May 25, 2017 Researchers at Howard Hughes Medical Institute developed a way to produce color-tagged, 3D, microscopic videos of organelles in a live cell. They came to Drexel's Andrew Cohen, PhD, to develop an algorithm that could process massive amounts of visual data to better understand the behavior of organelles as a group and individually. This technology will help them unlock cell behavior and response to drug treatment. Credit: Drexel University

Researchers at Howard Hughes Medical Institute and the Eunice Kennedy Shriver National Institute for Child Health and Human Development are getting a first glimpse at the inner-workings of live cells thanks to a new microscopy technique pioneered by Nobel laureate Eric Betzig with help from engineers at Drexel University. Their method uses grids of light that activate fluorescent color tags on each type of organellethe result is a 3-D video that gives researchers their best look at how cells function. It will allow scientists to better understand how cells react to environmental stressors and respond to drug treatment.

In a paper published today in Nature, the team lays out its methodology for using Betzig's lattice light sheet microscope in combination with image-tracking technology developed in Drexel's Computational Image Sequence Analysis Lab, led by Andrew Cohen, PhD, to produce 3-D time lapse videos of organelle movement and generate quantitative data on their interactions.

"The cell biology community has recognized for many years that the cytoplasm is full of many different types of organelles, and the field is recognizing more and more how significant cross-talk between these organelles is in the form of close contacts between these organelles," said Jennifer Lippincott-Schwartz, PhD, of HHMI's Janelia Research Campus, and senior author of the study. "When two organelles come close to each other they can transfer small molecules like lipids and calcium and communicate with each other through that transfer. But no one has been able to look at the whole set of these interactions at any particular time. This technology is providing a way to do that. But this paper is about a whole new technology, being able to tag six different objects with six different fluorophores, and unmixing the fluorophores so that you can observe the six different objects discretely."

Betzig's microscopy technique uses layers of light grids that interact with fluorescent protein-tagged cells to build a 3D microscopic image. At Janelia Research Campus, Betzig and Lippincott-Schwartz have refined that technology to produce a detailed look inside the cell by tagging each organelle type with its own color.

"The challenge is analyzing this data," Lippincott-Schwartz said. "It requires being able to simultaneously track these six different objects in 3D. What Andy Cohen and his group have done with the software system they have developed is enable us to really look at this in more quantitative ways than would be possible with conventional tools."

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Cohen's lab developed a tool called LEVER 3-D in 2015 to help researchers study 3-D images of neural stem cells. It applies an advanced image segmentation algorithm they developed that can identify boundaries of cells and track their movements. Prior to this technology being available to microbiologists, the processing of microscopic images and time-lapse footage would take massive amounts of time because they would have to create lineage trees by hand and attempt to follow cell changes by making their own observations when comparing images.

This process is even more involved when multiple objects are being tracked in three dimensions. Lippincott-Schwartz's group used a battery of computer programs to filter out all the different pieces of light spectra emitted by the organelles, to begin to bring the 3-D images and video into focus. The process, called "linear unmixing," required more than 32 cores of a computer work station to sift through 7 billion sets of six-color images, pixel by pixel.

Typically they would use expensive commercial software programs to stitch them into a 3-D volume to go about studying them. But these programs are expensive and time-consuming to use, and were not capable of the sophisticated analysis for tracking moving objects in order to make quantitative measurements of their behaviors and particularly how they interact.

Cohen's algorithm automates the entire process, which saves researchers a lot of time and it also lets them ask and answer more questions about what the cells are doing. He further verified the data by working with Drexel colleague Uri Herschberg, PhD, an associate professor in the School of Biomedical Engineering, Science and Health Systems and College of Medicine, to check it against 2-D images of the cells.

"It's some really impressive footage that gives biologists this ability to look deeper and deeper into live cells and see things they've never seen beforelike six different organelles in a living cell in true 3-D," said Cohen, a professor in Drexel's College of Engineering. "But it's also a lot of work to begin quantifying what they're seeingand that's where we can help, by using our program to automate big portions of that process and glean valuable data from it."

