Despite countless bruises, burns, blisters, cuts, and bug bites, our skin remains resilient. This is largely due to a number of bodily repair mechanisms in place that address the damage. Professor Shirin Bahmanyar and graduate student Lauren Penfield GRD ’20 of Yale University have been working to understand an analogous system on the subcellular level—that of nuclear envelope repair that protects DNA from harmful substances in the cell.
They learned more about the repair process in which a protein called lamin, a known structural support protein of the nuclear envelope, also acts to prevent holes from forming in the membrane and aids the repair process when disruptions of the membrane do occur. They further developed a timeline for the cellular repair process and opened the door to a new realm of nuclear envelope dynamics studies.
The security guard of the cell
The nuclear envelope is a two-sided membrane that encloses and protects genetic information from potential harmful substances inside cells. Mmuch like the security guards surrounding a government official in a crowd, the envelope forms a barrier and keeps away threats. The nuclear envelope is reinforced by lamins on the inner side of the membrane, forming a mesh-like framework that supports the envelope’s shape and structure.
By revealing more about its dynamic structure and functions, rrBahmanyar and her colleagues have challenged the misconception that the nuclear envelope is a static structure prior to cell divisionms. Two of its most fundamental roles are inherent in the way it encloses DNA. First, it must be able to allow RNA, a message carrier that is used to convert DNA into proteins, into the cell. This is accomplished via structured holes in the membrane known as nuclear pore complexes (NPCs). Secondly, it must be able to disintegrate during cell division, so that pairs of chromosomes can separate to opposite sides of the parent cell, and subsequently reform in each of the daughter cells.
In addition, recent research has led to better understanding of the specific roles played by the nuclear membrane in nuclear compartmentalization and tethering DNA to specific regions of the nucleus. The consequences of its massive importance in many cellular functions are dire for those with laminopathies, mutations or alterations in the genes encoding lamins. Muscular dystrophy, cardiomyopathy, and dermopathy are common examples of laminopathiese in which, in the absence of lamins, the tissues are easily damaged and destroyed. .
Sleeping on the job
Unfortunately, the study of these conditions is limited by poor overall understanding of how two important functions of lamin, supporting the structure of the nuclear envelope and managing the organization of the nucleus, work together. It is often difficult for researchers to identify which specific roles are responsible for certain defects. To isolate the structural role of lamin, a model system was developed in the embryos of Caenorhabditis elegans (C. elegans), a type of roundworm. Because these embryos, in their early stages, Amake little to no RNA out of DNA, this model can be used to isolate the effects of mutations affecting the structural role of lamins.
In studying the structural role of lamins in C. elegans embryos, Bahmanyar and Penfield found that a certain mutation would lead to the disappearance of the nuclear envelope prior to the embryo’s first cell division. Curiously, the nuclear envelope in the roundworms with this mutation appeared to act normally during the earliest stages of single-celled development when the two parental nuclei are separate.
However, r when the two parental nuclei of the embryo are pulled together during pronuclear migration, a later high-stress stage of development, the membrane disintegrated. In other words, the security guards of the nuclear envelope appeared to be doing their job, but, when a threat appeared, they didn’t do anything.
To tease out the effect of this lamin mutation on the permeability of the nuclear envelope, the researchers expressed a fluorescent tubulin molecule, a structural protein that luminesces when exposed to certain wavelength light, in the cell and used fluorescent microscopy to track whether the tubulin was being excluded and removed from the nucleus as it normally should be. They found that in mutant-lamin roundworm embryos, the fluorescent tubulin protein permeated into the nucleus.
They further recognized that the fluorescent tubulin molecule could penetrate the nucleus of roundworms with the mutant lamin even during the early stages of embryonic development. This suggests some disturbance of the mutant-lamin membrane even during times of low stress when the nuclear membrane appeared normal. Furthermore, they found that 82 percent of the mutant nuclei were able to remove the fluorescent protein following the initial penetration.
