Biology subjects

Rajfur, Z.

Publications and source records attributed to Rajfur, Z..

8 recordsLinked to original sources

bioRxiv2025

Small extracellular vesicles reflect senescence progression in human bone marrow-derived mesenchymal stem cells during hollow fiber bioreactor culture

Prolonged three-dimensional culture exposes stem cells to sustained microenvironmental and mechanical stresses that can promote aging- and senescence-associated phenotypic alterations. This study examined how long-term expansion of human bone marrow-derived mesenchymal stem cells (BMSCs) in a hollow fiber bioreactor (HFB) influences cellular senescence and the molecular composition of secreted small extracellular vesicles (sEVs). During extended HFB culture, BMSCs exhibited progressive morphological flattening and cytoskeletal disorganization, accompanied by increased senescence-associated {beta}-galactosidase activity and immunophenotypic remodeling characterized by reduced fluorescence intensity and spatial redistribution of canonical MSC markers, consistent with a stress-adapted, early senescence-associated cellular state. In parallel, sEVs were collected longitudinally over 40 days and characterized by nanoparticle tracking analysis, immunoblotting, and quantitative proteomics. While vesicle size, marker expression, and yield remained stable throughout culture, proteomic profiling revealed pronounced, phase-dependent remodeling of sEV cargo, including coordinated alterations in oxidative stress-related processes, lysosomal and extracellular matrix-associated pathways, and relative depletion of cytoskeletal and translational components. Notably, these vesicular signatures closely mirrored senescence-associated changes observed at the cellular level. The strong correspondence between cellular phenotypes and sEV proteomic profiles establishes vesicle analysis as a convergent and noninvasive readout of BMSC aging, enabling sensitive monitoring of senescence progression while reducing reliance on parallel, labor-intensive cellular assays. Collectively, these findings indicate that prolonged HFB culture promotes a controlled, stress-associated senescence program in BMSCs and position sEV proteomic profiling as a robust approach for assessing stem cell aging dynamics during long-term three-dimensional bioreactor culture.

molecular biology↗

bioRxiv2025

Substrate elasticity regulates cytoskeletal remodeling and mechanical behavior of U2OS osteosarcoma cells

Substrate elasticity plays a pivotal role in regulating the morphology, mechanical properties, and cytoskeletal organization of cancer cells. In this study, we examined the response of U2OS osteosarcoma cells to substrates of varying stiffness, with a particular focus on cytoskeletal remodeling, cell elasticity, and microparticle internalization. To simulate environments of moderate and high stiffness, cells were cultured on polyacrylamide (PA) hydrogels with a stiffness of 40 kPa and on rigid glass substrates, respectively. Changes in cell morphology and cytoskeletal organization were assessed using fluorescence microscopy, while cell mechanical properties were measured using atomic force microscopy (AFM). To investigate the relationship between substrate mechanics and endocytic activity, carboxylated fluorescent 2 {micro}m latex microspheres were introduced to the cell culture system. Our study showed that cell spreading increased with substrate stiffness. U2OS cells cultured on glass exhibited a significantly larger surface area, more actin stress fibers, and a more organized, stretched cytoskeletal architecture compared to cells grown on 40 kPa PA gels. AFM measurements further demonstrated that cells on glass were mechanically stiffer than those on PA substrates. Microparticle uptake was also strongly influenced by substrate stiffness. Cells cultured in the 40 kPa PA gels internalized a significantly greater number of fluorescent microspheres. Notably, these cells frequently formed distinct, cup-like structures composed of microtubules around the beads. Three-dimensional image reconstructions revealed that these structures encapsulate the particles in an asymmetrical manner, indicative of active cytoskeletal remodeling. To better understand the molecular composition of these microtubule-based structures, we analyzed the localization of selected microtubule-associated proteins (MAPs), including IQGAP1, CLIP1, and MARK2 (Conboy J.P. et al. 2024). Interestingly, only IQGAP1 was localized prominently to the microtubule cups on 40 kPa gels, often forming ring-like structures surrounding the beads. In some cases, these rings were observed independently of detectable microtubules, suggesting the involvement of an active, possibly microtubule-initiated, endocytic process. In conclusion, our findings demonstrate that substrate stiffness modulates multiple aspects of U2OS cell behavior, including morphology, cytoskeletal arrangement, mechanical properties, and microparticle uptake. These results underscore the mechanosensitive nature of osteosarcoma cells and highlight novel roles for microtubule structures and MAPs, particularly IQGAP1 in stiffness-dependent cellular uptake mechanisms.

