A (BAF)ling story of nuclear envelope repair

From the Hatch lab, Basic Sciences Division.

“What used to be a cell with components, the reality of which was often a matter of dispute and functions as a rule unknown is now a system of great organizational sophistication with units for the production of components essential to life and units for disposal of worn out parts and for defense against foreign organisms and substances.”

Nobel Prize Press release, 1974.

While we cannot know for sure who first saw the nucleus, much credit is given to Robert Brown for providing the earliest detailed accounts of the nucleus and for coining the term from the Latin word for seed or kernel. In the course of the past 200 years, large advances have been made in our understanding of the nucleus as the chief subcellular compartment and the major evolutionary event that separated eukaryotic from prokaryotic organisms. Key to the function of the nucleus is the separation of DNA from cytoplasmic processes via the nuclear envelope, a dynamic protein and membrane compartment that can lose and regain compartmentalization during cell division. The Hatch lab (Basic Sciences Division), studies the organization and dynamics of the nuclear envelope to understand how changes in this structure cause human genetic diseases and drive cancer development.

The nuclear envelope is frequently depicted as a static membrane barrier that separates the chromatin from the cytosol. However, this textbook depiction of the nuclear envelope is far from reality. Recent work from the Hatch lab and others supports a more dynamic model where membrane integrity loss is not only common but even beneficial. “The nucleus is compartmentalized by membranes to protect genetic processes in the cell, but these membranes can rupture in response to confining forces, such as migrating through a dense tissue, or as a result of disease linked mutations,” explains Dr. Ali Young, a postdoc in the Hatch lab. “It turns out that cells have robust nuclear membrane repair mechanisms, which are likely critical for the cell to survive these events,” she added.

Model of nuclear membrane repair. BAF binds to the exposed chromatin and recruits transmembrane proteins (NETs) in the ER to repair the gap. BAF indirectly recruits the ESCRT-III membrane remodeling complex, presumably to seal leftover holes. Depletion of BAF, ESCRT-III, or NETs leads to impaired membrane repair.
Model of nuclear membrane repair. BAF binds to the exposed chromatin and recruits transmembrane proteins (NETs) in the ER to repair the gap. BAF indirectly recruits the ESCRT-III membrane remodeling complex, presumably to seal leftover holes. Depletion of BAF, ESCRT-III, or NETs leads to impaired membrane repair. Adapted from Maciejowski and Hatch, Ann Rev Cell Dev Biol, in press.

Using live-cell imaging and other advanced light microscopy techniques, the Hatch lab seeks to dissect the molecular mechanisms underlying nuclear envelope rupture in cancer cells. In a recent BioRxiv preprint, they investigated the role of BAF (barrier to autointegration factor), in maintaining the integrity of mammalian nuclear envelope during interphase, the resting phase between two successive cell divisions. Dr. Young, the first author of the study, explained the significance of their findings. “This work provides new detail on nuclear envelope repair in cancer cells that will help flesh out pathways that are just beginning to be understood,” she said. “Our model is that the chromatin binding protein BAF is critical to recruit membrane from the ER to repair the nucleus rupture, but that other mechanisms can step in to repair these holes when BAF is absent. Interestingly, small nuclear membrane holes are less sensitive to BAF loss. We think the cell has multiple ways of repairing the nucleus depending on the size of the hole to be repaired and are interested in finding these other mechanisms.”

Dr. Young and her colleagues used a cancer cell line that has been genetically modified to undergo spontaneous membrane ruptures more frequently and found fluorescently labeled BAF at rupture sites. Genetic depletion of BAF delayed nuclear membrane repair, which was efficiently rescued by WT BAF, but not by a mutant lacking the LAP2, emerin, and MAN1 (LEM)-protein binding domain. Consistent with their findings and with the previously established role of LEM proteins in maintaining nuclear envelope integrity, genetic depletion of the LEM-domain nuclear transmembrane proteins (NETs), LEMD2 or emerin, phenocopied BAF depletion.

Dr. Young further commented on the study: “the most exciting part of this project was being able to watch the cells as they go through the nuclear membrane rupture and repair process using live cell microscopy and fluorescent proteins. The nucleus just bursts open spontaneously, and all of its soluble components come flooding out of it. Somehow the cells can repair these injuries even when they happen over and over again. It was motivating to discover more about how this process works because it could be a weakness in some cancers to be exploited for treatment. For example, targeting factors necessary for nuclear membrane repair could prevent some cancer cells from surviving migration.”

The study suggests that while BAF facilitates the repair of large membrane ruptures, in part by recruiting transmembrane nuclear envelope proteins, while small ruptures are repaired by a BAF-independent mechanism. Future work will focus on characterizing the different pathways for repairing the nucleus and defining the circumstances where each pathway is needed. “We and others have shown that in addition to BAF, several other proteins go to break sites in the nucleus. Some of these proteins depend on BAF to get to the break site, but we don’t know if this is true for all of the proteins involved. We predict that one or more of these proteins can repair the nucleus without BAF and we are excited to identify which ones work in their own pathway,” added Dr. Young.

Young A, Gunn A, Hatch E. 2020. BAF facilitates interphase nuclear envelope repair through recruitment of nuclear transmembrane proteins. bioRxiv.

UW/Fred Hutch Cancer Consortium member Emily Hatch contributed to this work.

This work was supported by funding from the National Institutes of Health.