Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.

• MeSH
• buněčná membrána metabolismus   MeSH
• nanotechnologie * MeSH
• receptory buněčného povrchu * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory buněčného povrchu * MeSH

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

• Klíčová slova
• auxin carriers, correlative microscopy, nanodomains, plasma membrane,
• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• buněčná membrána genetika   metabolismus   ultrastruktura   MeSH
• konfokální mikroskopie MeSH
• kovové nanočástice chemie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• mikroskopie elektronová rastrovací * MeSH
• počítačové zpracování obrazu MeSH
• protoplasty metabolismus   ultrastruktura   MeSH
• regulátory růstu rostlin genetika   metabolismus   MeSH
• tabák genetika   metabolismus   ultrastruktura   MeSH
• zlato chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH
• zlato MeSH

The interaction of T-cell receptors (TCRs) with self- and non-self-peptides in the major histocompatibility complex (MHC) stimulates crucial signaling events, which in turn can activate T lymphocytes. A variety of accessory molecules further modulate T-cell signaling. Of these, the CD4 and CD8 coreceptors make the most critical contributions to T cell sensitivity in vivo. Whereas, CD4 function in T cell development is well-characterized, its role in peripheral T cells remains incompletely understood. It was originally suggested that CD4 stabilizes weak interactions between TCRs and peptides in the MHC and delivers Lck kinases to that complex. The results of numerous experiments support the latter role, indicating that the CD4-Lck complex accelerates TCR-triggered signaling and controls the availability of the kinase for TCR in the absence of the ligand. On the other hand, extremely low affinity of CD4 for MHC rules out its ability to stabilize the receptor-ligand complex. In this review, we summarize the current knowledge on CD4 in T cells, with a special emphasis on the spatio-temporal organization of early signaling events and the relevance for CD4 function. We further highlight the capacity of CD4 to interact with the MHC in the absence of TCR. It drives the adhesion of T cells to the cells that express the MHC. This process is facilitated by the CD4 accumulation in the tips of microvilli on the surface of unstimulated T cells. Based on these observations, we suggest an alternative model of CD4 role in T-cell activation.

• Klíčová slova
• CD4, Lck, T lymphocytes, TCR coreceptor, cell-cell adhesion, microvilli,
• MeSH
• aktivace lymfocytů * MeSH
• CD4-pozitivní T-lymfocyty cytologie   imunologie   MeSH
• CD8-pozitivní T-lymfocyty cytologie   metabolismus   MeSH
• histokompatibilní antigeny imunologie   MeSH
• lidé MeSH
• receptory antigenů T-buněk imunologie   MeSH
• signální transdukce imunologie   MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• histokompatibilní antigeny MeSH
• receptory antigenů T-buněk MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH