Refine
Language
- English (4)
Has Fulltext
- yes (4)
Is part of the Bibliography
- yes (4)
Keywords
- stiffness (2)
- AFM (1)
- CD4+ T cell (1)
- IGF-1 receptor (1)
- Peak Force QNM (1)
- SARS-CoV-2 receptor binding domain (1)
- T cells (1)
- biophysics (1)
- calcium (1)
- cancer (1)
Scientific Unit
- Fellow (3)
The cytoskeletal protein vimentin is secreted under various physiological conditions. Extracellular vimentin exists primarily in two forms: attached to the outer cell surface and secreted into the extracellular space. While surface vimentin is involved in processes such as viral infections and cancer progression, secreted vimentin modulates inflammation through reduction of neutrophil infiltration, promotes bacterial elimination in activated macrophages, and supports axonal growth in astrocytes through activation of the IGF-1 receptor. This receptor is overexpressed in cancer cells, and its activation pathway has significant roles in general cellular functions. In this study, we investigated the functional role of extracellular vimentin in non-tumorigenic (MCF-10a) and cancer (MCF-7) cells through the evaluation of its effects on cell migration, proliferation, adhesion, and monolayer permeability. Upon treatment with extracellular recombinant vimentin, MCF-7 cells showed increased migration, proliferation, and adhesion, compared to MCF-10a cells. Further, MCF-7 monolayers showed reduced permeability, compared to MCF-10a monolayers. It has been shown that the receptor binding domain of SARS-CoV-2 spike protein can alter blood–brain barrier integrity. Surface vimentin also acts as a co-receptor between the SARS-CoV-2 spike protein and the cell-surface angiotensin-converting enzyme 2 receptor. Therefore, we also investigated the permeability of MCF-10a and MCF-7 monolayers upon treatment with extracellular recombinant vimentin, and its modulation of the SARS-CoV-2 receptor binding domain. These findings show that binding of extracellular recombinant vimentin to the cell surface enhances the permeability of both MCF-10a and MCF-7 monolayers. However, with SARS-CoV-2 receptor binding domain addition, this effect is lost with MCF-7 monolayers, as the extracellular vimentin binds directly to the viral domain. This defines an influence of extracellular vimentin in SARS-CoV-2 infections.
T cells are activated by target cells via an intimate contact, termed immunological synapse (IS). Cellular mechanical properties, especially stiffness, are essential to regulate cell functions. However, T cell stiffness at a subcellular level at the IS still remains largely elusive. In this work, we established an atomic force microscopy (AFM)-based elasticity mapping method on whole T cells to obtain an overview of the stiffness with a resolution of ~60 nm. Using primary human CD4+ T cells, we show that when T cells form IS with stimulating antibody-coated surfaces, the lamellipodia are stiffer than the cell body. Upon IS formation, T cell stiffness is enhanced both at the lamellipodia and on the cell body. Chelation of intracellular Ca2+ abolishes IS-induced stiffening at the lamellipodia but has no influence on cell-body-stiffening, suggesting different regulatory mechanisms of IS-induced stiffening at the lamellipodia and the cell body.
CD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.Graphical abstract: Overview of the experimental workflow.Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right.
T cells are activated by cognate target cells via an intimate contact, termed immunological synapse (IS). Cellular mechanical properties, especially stiffness, are essential to regulate cell functions, T cell stiffness at a subcellular level at the IS still remains largely elusive. In this work, we established an atomic force microscopy (AFM)-based elasticity mapping method on whole T cells to obtain an overview of the stiffness with a resolution of ~ 60 nm. Using Jurkat T-cells and primary human CD4+ T cells, we show that in the T cells in contact with functionalized surfaces, the lamellipodia are stiffer than the cell body. Upon IS formation, T cell stiffness is substantially enhanced both at the lamellipodia and in cell body. Chelation of intracellular Ca2+ abolishes IS-induced stiffening at the lamellipodia but has no influence on cell body-stiffening, suggesting different regulatory mechanism of IS-induced stiffening between the lamellipodia and the cell body.Competing Interest StatementThe authors have declared no competing interest.
