In apoptosis, cell death spreads through perpetuating waves

Inside a cell, death often occurs like the wave at a baseball game.

What starts with two hands flung skyward prompts another, and another, until the wave has rippled far and wide across the whole stadium. This kind of a rolling surge, spurred by the activity of one or a few things, is known as a trigger wave. A new study out of the Stanford University School of Medicine has found that this phenomenon guides one of the most well-known and widespread forms of cell death: apoptosis.

It’s not the first time trigger waves have been identified in the microcosms of life. The cell cycle, a cornerstone of cell biology in which cells divide to make new cells, regulates production via trigger waves, too. So do neuronal action potentials, which allow neurons to pass signals via electrical impulse. And it likely doesn’t end there.

“This work is another example of how nature makes use of these trigger waves — things that most biologists have never heard of — over and over again,” said James Ferrell, MD, PhD, professor of chemical and systems biology and of biochemistry at Stanford. “It is a recurring theme in cell regulation. I bet we’ll start to see it in textbooks soon.”

One of the better-understood forms of cell death, apoptosis still manages to mystify scientists. “Sometimes our cells die when we really don’t want them to — say, in neurodegenerative diseases. And sometimes our cells don’t die when we really do want them to — say, in cancer,” Ferrell said. “And if we want to intervene, we need to understand how apoptosis is regulated.”

The study will be published in Science Aug. 10. Ferrell is the senior author. Postdoctoral scholar Xianrui Cheng, PhD, is the lead author.

Spreads like wildfire

Trigger waves require two main elements: a positive feedback loop and a threshold think — falling dominoes. One domino collapses on another and triggers that domino to topple onto the next. The threshold is the force necessary to completely knock the tile over; a domino just shy of its threshold would teeter and rock back into a vertical position, whereas one that’s reached the threshold would fall. Trigger waves in an apoptotic cell are governed by that same phenomenon. Once cell death is initiated, by way of disease or something else, specific killer proteins in the cell, called caspases, activate. These proteins then float to other caspases and activate them; those follow suit until the entire cell has to pack it in.

“It spreads in this fashion and never slows down, never peters out,” Ferrell said. “It doesn’t get any lower in amplitude because every step of the way it’s generating its own impetus by converting more inactive molecules to active molecules, until apoptosis has spread to every nook and cranny of the cell.”

To see how death takes over a single cell, Cheng and Ferrell used Xenopus frog eggs. One egg is a single cell, and as cells go, these are enormous, making them a prime candidate to observe how death spreads from one end of the cell to the other, which can be done with the naked eye.

To start, the two scientists took fluid from the egg and inserted it into Teflon tubes, which were several millimeters long, and initiated apoptosis through a molecular “death signal.” By using a fluorescent technique linked to the activation of apoptosis, Ferrell and Cheng could watch as the bright green glow moved its way down the tube at a constant speed, indicating that apoptosis was spreading via trigger waves, as opposed to some other more rudimentary mechanism, such as diffusion, which slows down as it moves.

The question was, did apoptosis also spread like that in cells as they naturally occur?

Turning to fluorescence microscopy here proved more difficult, as intact frog eggs are quite opaque. However, Cheng and Ferrell noticed that when frog eggs die, a sort of ripple of pigmentation occurs at the egg’s surface. The scientists saw that during death, a dark ripple moved like a curved line across the egg at a constant speed from one side to the other. The speed of this surface wave, which was constant and did not slow down, tipped them off to trigger waves here too. So to further confirm, they analyzed individual dying eggs: Every egg that had undergone this surface wave contained activated caspase, whereas the eggs that had not yet undergone the waves did not — more evidence that trigger waves propagate cell death in an intact cell too.

A wave of trigger waves

So far, apoptosis is the only form of cell death in which trigger waves have been identified, but Ferrell is investigating other processes in biology to see if the continual waves might play a role.

Now, they’re looking into whether trigger waves might be responsible for how our innate immune response spreads from cell to cell. Viruses spread from cell to cell through trigger waves, so it makes sense that our initial line of immune defense might employ the same tactic.

“We have all this information on proteins and genes in all sorts of organisms, and we’re trying to understand what the recurring themes are,” Ferrell said. “We show that long-range communication can be accomplished by trigger waves, which depend on things like positive feedback loops, thresholds and spatial coupling mechanisms. These ingredients are present all over the place in biological regulation. Now we want to know where else trigger waves are found.”

