Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple ...units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties ...of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca
flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.
Understanding the mechanisms behind T cell dysfunctions during chronic diseases is critical in developing effective immunotherapies. As demonstrated by several animal models and human studies, T cell ...dysfunctions are induced during chronic diseases, spanning from infections to cancer. Although factors governing the onset and the extent of the functional impairment of T cells can differ during infections and cancer, most dysfunctional phenotypes share common phenotypic traits in their immune receptor and biophysical landscape. Through the latest developments in biophysical techniques applied to explore cell membrane and receptor-ligand dynamics, we are able to dissect and gain further insights into the driving mechanisms behind T cell dysfunctions. These insights may prove useful in developing immunotherapies aimed at reinvigorating our immune system to fight off infections and malignancies more effectively. The recent success with checkpoint inhibitors in treating cancer opens new avenues to develop more effective, targeted immunotherapies. Here, we highlight the studies focused on the transformation of the biophysical landscape during infections and cancer, and how T cell biomechanics shaped the immunopathology associated with chronic diseases.
Smart surfaces presenting both antifouling molecules with a charged functional group at their distal end, and molecules that are terminated by RGD peptides for cell adhesion, were fabricated and ...characterized (see picture). By applying potentials of +300 or −300 mV, the surfaces could be dynamically switched to make the peptide accessible or inaccessible to cells.
Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of ...epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.
The morphology and function of endothelial cells depends on the physical and chemical characteristics of the extracellular environment. Here, we designed silicon surfaces on which topographical ...features and surface densities of the integrin binding peptide arginine-glycine-aspartic acid (RGD) could be independently controlled. We used these surfaces to investigate the relative importance of the surface chemistry of ligand presentation versus surface topography in endothelial cell adhesion. We compared cell adhesion, spreading and migration on surfaces with nano- to micro-scaled pyramids and average densities of 6×10(2)-6×10(11) RGD/mm(2). We found that fewer cells adhered onto rough than flat surfaces and that the optimal average RGD density for cell adhesion was 6×10(5) RGD/mm(2) on flat surfaces and substrata with nano-scaled roughness. Only on surfaces with micro-scaled pyramids did the topography hinder cell migration and a lower average RGD density was optimal for adhesion. In contrast, cell spreading was greatest on surfaces with 6×10(8) RGD/mm(2) irrespectively of presence of feature and their size. In summary, our data suggest that the size of pyramids predominately control the number of endothelial cells that adhere to the substratum but the average RGD density governs the degree of cell spreading and length of focal adhesion within adherent cells. The data points towards a two-step model of cell adhesion: the initial contact of cells with a substratum may be guided by the topography while the engagement of cell surface receptors is predominately controlled by the surface chemistry.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times ...during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.
The initial steps of reverse cholesterol transport involve export of cholesterol from peripheral cells to plasma lipoproteins for subsequent delivery to the liver. The review discusses recent ...developments in our understanding of how these steps occur, with particular emphasis on the macrophage, the major site of cellular cholesterol accumulation in atherosclerosis.
ATP binding cassette transporter (ABC) A1 exports cholesterol and phospholipid to lipid-free apolipoproteins, while ATP binding cassette transporter G1 and scavenger receptor BI export cholesterol to phospholipid-containing acceptors. ABCA1-dependent cholesterol export involves an initial interaction of apolipoprotein AI with lipid raft membrane domains, although ABCA1 and most exported cholesterol are not raft associated. ABCG1 exports cholesterol to HDL and other phospholipid-containing acceptors. These include particles generated during lipidation of apoAI by ABCA1, suggesting that the two transporters cooperate in cholesterol export. Scavenger receptor BI is atheroprotective, mediating clearance of HDL cholesterol by the liver. The relative contributions of scavenger receptor BI and ABCG to cholesterol export to HDL from macrophages is unclear and may depend on cellular cholesterol status and the cholesterol gradient between cell and acceptor.
The presence of distinct pathways for cholesterol efflux to lipid-free apolipoprotein AI and phospholipid-containing HDL species clarifies our understanding of reverse cholesterol transport, and provides new opportunities for its therapeutic manipulation.
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