Developmental Biology - Cell Death|
Star Wars 'Burst' of Activity Before Cell Death
A novel platform reveals dynamic movement in cells just before death...
Studying the movement of tiny cells is no small task. For chromatin, the group of DNA, RNA, and protein macromolecules packed within our genome — motion is integral to the regulation of how our genes get expressed or repressed.
"Understanding macromolecular motion is critical, but scientists know very little about it. Part of the reason is because we lack instrumental techniques to observe those processes."
Vadim Backman PhD, Walter Dill Scott Professor of Biomedical Engineering; The Center for Advanced Regenerative Engineering; and, The Center for Physical Genomics and Engineering, Northwestern University, Evanston, Illinois, USA.
Now, a research team at the McCormick School of Engineering led by Backman has developed a new optical technique to study the movement of cells without using labels or dyes to track them. The innovative method has also revealed an undiscovered phenomenon that may play a role in the earliest stages of cell death.
The team's insights were published on April 10 in the journal Nature Communications.
While scientists can currently track the movement of cells using molecular dyes or labels, the practice comes with limitations. Dyes are toxic and alter the behavior of cells before, eventually, killing them. Labels are attached to cells, can be toxic or result in photobleaching, and may alert the motion of the very molecules they label.
The new technique, called dual-PWS, is label-free and can image and measure macromolecular motion without using dyes.
Building off of a quantitative imaging technique previously created by Backman called Partial Wave Spectroscopy (PWS), the platform uses interference and pattern change from backscattered light to monitor both the macromolecular structure of cells along with their dynamic movement.
"Critical processes like the transcription of a gene or the repair of damaged proteins requires the movement of many molecules simultaneously within a highly packed, complex environment," explains Scott Gladstein, a PhD student in Backman's lab and the study's first author.
"As an imaging platform with the capability to measure both intracellular structure and macromolecular dynamics in living cells, with a sensitivity to structures as small as 20nm with millisecond temporal resolution, the dual-PWS is uniquely suited to allow us to study these processes."
Researchers applied dual-PWS by studying the nanoscale structural and dynamic changes of chromatin in eukaryotic cells in vitro. Using ultraviolet light to induce cell death, they measured how movement of cell chromatin changed.
"It makes sense that as cells are about to die, their dynamics lessen," Backman explains. "The facilitative motion that exists in live cells to help express genes and change their expression in response to stimuli disappear. We expected that."
What researchers didn't expect was to witness a biological phenomenon for the first time. According to Backman, a cell reaches a "point of no return" during decay, where even if the source of cellular damage is stopped, the cell is unable to repair itself back into a functioning state.
Using dual-PWS, researchers observed that just prior to this turning point, the cells' genomes burst with fast, instantaneous motion, with different parts of the cell moving seemingly at random.
"Every cell we tested that was destined to die experienced this paroxysmal jerk. None of them could return to a viable state after it took place," said Backman, who leads Northwestern's new Center for Physical Genomics and Engineering.
The team is unclear why or how the phenomenon, called "cellular paroxysm", occurs. Backman originally wondered if the movement could be due to ions entering the cell, but such a process would have taken too long. The uncoordinated motions of the cellular structures occurs in milliseconds.
"There's simply nothing in biology that moves that fast." Members of his lab were so surprised by the results, they joked the phenomenon could be explained as "Midichlorians" leaving the cell — a reference to the chemical embodiment of "The Force" in Star Wars films.
Vadim Backman PhD.
While cellular paroxysms remain a mystery for now, Backman believes the team's findings highlight the importance of studying the macromolecular behavior of live cells. The more insights researchers can gain about chromatin, the more likely they can one day be able to regulate gene expression, which could change how people are treated for diseases like cancer and Alzheimer's.
"Every single biological process you can imagine involves some sort of macromolecular rearrangement," Backman adds. "As we expand our research, I can't help but wonder, 'What will we find next?'"
Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells.
Scott Gladstein, Luay M. Almassalha, Lusik Cherkezyan, John E. Chandler, Adam Eshein, Aya Eid, Di Zhang, Wenli Wu, Greta M. Bauer, Andrew D. Stephens, Simona Morochnik, Hariharan Subramanian, John F. Marko, Guillermo A. Ameer, Igal Szleifer and Vadim Backman.
Northwestern Engineering's Guillermo Ameer, Daniel Williams Hale Professor of Biomedical Engineering, and Igal Szleifer, Christina Enroth-Cugell Professor of Biomedical Engineering, also contributed to the research.
This work was supported by fellowships and grants from the National Institutes of Health grant R01-GM105847, R01 CA200064, R33CA225323, 1R01CA228272, R01CA225002, R01EB016983, R01CA165309, K99 GM123195, the National Cancer Institute grant U54-CA193419 (CR-PS-OC), the NIH Chemistry of Life Processes Pre-doctoral Training grant #5T32GM105538-03 and the National Science Foundation grant CBET-1240416. An award of computer time was provided by the INCITE program. This research used resources of the Argonne Leadership Computing Facility, which is a DOE Office of Science User Facility supported under Contract DE-AC02-06CH11357.
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"Midichlorians" leaving the cell
as in Star Wars. Control cells (top) and UV irradiated cells (bottom) reflect how UV irradiation stalls DNA replication. CREDIT: Vadim Backman PhD.