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New lung cell type discovered: A previously unknown airway cell type may be a key to efforts to cure cystic fibrosis

In separate studies published online in Nature on Aug. 1, two independent research teams report the discovery of a new, rare type of cell in the human airway. These cells appear to be the primary source of activity of the CFTR gene, mutations to which cause cystic fibrosis, a multiorgan disease that affects more than 70,000 people worldwide.

Despite decades of study on CFTR and progress in treating the disease, there is still no cure. The new findings show that CFTR activity is concentrated in a small, previously unknown population of cells, which serve as promising targets for future therapeutic strategies against cystic fibrosis.

The researchers named the cells “pulmonary ionocytes” due to similarities with ionocytes, a type of cell found in the gills of freshwater fish and frog skin, which regulate salt balance.

One group was led by scientists from Harvard Medical School (HMS) and the Novartis Institutes for Biomedical Research (NIBR). The other group was led by HMS researchers based at Massachusetts General Hospital and scientists at the Broad Institute of MIT and Harvard.

“As researchers work toward cures for cystic fibrosis, knowing you are looking at 1 percent of the cell population seems essential for any type of trouble shooting to improve a therapy or develop new therapies,” said Allon Klein, co-corresponding author of one of the Nature studies and assistant professor of systems biology at HMS.

The studies also revealed the characteristics of other new, rare and poorly understood cell types, which expands the current understanding of lung biology and disease.

“Cystic fibrosis is an amazingly well-studied disease, and we’re still discovering completely new biology that may alter the way we approach it,” said Jayaraj Rajagopal, co-corresponding author of the second study and HMS professor of medicine at Mass General. “We have the framework now for a new cellular narrative of lung disease.”

Single-cell surprise

Both teams set out to build an atlas of the cells that make up the airway. Using single-cell sequencing technology, they analyzed gene expression in tens of thousands of individual cells isolated from human and mouse airways — one cell at a time.

Comparing patterns of gene expression and using previously described cells as references, the teams created comprehensive catalogues of different cell types and states, as well as their abundance and distribution.

The teams’ analyses mapped out the genetic identities of both known and previously undescribed cell types. One new cell type, which they named pulmonary ionocytes, was particularly striking as these cells expressed higher levels of CFTR than any other cell.

Identified in the late 1980s, CFTR (cystic fibrosis transmembrane conductance regulator) codes for a protein that transports chloride ions across cell membranes. Mutations to CFTR can lead to the buildup of thick mucus in the lung, pancreas and other organs, which in turn leads to frequent respiratory infections and other symptoms that characterize cystic fibrosis. Scientists have long assumed that CFTR is expressed at low levels in ciliated cells, a common airway cell type.

The new studies, however, suggest that the majority of CFTR expression occurs in pulmonary ionocytes, which make up only around 1 percent of airway cells. Klein, along with Aron Jaffe, a co-leader of respiratory disease research at NIBR, and colleagues additionally showed that the activity of CFTR, not just its expression, relates to the number of pulmonary ionocytes in the tissue.

“With single-cell sequencing technology, and dedicated efforts to map cell types in different tissues, we’re making new discoveries — new cells that we didn’t know existed, cell subtypes that are rare or haven’t been noticed before, even in systems that have been studied for decades,” said Broad core institute member and MIT professor of biology Aviv Regev, co-corresponding author of the study with Rajagopal.

When Regev, Rajagopal and colleagues disrupted a critical molecular process in pulmonary ionocytes in mice, they observed the onset of key features associated with cystic fibrosis, notably the formation of dense mucus. This finding underscores how important these cells are to airway-surface regulation, the researchers said.

Together, the teams’ discoveries point to new strategies for treating cystic fibrosis, such as increasing the amount of pulmonary ionocytes to increase the amount of CFTR activity, said Jaffe, who is co-corresponding author of the study with Klein.

The identification of these cells can also help guide teams trying to use gene therapy to correct CFTR mutations, the authors suggest.

“We can use this information to be a bit more clever when we devise therapeutic approaches to cystic fibrosis,” Jaffe said.

Here and rare

The teams’ analyses also shed light on new, rare or poorly described cell states and subtypes, and characterized changes to certain cells after injury or during development.

Klein, Jaffe and colleagues, for example, identified cell states that emerge or expand following damage or injury by studying regenerating tracheas. Using InDrops, a single-cell sequencing technology developed at HMS by Klein and colleagues, at multiple points during regeneration, the team could track how cell states specific to the injury response transitioned over time.

Rajagopal, Regev and colleagues developed a new method called Pulse-Seq to monitor development of cell types from their progenitors in the mouse airway, which were verified in human tissue. By adding a label to progenitor cells, they showed that mature cells in the airway arise from a common progenitor known as basal cells.