biophysics↗

bioRxiv2025

Software for semi-automatic analysis of microscopic images of adhesion structures and protein colocalization in cells

Background and ObjectiveAdhesion structures, such as focal and fibrillar adhesions, are protein complexes that mediate cell attachment to extracellular matrix via integrins. These structures participate in the mechanosensing process, transmitting forces from the cellular microenvironment to the cytoskeleton. The interaction between cells and their environment, along with the role of focal adhesion proteins, are areas of significant research interest. Accurate, quantitative analysis of adhesions structures in microscopic images is essential for advancing our understanding of the subject. However, the high variability and complexity of those structures in cells makes image analysis challenging, both in terms of subjectivity and time consumption. MethodsWe present a novel semi-automatic script for the detection of adhesion structures and analysis of their parameters, developed in MATLAB 2021a, to address the challenge of accurate image analysis of focal and fibrillar adhesions in cells. ResultsOur script offers a more time-efficient and less subjective alternative to manual analysis, while still allowing the user to retain control over the analytical process. It detects adhesion structures in confocal images and measures key parameters such as shape, orientation, and spatial distribution within cells, additionally providing visual label maps of the identified adhesion structures. The second add-on enables the calculation of correlation coefficients between two confocal microscopy image channels representing different stained cellular structures within the same cell, and generates visual colocalization maps, further enhancing the analysis of cellular architecture. ConclusionsThe presented open-source script offers a robust solution for the comprehensive, quantitative analysis of adhesion structures in microscopic images, based on user-defined analysis parameters. It is available online at https://github.com/patrycja-twardawa/FA-Colocalization.git.

bioinformatics↗

bioRxiv2025

Methodological study of fibroblasts MEF 3T3 heterogeneity cultured on hydrogel substrates with different surface functionalization

Recently, cellular heterogeneity has gained attention in several scientific studies. Its a phenomenon of the occurrence of differences in cellular parameters, for example, morphology or migration parameters of cells from a single cell line. It has been studied primarily in cancer cells; however, normal cells exhibit analogous behavior. This study investigated morphological and migratory parameters heterogeneity in mouse embryonic fibroblast (MEF 3T3) cells cultured on polyacrylamide (PA) substrates with elasticity of 20 and 40 kPa, which were functionalized with fibronectin solutions in 1, 5, 10 and 20 g/ml concentrations. Two-hour time lapse microscopy made it possible to quantify the cells spread area and velocity. Based on these parameters and observations of cellular migration strategies, it was possible to classify normal cells into four subpopulations - mesenchymal, amoeboid, slow amoeboid and polygonal/bigonal. Cells grown on softer substrates (20 kPa) showed a greater sensitivity to fibronectin than those grown on stiffer substrates. Cells area was maximal, with the lowest velocity for cells grown on PA substrates functionalized with a 10 g/ml solution, making their parameters closest to those grown on glass. Cells underwent spontaneous transitions between subpopulations, but no direct transition between mesenchymal and amoeboid or slow amoeboid has been observed. Overall, substrate elasticity and concentration of fibronectin in the functionalizing solution impact the morphology and migration parameters of cells. These findings demonstrate the occurrence of heterogeneity phenomenon in normal mouse fibroblasts. This study also shows the impact of the concentration of fibronectin in the functionalization solution on cells behavior.

biophysics↗

bioRxiv2025

Talin1 adhesions' morphology is largely unaffected by polyacrylamide substrate stiffness

