Significance: Interactions between the cell nucleus and cytoskeleton regulate cell mechanics and are facilitated by the interplay between the nuclear lamina and linker of nucleoskeleton and cytoskeleton (LINC) complexes. To date, the specific contribution of the four lamin isoforms to nucleocytoskeletal connectivity and whole-cell mechanics remains unknown. We discover that A- and B-type lamins distinctively interact with LINC complexes that bind F-actin and vimentin filaments to differentially modulate cortical stiffness, cytoplasmic stiffness, and contractility of mouse embryonic fibroblasts (MEFs). We propose and experimentally verify an integrated lamin–LINC complex–cytoskeleton model that explains cellular mechanical phenotypes in lamin-deficient MEFs. Our findings uncover potential mechanisms for cellular defects in human laminopathies and many cancers associated with mutations or modifications in lamin isoforms.

Significance: Filamentous actin (F-actin) and vimentin intermediate filaments (VIFs) are two major cytoskeletal components; they are generally thought to be spatially compartmentalized and to have distinctly different and independent functions. Here we combine two imaging methods, high-resolution structured illumination microscopy and cryo-electron tomography, as well as functional characterizations, to show that unexpectedly, VIFs and F-actin have extensive structural interactions within the cell cortex and form interpenetrating networks. These interactions have very important functional consequences for cells, which are broadly significant given the wide range of processes attributed to F-actin. These results profoundly alter our understanding of the contributions of cytoskeletal components and counter the common belief that VIFs and F-actin are independent in both structure and function.

Large-scale profiling assays capture a cell population’s state by measuring thousands of biological properties per cell or sample. However, evaluating profile strength and similarity remains challenging due to the high dimensionality and non-linear, heterogeneous nature of measurements. Here, we develop a statistical framework using mean average precision (mAP) as a single, data-driven metric to address this challenge. We validate the mAP framework against established metrics through simulations and real-world data, revealing its ability to capture subtle and meaningful biological differences in cell state. Specifically, we use mAP to assess a sample’s phenotypic activity relative to controls, as well as the phenotypic consistency of groups of perturbations (or samples). We evaluate the framework across diverse datasets and on different profile types (image, protein, mRNA), perturbations (CRISPR, gene overexpression, small molecules), and resolutions (single-cell, bulk). The mAP framework, together with our open-source software package copairs, is useful for evaluating high-dimensional profiling data in biological research and drug discovery.

Retinoblastoma (RB), a childhood cancer, is caused by biallelic mutation of the RB1 gene, but its development is not clearly understood. Furthermore, the presence of a cancer stem cell subpopulation in RB might impact its treatment. PIWI protein, known for its role in stem cell self-renewal, is aberrantly expressed in cancers. We examined the role of the PIWI-like protein HIWI2 in RB and its effect on the stem cell markers in cells of the RB line, Y79. The expression of HIWI2 is significantly increased in Y79 compared with its level in HeLa and ARPE19 cells. The stem cell markers Oct-3/4, Nanog and Sox-2 were not altered upon HIWI2 knockdown in Y79 cells. Interestingly, OTX2 was significantly downregulated in the absence of HIWI2. Otx2 transcripts also decreased in HIWI2-silenced Y79 and ARPE19 cells. Moreover, silencing HIWI2 in Y79 accumulated the cells at G2–M phase and reduced the levels of proliferating cell nuclear antigen (PCNA) and the tumor suppressor, p16. Our results demonstrate that HIWI2 is aberrantly expressed in Y79 cells and silencing of HIWI2 downregulates OTX2, suggesting that HIWI2 might play a role in the progression of RB.