“The lamin mutant nuclear membrane must have had transient gaps opening and closing,” Penfield said. However, never before had it been shown that live organisms could reestablish the nuclear membrane barrier following disruption. “We were the first to show evidence of repair after sudden and complete loss of nuclear compartmentalization in an intact organism,” Bahmanyar said.
To better understand these holes and learn more about the mechanism by which normal lamin proteins and a healthy nuclear envelope respond to them, the researchers artificially punctured the nuclear envelope using laser light. Again, the permeability of the nuclear membrane to the fluorescently-tagged tubulin molecule was used as a marker. The punctures caused an initial equilibration of tubulin between the cytosol and nucleus followed by a decrease in nuclear tubulin, indicating the recovery of the nuclear envelope barrier.
This demonstrates that there must be a mechanism of repair and restructuring of the nuclear envelope. In the lamin mutants, this mechanism is sufficient to maintain the envelope’s integrity following the initial transient disturbances before pronuclear migration. “As long as they can repair these ruptures, they can still survive,” Bahmanyar said. However, when the forces pulling the nuclei together create too much strain on the nuclear envelope, the mutant-laminss tear, leading to chromosome scattering and loss of the nuclear membrane barrier.
Calling the police
To understand the interactions between lamin and other proteins involved in repairing holes in the membrane, Bahmanyar and her team investigated various proteins on the nuclear membrane in the same embryo system. First, nuclear pore complex proteins were visualized during early embryonic development. In most of the lamin mutants, a distinct section of the nuclear membrane lacking nuclear pore complexes developed. Near this gap of nuclear pore complexes, which was verified to be where the envelope ruptures occur, chromatin, the regular state of DNA in the nucleus, was seen to condense rapidly, potentially leading to DNA damage and later issues with nuclear organization. This strongly suggested that lamin plays a critical role in organization and distribution of nuclear membrane proteins, which is in turn related to membrane stability.
To test this idea, a protein called Endosomal Sorting Complexes Required for Transport-III (ESCRT-III), which is known to be involved in nuclear repair, was fluorescently labeled so that its movement in the cells could be tracked. As expected, ESCRT-III accumulated near the damaged areas that lacked nuclear pore complexes. A nuclear envelope protein known to bring ESCRT-III to seal holes during regular cell division, called LEM-2, amassed at the same location. Although lamin was not required for this repair process, it was also found in the same region, and likely is involved in stabilizing the rupture while repair occurs. However, in these lamin-depleted mutants, these attacks occur more frequently and do more damage to the precious cargo within.
Hiring the Secret Service?
This research has promising potential for people with laminopathies, which are often life-threatening or fatal. It may be possible to synthetically engineer normal lamin and transport it to the nuclear membrane of cells. Alternatively, with developing genetic engineering technologies, researchers might be able to manipulate cells to produce fully functional lamin instead of the mutated version. It is not unreasonable to predict that, soon, we may understand the nuclear envelope system well enough to synthetically engineer improved lamin to increase the security it provides for our DNA—essentially, hiring the secret service to stand guard.
Furthermore, it is hypothesized that migrating cancer cells would overuse this nuclear rupture-and-repair process to fit through small blood vessels when traveling through the bloodstream. These migrating cancer cells are critical for metastasis formation—the main cause of cancer deaths. “If we have molecular markers for nuclear envelope rupture and repair, we might be able to better detect migrating cancer cells,” Penfield said. Then, the immune system might be programmed to attack and kill these cells before they turn into new tumors or they could be removed via other methods.
Finally, knowing that the membrane plays an important role in accepting viral DNA and reforming after incorporating it, understanding lamins and the nuclear envelope could be helpful in the field of genetic engineering. A better knowledge of lamin proteins, how the nuclear envelope works to repair itself, and what treatments might increase the efficiency of DNA incorporation would take us one step closer to a genome editing therapy. While a challenge to study due to its rare activity, the nuclear envelope is nevertheless a fundamental cellular component. Future biomedical technologies will require an understanding of this complicated and fascinating organelle, a challenge Bahmanyar’s group shows no signs of shying away from.