A comprehensive atlas of cell types and their genetic fingerprints, both in normal conditions and in development and regeneration, serves as potential baseline data for future studies of diseases and other health-related conditions, according to the research teams.

Rajagopal, Regev and colleagues, for example, found that a gene linked to asthma development is specifically expressed by ciliated cells. Another gene linked with asthma is expressed in tuft cells, which separated into at least two groups — one that senses chemicals in the airway and another that produces inflammation. These findings may inform understanding and treatment of the disease.

“We’ve uncovered a whole distribution of cell types that seem to be functionally relevant,” Rajagopal said. “What’s more, genes associated with complex lung diseases can now be linked to specific cells that we’ve characterized. The data are starting to change the way we think about lung diseases such as cystic fibrosis and asthma.”

Lindsey Plasschaert, postdoctoral researcher at NIBR, and Rapolas Žilionis, graduate student at Vilnius University, Lithuania and visiting scholar at HMS, are co-first authors, and Klein and Jaffe are co-corresponding authors of the study “A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.”

Additional authors include Rayman Choo-Wing, Virginia Savova, Judith Knehr and Guglielmo Roma. The study was supported by an award by the Burroughs Wellcome Fund, an Edward Mallinckrodt Jr. Foundation Grant, the Lithuanian Education Exchanges Support Foundation and the National Cancer Institute (R33CA212697-01).

Daniel Montoro, graduate student at HMS, and Adam Haber and Moshe Biton, both postdoctoral fellows at the Broad, are co-first authors, and Rajagopal and Regev are co-corresponding authors of the study “A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.”

Additional authors include Vladimir Vinarsky, Brian Lin, Susan Birket, Feng Yuan, Sijia Chen, Hui Min Leung, Jorge Villoria, Noga Rogel, Grace Burgin, Alexander Tsankov, Avinash Waghray, Michal Slyper, Julia Waldman, Lan Nguyen, Danielle Dionne, Orit Rozenblatt-Rosen, Purushothama Rao Tata, Hongmei Mou, Manjunatha Shivaraju, Hermann Bihler, Martin Mense,

Guillermo Tearney, Steven Rowe and John Engelhardt. The study was supported in part by the Klarman Cell Observatory at the Broad, the Manton Foundation, Howard Hughes Medical Institute, New York Stem Cell Foundation, the Harvard Stem Cell Institute, the Human Frontier Science Program and the National Institutes of Health.

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New method discovered to view proteins inside human cells: Scientists develop tagging device using Ferritin

Scientists at the University of Warwick have created a new way to view proteins that are inside human cells.

Using Ferritin, a large protein shell that our cells use to store iron, the researchers have found a method they have called FerriTag that allows an electron microscope (EM) to view proteins precisely unlike current methods.

The method allowed the scientists to enable the cell to make the tag itself avoiding damage caused by placing it from the outside of the cell. Their paper FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy is published in the journal Nature Communications.

The team set out to precisely localise a protein found in clathrin-coated pits. These are 100 nm wide entry points used by viruses to invade cells and infect them. Using FerriTag, the team were able to see where the protein is found in the pit and on the inside face of the cell’s surface.

The team was led by Dr Stephen Royle Associate Professor and Senior Cancer Research UK Fellow at Warwick Medical School. He said: “Proteins do almost all of the jobs in cells that scientists want to study. We can learn a lot about how proteins work by simply watching them down the microscope. But we need to know their precise location.”

Although light microscopy can be used to view proteins move around the resolution is low, so seeing a protein’s precise location is impossible. This can be overcome by using electron microscopy which gives a higher resolution.

To allow proteins to be viewed by both microscopes and correlate them, the research team developed a method of tagging the proteins so that they can be seen by both types of equipment.

Tagging is widely used and several tags are available however they have established drawbacks; some are not precise enough, or they don’t work on single proteins. To overcome this Dr Royle’s lab created a new tag and fused it with a fluorescent protein.

Dr Royle’s team named the new technique FerriTag because it is based on ferritin which can be viewed by an electron microscope because iron scatters electrons.

Dr Royle said his lab had to defeat another obstacle: “When Ferritin is fused to a protein, we end up with a mush. So, we altered Ferritin so that it could be attached to the protein of interest by using a drug.

“This meant that we could put the FerriTag onto the protein we want to image in a few seconds.

“The cool thing about FerriTag is that it is genetically encoded. That means that we get the cell to make the tag itself and we don’t have to put it in from outside which would damage the cell.”

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