Vimentin intermediate filaments and filamentous actin form unexpected interpenetrating networks that redefine the cell cortex

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin

PIWI-like protein, HIWI2 is aberrantly expressed in retinoblastoma cells and affects cell-cycle potentially through OTX2

Abstract 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

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures

Bridging imaging users to imaging analysis - A community survey

The “Bridging Imaging Users to Imaging Analysis” survey was conducted in 2022 by the Center for Open Bioimage Analysis (COBA), Bioimaging North America (BINA), and the Royal Microscopical Society Data Analysis in Imaging Section (RMS DAIM) to understand the needs of the imaging community. Through multi-choice and open-ended questions, the survey inquired about demographics, image analysis experiences, future needs, and suggestions on the role of tool developers and users. Participants of the survey were from diverse roles and domains of the life and physical sciences. To our knowledge, this is the first attempt to survey cross-community to bridge knowledge gaps between physical and life sciences imaging. Survey results indicate that respondents' overarching needs are documentation, detailed tutorials on the usage of image analysis tools, user-friendly intuitive software, and better solutions for segmentation, ideally in a format tailored to their specific use cases. The tool creators suggested the users familiarize themselves with the fundamentals of image analysis, provide constant feedback, and report the issues faced during image analysis while the users would like more documentation and an emphasis on tool friendliness. Regardless of the computational experience, there is a strong preference for ‘written tutorials’ to acquire knowledge on image analysis. We also observed that the interest in having ‘office hours’ to get an expert opinion on their image analysis methods has increased over the years. The results also showed less-than-expected usage of online discussion forums in the imaging community for solving image analysis problems. Surprisingly, we also observed a decreased interest among the survey respondents in deep/machine learning despite the increasing adoption of artificial intelligence in biology. In addition, the community suggests the need for a common repository for the available image analysis tools and their applications. The opinions and suggestions of the community, released here in full, will help the image analysis tool creation and education communities to design and deliver the resources accordingly

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole-cell mechanics.

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the lamin–LINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics

Vimentin protects cells against nuclear rupture and DNA damage during migration

Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration