cell contacts (green). © University of Copenhagen


Reference: Balasubramaniam, L., Doostmohammadi, A., Saw, T.B. et al. Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers. Nat. Mater. (2021). https://doi.org/10.1038/s41563-021-00919-2


Provided by University of Copenhagen

Lab Study Solves Textbook Problem: How Cells Know Their Size (Biology)

The answer to a basic science question could unlock the key to complex medical challenges

Scientists have searched for years to understand how cells measure their size. Cell size is critical. It’s what regulates cell division in a growing organism. When the microscopic structures double in size, they divide. One cell turns into two. Two cells turn into four. The process repeats until an organism has enough cells. And then it stops. Or at least it is supposed to.

The complete chain of events that causes cell division to stop at the right time is what has confounded scientists. Beyond being a textbook problem, the question relates to serious medical challenges: Cells that stop dividing too soon can cause defects in growing organisms. Uncontrolled cell growth can lead to cancers or other disorders.

A study from Dartmouth, published in Current Biology, provides a new answer to the question by tackling the problem in reverse: The research focused on large cells that reduce their size through division until enough cells are formed to move to other stages of development.

“The early embryo is an ideal place to study cell size control,” said Amanda Amodeo, an assistant professor of biology at Dartmouth and the lead researcher. “The cells we work with are eggs that are visible to the eye. They don’t need to grow before dividing, so it allows us to look at connections that are obscured in adult cells.”

According to the study, a set amount of the protein histone H3 is loaded into an embryo before fertilization and is used up as the embryo divides into more cells. As histones are consumed to accommodate the growing number of nuclei, they release the enzyme Chk1 to bind with another protein, CDC25, to stop the multiplication of cells.

The research is technical, but the mechanism is relatively straightforward: With histone H3 out of the way in a growing cell, the stop enzyme Chk1 finds and disables the protein that triggers cell cycle progression, CDC25.

“The key to our research result was coming up with the possibility that unusually large amounts of histone H3 may feed into the stop enzyme,” said Yuki Shindo a postdoctoral research fellow at Dartmouth and first author of the paper. “Once we noticed that, we were able to test this idea in our living test tube, fruit fly eggs.”

The new research builds on earlier studies which found that a biological constant exists between the size of a genome and the size of a cell. Researchers knew that once a balance point was achieved, cells would stop duplicating, but didn’t understand how cells could determine the ratio.

To find the answer to the long-running question, the research team studied fruit fly eggs. Because of their large size compared to other cells, the team was able to get a different perspective on the cell cycle.

“We’ve had all of the pieces for years but couldn’t quite get them to fit together,” said Amodeo. “Once we recognized that H3 interacts directly with both DNA and Chk1, the work went very fast. Everything worked the first time, which is a good sign that the hypothesis is right.”

Since the same molecules that control cell division–histone H3, CDC25 and Chk1–are all identified in cancer and other ailments, the finding can help researchers that are seeking answers to questions related to development and disease.

“We were originally curious about a basic biological question on how cells in a growing egg make a decision to stop at the correct timing,” said Shindo. “We are now excited that our findings may also have an important implication for a broader context such as disease.”

Research conducted for this study was performed at and supported by the Lewis-Sigler Institute at Princeton University as well as by grants from the Japan Society for the Promotion of Science, the Uehara Memorial Foundation, and the Japanese Biochemical Society.

Featured image: New research describes how cells judge their size to know when to stop dividing. In this digital microscopy image, waves of cell division sweep through a fruit fly embryo to reduce cell size. © Image from the Amodeo Lab/Dartmouth College.


Reference: Yuki Shindo, Amanda A. Amodeo et al., “Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo”, Current Biology, 2021. DOI: https://doi.org/10.1016/j.cub.2021.03.035


Provided by Dartmouth College


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New Method Created By Scientists Will Transform How Cells Are Studied (Biology)

Scientists at Oxford Brookes University have developed a new single-cell transcriptomic method which will aid multiple fields of biology, including the study of human health, disease and injury.

Single-cell transcriptomic methods allow scientists to study thousands of individual cells from living organisms, one-by-one, and sequence each cell’s genetic material. Genes are activated differently in each cell type, giving rise to cell types such as neurons, skin cells and muscle cells.

Single-cell transcriptomics allows scientists to identify the genes that are active in each individual cell type, and discover how these genetic differences change cellular identity and function. Careful study of this data can allow new cell types to be discovered, including previously unobserved stem cells, and help scientists trace complex developmental processes.

“Single-cell transcriptomics have revolutionised biology but are still an area in active development,” explains Helena Garcia Castro, a PhD student in the Department of Biological and Medical Science at Oxford Brookes University and co-author of the paper.

“Current methods use cell dissociation protocols with ‘live’ tissues, which put cells under stress, causing them to change, and limiting accurate investigations.”

To solve this problem, the research team used historical research and revived a process from the 19th and 20th centuries to create the ACME (ACetic acid MEthanol dissociation) method.

“This means scientists can now exchange samples between labs, preserve the cell material and large sample sets can be frozen in order to be analysed simultaneously, without destroying the integrity of the genetic material in the cell.”

— Dr Jordi Solana, Research Fellow, Oxford Brookes University

Scientists realised that with this method, cells did not suffer from the dissociation as it stops their biological activity and ‘fixes’ them from the very beginning of the investigation.

The ACME method then allows cells to be cryopreserved, one or several times throughout the process, either immediately after the dissociation process, in the field or when doing multi-step protocols.

Dr Jordi Solana, Research Fellow at Oxford Brookes University adds: “This means scientists can now exchange samples between labs, preserve the cell material and large sample sets can be frozen in order to be analysed simultaneously, without destroying the integrity of the genetic material in the cell.

“We took the method from the old papers and repurposed it to make it work with current single-cell transcriptomic techniques. With our new method, we will now set out to characterise cell types in many animals.”

Scientists are now able to collaborate with other laboratories and research a wider variety of animal cells, thanks to the ACME method. This would not have been possible without the technology to dissociate and freeze live cell tissues.

The paper, ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics, is published in Genome Biology.

Image: Flatworm cell map generated using the new ACME protocol. Each dot is an individual cell, and each colour indicates a particular type of cell – close colours are related in function. Using these maps, scientists can compare cell type abundance between different species, and query the underlying data to discern more about their identity and function. © Oxford Brookes University


Reference: García-Castro, H., Kenny, N.J., Iglesias, M. et al. ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics. Genome Biol 22, 89 (2021). https://genomebiology.biomedcentral.com/articles/10.1186/s13059-021-02302-5 https://doi.org/10.1186/s13059-021-02302-5


Provided by Oxford Brookes University

Scientists Identify Molecular Pathway That Helps Moving Cells Avoid Aimless Wandering (Biology)

Findings from Johns Hopkins Medicine study have potential implications for understanding cancer cell spread

Working with fruit flies, scientists at Johns Hopkins Medicine say they have identified a new molecular pathway that helps steer moving cells in specific directions. The set of interconnected proteins and enzymes in the pathway act as steering and rudder components that drive cells toward an “intended” rather than random destination, they say.

In a report on the work, published March 2 in Cell Reports, these same molecular pathways, say the scientists, may drive cancer cells to metastasize or travel to distant areas of the body and may also be important for understanding how cells assemble and migrate in an embryo to form organs and other structures.

The team of scientists was led by Deborah Andrew, Ph.D., professor of cell biology and associate director for faculty development for the Institute for Basic Biomedical Sciences at the Johns Hopkins University School of Medicine.

Andrew and her colleagues began this research while studying a gene called Tre1 and its role in the development of salivary glands in fruit flies. The tools to study the effects of turning the gene on and off weren’t ideal, she says. So, two of the team members, Caitlin Hanlon, Ph.D., of Quinnipiac University and JiHoon Kim, Ph.D., of Johns Hopkins, generated fruit flies that lack the protein-coding portion of the Tre1 gene. The pair also put a fluorescent tag on the Tre1 protein to learn where it localized during key steps in development.

In experiments with fruit fly embryos carrying an intact Tre1 gene, cells that produce future generations of the organism, called germ cells, migrate correctly to the sex organ, known as the gonad.

“Without the Tre1 gene, however, most of the germ cells failed to meet up with other nongerm cells, or somatic cells, of the gonad,” says Andrew. “Correct navigation of germ cells is important to ensure that future generations of the organism will happen.”

This is not the first time that scientists noted Tre1’s importance in germ cell navigation. Two research teams from Indiana University and the Massachusetts Institute of Technology had previously made the link. However, says Andrew, questions remained about what happens inside germ cells to get cells to the right place once Tre1 activates.

It was already known that the Tre1 gene encodes a protein that spans the cell membrane multiple times and pokes out onto the cell’s surface. It’s a member of a large family of proteins called G protein-coupled receptors, which enable cells to communicate and respond to signals from other cells and light and odor cues. Nearly 35% of FDA approved medicines target G protein-coupled receptors, says Andrew.

To more precisely track the molecular events downstream of Tre1, Kim, a research associate and postdoctoral fellow at the Johns Hopkins University School of Medicine, used tissue cultures of fruit fly cells to find the location of fluorescently tagged molecules that are potentially triggered by the activated Tre1 protein. In the tissue cultures and germ cells of living flies, Kim uncovered the downstream genetic pathway.

He found that Tre1 functions as the cell’s helmsman, controlling steering of the cell. Tre1 activates the cell’s steering and rudder components by spurring on a cascade of proteins and enzymes, including a phospho-inositol kinase, PI(4,5)P2, dPIP5K, dWIP and WASp.

At the end of the molecular cascade, a chain of actin proteins forms in a protrusion at the cell’s leading edge to exert mechanical forces for movement.

The scientists also searched for the upstream signal that activates Tre1. They used a genetically engineered protein made by researchers at the University of California, San Francisco to track the location of a signaling protein called Hedgehog, which has previously been linked to germ cell migration, although its role in this process has been disputed.

In germ cells, Hedgehog signaling increases the membrane levels of a protein called Smoothened, which is found in the cells’ leading edge protrusion where Tre1 is also found.

The scientists plan to continue studying the pathways surrounding Tre1 and connections between the proteins and enzymes involved in the pathway.

“A deeper understanding of how moving cells navigate and spread has the potential to provide more targets for interrupting the spread of cancer cells,” says Andrew.

Funding for the research was provided by the National Institutes of Health (RO1DE013899, R35GM118177 and F31DE022233).

In addition to Andrew, Hanlon and Kim, other scientists who contributed to the research include Sunaina Vohra and Peter Devreotes of Johns Hopkins.

Featured image: Migrating germ cells in a fruit fly embryo. © Deborah Andrew and JiHoon Kim, Johns Hopkins Medicine.


Reference: Ji Hoon Kim, Caitlin Hanlon et al., “Hedgehog signaling and Tre1 regulate actin dynamics through PI(4,5)P2 to direct migration of Drosophila embryonic germ cells”, 34(9), mar 2, 2021. DOI: https://doi.org/10.1016/j.celrep.2021.108799


Provided by Johns Hopkins Medicine