Each cell contains well-defined compartments, each of which has a specific function. The nuclear envelope, for example, is a highly regulated membrane barrier that separates the nucleus from the cytoplasm; therefore, maintaining the integrity of the nuclear envelope is essential. In response to different stressors, such as cell migration, nuclear membranes can rupture, resulting in the loss of compartmentalization of the nucleus. “Nuclear membrane rupture is defined as the rapid loss of nucleus compartmentalization in interphase resulting in the mixing of nuclear and cytoplasmic proteins and organelles” the authors said. These ruptures must be repaired within minutes to restore a functional nucleus. Although the nuclear membrane can repair itself efficiently, ruptures can still cause DNA damage and changes in cellular signaling. The mechanism by which nuclear membrane rupture is regulated remains unclear, and there are few factors known to regulate nuclear membrane stability. A mechanistic understanding of nuclear membrane integrity is the goal pursued by Dr. Emily Hatch, Assistant Professor in the Basic Sciences Division, in a new publication from her lab. A study led by Dr. Amanda L. Gunn, a former Hatch lab staff scientist, developed a new automated analysis pipeline to quantify nuclear membrane rupture frequency in fixed cells in conjunction with a siRNA screening. Using this approach, the protein CTDNEP1 was identified as a key nuclear stability factor.
To study nuclear membrane rupture during interphase, Dr. Hatch's team developed a nuclear membrane rupture reporter called RFP-Cyto. They took advantage of the fact that large proteins remain mislocalized following nuclear membrane rupture because they are not able to diffuse back to the cytosol through the nuclear pores following nuclear membrane repair. In addition, the RFP-Cyto reporter lacks a nuclear localization signal, resulting in its mislocalization following the rupture of the nuclear membrane. Thus, the reporter is localized in the cytosol of the cells and its mislocalization can be used as a marker of nuclear membrane rupture. As a proof of principle, the team co-expressed RFP-Cyto in U2OS RuptR cells that also express GFP-Nuc, a nuclear protein whose localization can be used to study nuclear membrane rupture events.Essentially, the intensity of GFP in the cytoplasm and RFP in the nucleus (which only occurs during nuclear membrane rupture) provides an indication of the frequency of nuclear membrane rupture in that cell. Next, cells were transfected with a shRNA against LMNB1, which resulted in a modest reduction of the protein Lamin B1, to increased nuclear rupture frequency and allowed identification of proteins that inhibit nuclear membrane rupture. Live-cell imaging revealed that RFP-Cyto intensity increased in the nucleus after nuclear membrane rupture and remained there after nuclear membrane repair. This was confirmed by nuclear co-localization of RFP-Cyto with GFP-Nuc. Based on these findings, RFP-Cyto nuclear localization accurately predicted ruptures of nuclear membranes.
Having demonstrated that RFP-Cyto can be used to analyze nuclear membrane rupture, the team developed a high throughput analysis using CellProfiler, an open-source software program for image analysis. As part of the workflow developed by the team, U2OS RuptR cells were transfected with siRNAs, arrest for 24 hours (to increase nuclear membrane rupture frequency) and staining with Hoechst (a nuclear staining). Nuclei and cells were segmented using the Hoechst and RFP-Cyto signals in CellProfiler. CellProfiler's segmentation strategy was complemented by a set of post-segmentation intensity filters to eliminate data from cells with low RFP-Cyto expression, mitotic cells, dead cells, or cells out of focus and morphological filters to accommodate variations in nucleus shape and size during screening. By calculating the mean intensity ratio between the nucleus and the cytoplasm, RFP-Cyto localization was determined. By knocking down LMNB1, SUN1 (to suppress the frequency of nuclear membrane rupture) and BAF (required for efficient membrane repair), the pipeline was validated. In fact, these results indicated that this pipeline can provide accurate information regarding relative rupture frequencies and with a high degree of sensitivity.
After establishing high throughput analysis, the team conducted a plate-based siRNA screen to identify new regulators of nuclear membrane rupture. From the screen, 14 hits resulted in reduced rupture frequency when depleted and 8 hits resulted in an increase in rupture frequency, indicating that the pipeline along with the shRNA screen provides an unbiased method for determining nuclear membrane stability factors in a highly accurate manner. First, the team investigated hits that were associated with the repair of nuclear membranes or the frequency of nuclear membrane ruptures. Only RB1 and STK11 depletion increased the number of rupture events per cell, indicating that both of them play a role in maintaining nuclear integrity. Additionally, the team investigated hits that altered nuclear size, such as the well-known LMNB1. One of them was CTDNEP1, a phosphatase located on the nuclear membrane and regulates lipid composition. Both CTDNEP1 and LMNB1 depletion increased nuclear rupture. CTDNEP1 depletion caused the greatest increase in nuclear rupture, independent of lamin B1 depletion. In spite of this, it remains unclear how the loss of CTDNEP1 increases rupture frequency. CTDNEP1 loss does not significantly alter nuclear lamina gap frequency, nuclear pore density, or nuclear confinement, strongly suggesting that it is a new mechanism of membrane stabilization. Going forward, the team aims to define the molecular mechanisms by which CTDNEP1 affects nuclear membrane stability. Dr. Hatch's team hopes to provide new insights as to how lipid composition could influence nuclear membrane stability or how signaling pathways affect nuclear envelope structure.
This spotlighted research was supported by the National Institutes of Health, the Cellular and Molecular Biology training grant and the Cellular Imaging and Flow Cytometry Shared Resources of the Fred Hutch/University of Washington Cancer Consortium, and Fred Hutch Scientific Computing grant.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Dr. Emily Hatch contributed to this work.
Gunn AL, Yashchenko AI, Dulbrulle J, Johnson J, Hatch EL. A high-content screen reveals new regulators of nuclear membrane stability. bioRxiv [Preprint]. 2023 Sep 10:2023.05.30.542944. doi: 10.1101/2023.05.30.542944. PMID: 37398267; PMCID: PMC10312541.