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Using the new technology to simultaneously look at six sets of organelles, Lippincott-Schwartz's teams at Janelia and at the National Institutes of Health are making exciting new observations. They are looking at how the organelles distribute themselves inside the cell, how often they interact with each other and where, when and how fast they move during various times in the cell's lifecycle.

"One very interesting outcome is that we found the largest organelle in the cell, which is the ER [endoplasmic reticulum], at any particular time point will be occupying about 25 percent of the volume of the cytoplasm, excluding the nucleus. But if you track the way it disperses through the cytoplasm over a short period of time, like 15 minutes, you see that it explores 95 percent of the whole cytoplasm during that time period," Lippincott-Schwartz said. "We can do this for all of the other organelles at the same time to see how the cytoplasm is being sensed through the dynamic motions of dispersive activities of these organelles."

Observing sub-cellular behavior is just the first application of this technology. Now that it has proven to generate usable data, the team will forge ahead to study what happens inside a cell when it is exposed to drug treatments and other common stresses on the system. The researchers suggest that it could be used to study many more than six types of microscopic objects. And it could help dig even deeper into the building blocks of lifeinto interactions of RNA particles and other proteins that play a role in a cell's function and the behavior of diseased cells.

"As these tools continue to improve they will give researchers both a better look at cell behavior and many options for gathering and analyzing that data," Cohen said. "They will be able to ask and answer increasingly complicated questions and that's going to lead to some very exciting and important discoveries."

Explore further: How plant cell compartments change with cell growth

More information: Alex M. Valm et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome, Nature (2017). DOI: 10.1038/nature22369

Journal reference: Nature

Provided by: Drexel University

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Researchers help provide first glimpse of organelles in action inside living cells - Phys.Org

May 25, 2017

Researchers at the University of Iowa have discovered that a molecule which can sense the swelling of fat cells also controls a signaling pathway that allows fat cells to take up and store excess glucose. Mice missing this protein, known as SWELL1, gain less weight (fat) than normal mice on a high-fat diet, but also develop diabetes.

"Although we have created a mouse that is resistant to weight gain by removing the SWELL1 protein, the mouse is not healthy; it has insulin resistance and glucose intolerance," says Rajan Sah, MD, PhD, assistant professor of internal medicine at the University of Iowa Carver College of Medicine and senior author of the study.

Type 2 diabetes is one of the more serious health problems associated with obesity. The disease makes cells less sensitive to insulin and causes blood sugar levels to become abnormally high. It is healthier for the body to store excess glucose as fat rather than have it circulating in the blood where it can damage blood vessels and nerves.

In healthy people, insulin released in response to high glucose levels acts on many different tissues to coordinate use or storage of the glucose. It triggers fat cells to take up excess glucose and store it as fat.

Sah's study, which was published recently in Nature Cell Biology, found that removing SWELL1 from fat cells in mice disrupts this insulin signaling pathway and prevents fat cells from taking up glucose.

Sah and his team homed in on SWELL1 because of several pieces of converging evidence. Fat cells have a tremendous capacity to expand - up to 30 times their normal volume in the context of obesity. It's also long been known that changes in fat cell size alters fat cell signaling.

Through exploratory experiments investigating cell swelling in fat cells from lean and obese mice as well as fat cells obtained from bariatric surgery patients, Sah and his team serendipitously identified SWELL1 protein as an essential component of fat cells' volume-sensing mechanism. From unrelated work by other researchers, they also knew that this protein was involved in a signaling pathway common to all cells. In fat cells this pathway regulates glucose uptake in response to insulin.

"We thought maybe this SWELL1 protein is what links the two pieces together - the size-sensing mechanism and the signaling pathway that responds to size changes by altering insulin sensitivity," explains Sah, who also is a member of the Fraternal Order of Eagles Diabetes Research Center, and the Abboud Cardiovascular Research Center at the UI.