Cells sense the stiffness of their extracellular matrix (ECM) and adapt their behavior accordingly. We investigated how ECM stiffness affects the spatial organization of talin1, a key mechanosensitive focal adhesion protein. Using polyacrylamide (PA) hydrogels with tunable stiffnesses (0.2-188 kPa), we analyzed cell morphology, migration, talin1 distribution, colocalization with tensin3, and fibronectin deposition. Softer substrates enhanced filopodia activity and altered migration behavior. On softer ECMs, talin1 displayed a more even intracellular distribution, whereas on stiffer matrices it localized to the cell periphery. PA gels supported elongated talin1-based adhesions, whose morphology showed minimal variation across the 3-188 kPa stiffness range. Talin1-tensin3 colocalization was maintained regardless of stiffness, indicating a stable interaction. Notably, cells deposited more fibronectin on softer substrates. While talin1 adhesion morphology varied little with stiffness, cell migration behavior changed markedly. Combined with prior studies, our data suggests that ECM stiffness regulates talin1 primarily through conformational changes rather than macroscopic adhesion remodeling. These findings highlight talin1s central role in translating mechanical cues into dynamic cellular responses. Summary statementTalin1 forms elongated adhesions and robustly colocalizes with tensin3 across varying matrix stiffnesses, showing that their spatial organization is largely insensitive to mechanical cues.

cell biology↗

bioRxiv2021

Ectosome effect on endothelial monolayers in hyperglycemic and normoglycemic conditions

Extracellular vesicles, namely those larger ones - Ectosomes (Ect), are thought to be important cell-to-cell communication medium. Ect are considered as a potential therapeutic for type-1 and type-2 diabetes mellitus. Ect can be internalized by endothelial cells and, owing to their cargo, they modulate targeted cell behavior. Under hyperglycemic conditions (HGC), endothelial cells changed their properties and became stiffer and less mobile which causes endothelial dysfunction and abnormalities in micro- and macrovascular systems. The aim of this study was to find whether Ect restore mobility and motility of macrovascular endothelial cells under HGC. Uptake of Ect, cell morphology, cytoskeleton organization and membrane stiffness (by atomic force microscopy) were analyzed after the exposure to isolated Ect. To find which cellular pathways were deregulated by HGC and whether Ect could potentially restore gene expression profile, transcriptome analysis was done. We observed that endothelial cells internalized more Ect under normoglycemic conditions (NGC) then HGC. Hyperglycemic cells (HG) were bigger and showed the stiffer surface with denser actin cytoskeleton in comparison to normoglycemic cells. Number of metabolic pathways was influenced under HGC, especially those related to intracellular transport, metabolism and cellular component organization and Ect did not restore HGC impaired cell signaling. Ectosomes cannot reverse this harmful effect of hyperglycemia in endothelial cells, which can have clinical implication in use Ect as therapeutic target in diabetes treatment.

cell biology↗

bioRxiv2020

Replication of SARS-CoV-2 in human respiratory epithelium

SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost importance to understand the virus biology and to rapidly assess the potential of existing drugs and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in the Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2. Further, we also provide basic characteristics of the system.

microbiology↗

bioRxiv2019

RHOA mediated mechanical force generation through Dectin-1

Dectin-1 is an innate immune pattern recognition receptor which recognizes {beta}-glucan on the Candida albicans (C. albicans) cell wall. Recognition of {beta}-glucan by immune cells leads to phagocytosis, oxidative burst, cytokine and chemokine production. We looked for specific mechanisms that coordinate phagocytosis downstream of Dectin-1 leading to actin reorganization and internalization of fungus. We found that stimulation of Dectin-1 by soluble {beta}-glucan leads to mechanical force generation and areal contraction in Dectin-1 transfected HEK-293 cells and M1 macrophages. With inhibitor studies, we found this force generation is a SYK-independent, SFK (SRC Family Kinase)-dependent process mediated through the RHOA-ROCK-MLC pathway. We confirmed activation of RHOA downstream of Dectin-1 using G-LISA and stress fiber formation. Through phagocytosis assays, we found direct evidence for importance of RHOA-ROCK-MLC pathway in process of phagocytosis of C. albicans. In conclusion, we found evidence for RHOA-ROCK-MLC mediated mechanical force generation downstream of Dectin-1 for C. albicans phagocytosis.

immunology↗