Biomedical subjects

Zenon Rajfur

Publications and source records attributed to Zenon Rajfur.

4 recordsLinked to original sources

PubMed2026

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 sustain 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 β-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.

Mesenchymal Stem Cells↗

PubMed2003

JNK phosphorylates paxillin and regulates cell migration.

The c-Jun amino-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. Accordingly, its substrates are transcription factors and anti-apoptotic proteins. However, JNK has also been shown to be required for Drosophila dorsal closure, and MAP kinase/ERK kinase kinase 1, an upstream kinase in the JNK pathway, has been shown to be essential for cell migration. Both results imply that JNK is important in cell migration. Here we show that JNK1 is required for the rapid movement of both fish keratocytes and rat bladder tumour epithelial cells (NBT-II). Moreover, JNK1 phosphorylates serine 178 on paxillin, a focal adhesion adaptor, both in vitro and in intact cells. NBT-II cells expressing the Ser 178 --> Ala mutant of paxillin (Pax(S178A)) formed focal adhesions and exhibited the limited movement associated with such contacts in both single-cell-migration and wound-healing assays. In contrast, cells expressing wild-type paxillin moved rapidly and retained close contacts as the predominant adhesion. Expression of Pax(S178A) also inhibited the migration of two other cell lines. Thus, phosphorylation of paxillin by JNK seems essential for maintaining the labile adhesions required for rapid cell migration.

Amino Acid Sequence↗

PubMed2002

Microscope-based techniques to study cell adhesion and migration.

Modern light microscopy has evolved to provide a variety of quantitative imaging techniques and also the capability to perturb structure-function relationships in living cells. The advances have been especially useful in the study of cell adhesion and migration. This review will focus on how such microscopy-based techniques can be useful in situ to study the molecular interactions and dynamics, to locally perturb actin-based structures and to measure the traction forces exerted by motile cells.

Actins↗

PubMed2002

Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins within cells. Here, we demonstrate that GFP can be used as a CALI reagent to locally inactivate proteins in living cells. We show that focused laser irradiation of EGFP-alpha-actinin expressed in Swiss 3T3 fibroblasts results in the detachment of stress fibres from focal adhesions (FAs), whereas the integrity of FAs, as determined by interference reflection microscopy (IRM), is preserved. Moreover, consistent with a function for focal adhesion kinase (FAK) in FA signalling and not FA structure, laser irradiation of EGFP-FAK did not cause either visible FA damage or stress fibre detachment, although in vitro CALI of isolated EGFP-FAK decreased its kinase activity, but not its binding to paxillin. These data indicate that CALI of specific FA components may be used to precisely dissect the functional significance of individual proteins required for the maintenance of this cytoskeletal structure. In vitro CALI experiments also demonstrated a reduction of EGFP-alpha-actinin binding to the cytoplasmic domain of the beta(1) integrin subunit, but not to actin. Thus, alpha-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in alpha-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.

3T3 Cells↗