Abstract:The tyrosine phosphatase CD45 is a major gatekeeper for restraining T cell activation. Its exclusion from the immunological synapse (IS) is crucial for T cell receptor (TCR) signal transduction. Here, we use expansion super-resolution microscopy to reveal that CD45 is mostly pre-excluded from the tips of microvilli (MV) on primary T cells prior to antigen encounter. This pre-exclusion is diminished by depleting cholesterol or by engineering the transmembrane domain of CD45 to increase its membrane integration … Show more
“…7e ). These data are in agreement with recently reported observations that CD45 segregates from microvilli tips in resting and activated T cells 76 , 77 . Fig.…”
Section: Resultssupporting
confidence: 94%
“…Our data thus indicate that CD45 segregates from signaling molecules by localizing to the base of the plasma membrane. A similar observation was recently reported by the group of Klaus Ley 76 . They further demonstrate that such localization is determined by the transmembrane domain of CD45.…”
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.
“…7e ). These data are in agreement with recently reported observations that CD45 segregates from microvilli tips in resting and activated T cells 76 , 77 . Fig.…”
Section: Resultssupporting
confidence: 94%
“…Our data thus indicate that CD45 segregates from signaling molecules by localizing to the base of the plasma membrane. A similar observation was recently reported by the group of Klaus Ley 76 . They further demonstrate that such localization is determined by the transmembrane domain of CD45.…”
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.
“…On the other hand, there is controversy as to whether the appearance of TCR clusters in super-resolution studies is significantly different from a random arrangement of receptors 115 . Jung et al suggest that TCR nanoclusters are associated with tips of microvilli, supporting the argument that small clusters are not random 116 . It should be noted that these configurations, including pre-segregation of TCR from CD45 116 , are based on fixed snapshots and may be subject to rearrangements on a subsecond time scale to prevent spontaneous signaling while keeping the system poised to respond rapidly.…”
Section: Organization Of Membrane Proteins Involved In T Cell Activationmentioning
T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.
“…It was also shown experimentally that signalling could be impaired when the sorting out of bulky molecules was prevented by replacing native adhesion molecules with engineered molecular linkers of increased length [ 103 , 104 ]. Second, as often occurs in the domain of cell biology, this may not be the whole story since the sorting out of molecules such as CD43 [ 105 ] or CD45 [ 106 ] may involve active cell phenomena other than mere steric exclusion. Also, it must be emphasized that topographical reorganization of cell membrane molecules such as integrins have long been shown to influence cell function [ 107 ].…”
Section: How Can Mechanical Phenomena Generate or Influence Signalling Cascades?mentioning
How do cells process environmental cues to make decisions? This simple question is still generating much experimental and theoretical work, at the border of physics, chemistry and biology, with strong implications in medicine. The purpose of mechanobiology is to understand how biochemical and physical cues are turned into signals through mechanotransduction. Here, we review recent evidence showing that (i) mechanotransduction plays a major role in triggering signalling cascades following cell–neighbourhood interaction; (ii) the cell capacity to continually generate forces, and biomolecule properties to undergo conformational changes in response to piconewton forces, provide a molecular basis for understanding mechanotransduction; and (iii) mechanotransduction shapes the guidance cues retrieved by living cells and the information flow they generate. This includes the temporal and spatial properties of intracellular signalling cascades. In conclusion, it is suggested that the described concepts may provide guidelines to define experimentally accessible parameters to describe cell structure and dynamics, as a prerequisite to take advantage of recent progress in high-throughput data gathering, computer simulation and artificial intelligence, in order to build a workable, hopefully predictive, account of cell signalling networks.
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