The team's study showed that swelling of mouse or human fat cells, either artificially in a petri dish, or because the cells have expanded due to obesity, activates SWELL1 signaling. Removing SWELL1 from mouse fat cells knocks out this volume-sensing signal and disrupts the insulin signaling pathway used by fat cells to take up and store excess glucose. Mice missing SWELL1 have smaller fat cells, but also develop insulin resistance and glucose intolerance.

Interestingly, on a regular diet, mice missing SWELL1 had body weights, fat composition, and metabolism that were all essentially the same as a normal mouse. The only difference was they had no SWELL1 activity in their fat cells, as well as reduced ability to clear glucose from the blood and impaired insulin sensitivity (insulin resistance).

When the mice were put on a high-fat diet, the mice missing SWELL1 did not gain weight as fast as the normal mice but the insulin resistance and glucose intolerance became worse.

"The idea that fat is bad is not necessarily true," Sah says. "Too much fat is bad, and fat in the wrong places is bad, but fat in the right place and allowed to expand normally may be somewhat protective against diabetes.

"If fat cells can sense their own expansion, then SWELL1 protein might be the mechanism for that," he continues. "What we see here is what the cell does with the information that it is getting bigger. It turns on a signaling pathway that modulates glucose uptake and insulin sensitivity. From this discovery, we can start to look at whether we can target this modulation of insulin sensitivity in a therapeutic way."

Explore further: Your muscles can 'taste' sugar, research finds

More information: Yanhui Zhang et al, SWELL1 is a regulator of adipocyte size, insulin signalling and glucose homeostasis, Nature Cell Biology (2017). DOI: 10.1038/ncb3514

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Can fat 'feel' fat? Size-sensing protein controls glucose uptake and storage in fat cells - Phys.Org

Researchers from Princeton Universitys Department of Molecular Biology have identified a small RNA molecule that helps maintain the activity of stem cells in both healthy and cancerous breast tissue. The study, which will be published in the June issue of Nature Cell Biology, suggests that this microRNA promotes particularly deadly forms of breast cancer and that inhibiting the effects of this molecule could improve the efficacy of existing breast cancer therapies.

Stem cells give rise to the different cell types in adult tissues but, in order to maintain these tissues throughout adulthood, stem cells must retain their activity for decades. They do this by self-renewing dividing to form additional stem cells and resisting the effects of environmental signals that would otherwise cause them to prematurely differentiate into other cell types.

Many tumors also contain so-called cancer stem cells that can drive tumor formation. Some tumors, such as triple-negative breast cancers, are particularly deadly because they contain large numbers of cancer stem cells that self-renew and resist differentiation.

To identify factors that help non-cancerous mammary gland stem cells (MaSCs) resist differentiation and retain their capacity to self-renew, Yibin Kang, the Warner-Lambert/Parke-Davis Professor of Molecular Biology, and colleagues searched for short RNA molecules called microRNAs that can bind and inhibit protein-coding messenger RNAs to reduce the levels of specific proteins. The researchers identified one microRNA, called miR-199a, that helps MaSCs retain their stem-cell activity by suppressing the production of a protein called LCOR, which binds DNA to regulate gene expression. The team showed that when they boosted miR-199a levels in mouse MaSCs, they suppressed LCOR and increased normal stem cell function. Conversely, when they increased LCOR levels, they could curtail mammary gland stem cell activity.

Kang and colleagues found that miR-199a was also expressed in human and mouse breast cancer stem cells. Just as boosting miR-199a levels helped normal mammary gland stem cells retain their activity, the researchers showed that miR-199a enhanced the ability of cancer stem cells to form tumors. By increasing LCOR levels, in contrast, they could reduce the tumor-forming capacity of the cancer stem cells. In collaboration with researchers led by Zhi-Ming Shao, a professor at Fudan University Shanghai Cancer Center in China, Kangs team found that breast cancer patients whose tumors expressed large amounts of miR-199a showed poor survival rates, whereas tumors with high levels of LCOR had a better prognosis.

Kang and colleagues found that LCOR sensitizes cells to the effects of interferon-signaling molecules released from epithelial and immune cells, particularly macrophages, in the mammary gland. During normal mammary gland development, these cells secrete interferon-alpha to promote cell differentiation and inhibit cell division, the researchers discovered. By suppressing LCOR, miR-199a protects MaSCs from interferon signaling, allowing MaSCs to remain undifferentiated and capable of self-renewal.

The microRNA plays a similar role during tumorigenesis, protecting breast cancer stem cells from the effects of interferons secreted by immune cells present in the tumor. This is a very nice study linking a normal and malignant mammary gland stem cell program to protection from immune modulators, said Michael Clarke, the Karel H. and Avice N. Beekhuis Professor in Cancer Biology at Stanford School of Medicine, Institute of Stem Cell Biology and Regenerative Medicine, who first discovered breast cancer stem cells but was not involved in this study. It clearly has therapeutic implications for designing strategies to rationally target the breast cancer stem cells with immune modulators.

Toni Celi-Terrassa, an associate research scholar in the Kang lab and the first author of the study, said, This study unveils a new property of breast cancer stem cells that give them advantages in their interactions with the immune system, and therefore it represents an excellent opportunity to exploit for improving immunotherapy of cancer.

Interferons have been widely used for the treatment of multiple cancer types, Kang said. These treatments might become more effective if the interferon-resistant cancer stem cells can be rendered sensitive by targeting the miR-199a-LCOR pathway.

This article has been republished frommaterialsprovided byPrinceton University. Note: material may have been edited for length and content. For further information, please contact the cited source.

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RNA Molecule that Shields Breast Cancer Stem Cells from Immune System Identified - Technology Networks

The best part of high school biology was the movies. Some of them basically amounted to weird close-up fetish porn, sure. Other high school biology videos were actually educational, including the only one that ever taught me anything about the human cell, Inner Life of a Cell. But a new video puts them all to shame.

Rather than using boring old computer-generated graphics, a team of American scientists made what might be the most complex video of a cell in action yet. Its all based on a real monkey cell, analyzed with a series of proteins, dyes and a special kind of microscope.

Sure, other microscopes have made videos of cells moving, or pairs of cell parts, called organelles, interacting. But this is the first time were doing this many compartments in live cells, Sarah Cohen, scientist at the National Institutes of Health, told Gizmodo.

The video shows the chaotic movement of lipid (fat) droplets traveling through different parts of the cell. You might remember that the cell is a factory, where the mitochondria is the power plant. The endoplasmic reticulum makes stuff like proteins and lipids, and the golgi apparatus packages them. The lysosomes and peroxisomes break things down. Now, you can see all those organelles doing their jobs simultaneously, thanks to the research published today in the journal Nature.

Getting such a detailed look at the cells function was surprisingly intuitive. The researchers tagged the different organelles with proteins that fluoresce in response to different colors of light. They also soaked the cell in a dye that sticks to lipid molecules so they could visualize how they traveled between organelles. A special microscope looks at the cell in slices, rather than viewing the entire thing at once, which prevents the light from killing it. The study added evidence that the endoplasmic reticulum serves as the central hub of the cell, and looks like a mesh, interacting with nearly everything else.

Other researchers are excited about the new tool. [The study authors] breakthrough opens up wide-ranging opportunities for exploring the molecular mechanisms that underpin the organelle communitys dance, Sang-Hee Shim, assistant chemistry professor at Korea University wrote in commentary for Nature.

Like any scientific method, there are limitations to this one. Too much time under the microscope could harm the cell, for example. Plus, the resolution isnt as good as more invasive microscope techniques, so it might muddle some of the finer details, like the interaction point between the mitochondrion and the endoplasmic reticulum, said Shim.

But the method will definitely help scientists figure out exactly how the cell works and how molecules move around inside of it. This could be useful for things like drug screening or personalized medicine, applications where you need to know exactly where and how a molecule is moving down to the cellular level, said Cohen.

Really, its just cool as hell.

[Nature]

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This Is the Most Complex Video of a Real Cell Ever Made - Gizmodo