Bayesian metamodeling of early T-cell antigen receptor signaling accounts for its nanoscale activation patterns.

T cells respond swiftly, specifically, sensitively, and robustly to cognate antigens presented on the surface of antigen presenting cells. Existing microscopic models capture various aspects of early T-cell antigen receptor (TCR) signaling at the molecular level. However, none of these models account for the totality of the data, impeding our understanding of early T-cell activation.

Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions.

T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we studied the nanoscale organization of CARs targeting CD138 proteoglycans in such fixed and live interfaces, generated optimally for single-molecule localization microscopy.

Cell Surface Vibrations Distinguish Malignant from Benign Cells.

The mechanical properties of living cells, including their shape, rigidity, and internal dynamics play a crucial role in their physiology and pathology. Still, the relations between the physiological cell state and its rigidity and surface vibrations remain poorly understood. Here, we have employed AFM measurements on T cells and found a negative relation between cell surface stiffness and its vibrations.

High- and Super-Resolution Imaging of Cell-Cell Interfaces.

Physical interfaces mediate interactions between multiple types of cells. Despite the importance of such interfaces to the cells' function, their high-resolution optical imaging has been typically limited due to poor alignment of the interfaces relative to the optical plane of imaging. Here, we present a simple and robust method to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips and bringing them into contact in a controlled and stable fashion.

NRas activity is regulated by dynamic interactions with nanoscale signaling clusters at the plasma membrane.

NRas is a key mediator of the mitogenic pathway in normal cells and in cancer cells. Its dynamics and nanoscale organization at the plasma membrane (PM) facilitate its signaling. Here, we used two-color photoactivated localization microscopy to resolve the organization of individual NRas and associated signaling proteins in live melanoma cells, with resolution down to ∼20 nm.

Nano-scale architecture of blood-brain barrier tight-junctions.

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs.

Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles.

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca probe as a small and mobile tracer in live T cells.

Spectral Analysis of ATP-Dependent Mechanical Vibrations in T Cells.

Mechanical vibrations affect multiple cell properties, including its diffusivity, entropy, internal content organization, and thus-function. Here, we used Differential Interference Contrast (DIC), confocal, and Total Internal Reflection Fluorescence (TIRF) microscopies to study mechanical vibrations in live (Jurkat) T cells. Vibrations were measured via the motion of intracellular particles and plasma membrane.

Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces.

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips.

MEK Inhibition Reverses Aberrant Signaling in Melanoma Cells through Reorganization of NRas and BRAF in Self Nanoclusters.

Hotspot mutations of the oncogenes BRAF and NRas are the most common genetic alterations in cutaneous melanoma. Still, the nanoscale organization and signal coupling of these proteins remain incompletely understood, particularly upon expression of oncogenic NRas mutants. Here we employed single-molecule localization microscopy to study the nanoscale organization of NRas and BRAF at the plasma membrane (PM) of melanoma cells.

Time-correlated single molecule localization microscopy enhances resolution and fidelity.

Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samples down to ~ 10-20 nm and in single molecule detail. However, common SMLM reconstruction largely disregards information embedded in the entire intensity trajectories of individual emitters. Here, we develop and demonstrate an approach, termed time-correlated-SMLM (tcSMLM), that uses such information for enhancing SMLM reconstruction.

Fast and synchronized fluctuations of cortical actin negatively correlate with nucleoli liquid-liquid phase separation in T cells.

Liquid-liquid phase separation is an important mechanism by which eukaryotic cells functionally organize their intracellular content and has been related to cell malignancy and neurodegenerative diseases. These cells also undergo ATP-driven mechanical fluctuations, yet the effect of these fluctuations on the liquid-liquid phase separation remains poorly understood. Here, we employ high-resolution microscopy and atomic force microscopy of live Jurkat T cells to characterize the spectrum of their mechanical fluctuations, and to relate these fluctuations to the extent of nucleoli liquid-liquid phase separation (LLPS).

Elevated basal transcription can underlie timothy channel association with autism related disorders.

Timothy syndrome (TS) is a neurodevelopmental disorder caused by mutations in the pore-forming subunit α1.2 of the L-type voltage-gated Ca-channel Cav1.2, at positions G406R or G402S.

T Cell Activation through Isolated Tight Contacts.

T cells engage antigen-presenting cells in search for cognate antigens via dynamic cell protrusions before forming a tight immune synapse. The spatiotemporal events that may lead to rapid TCR triggering and signal amplification in microvilli-driven isolated contacts, and in subsequent, more uniform contacts, remain poorly understood. Here, we combined interference-reflectance microscopy and single-molecule localization microscopy in live cells to resolve TCR-dependent signaling at tight cell contacts.

Nanoscale Topography-Rigidity Correlation at the Surface of T Cells.

The mechanical properties of cells affect their function, in sensing, development, and motility. However, the rigidity of the cell surface and its correlation to its local topography remain poorly understood. Here, we applied quantitative imaging AFM to capture high-resolution force maps at the surface of nonadherent T cells.

InterCells: A Generic Monte-Carlo Simulation of Intercellular Interfaces Captures Nanoscale Patterning at the Immune Synapse.

Molecular interactions across intercellular interfaces serve to convey information between cells and to trigger appropriate cell functions. Examples include cell development and growth in tissues, neuronal and immune synapses (ISs). Here, we introduce an agent-based Monte-Carlo simulation of user-defined cellular interfaces.

Synergizing superresolution optical fluctuation imaging with single molecule localization microscopy.

Single-molecule-localization-microscopy (SMLM) and superresolution-optical-fluctuation-imaging (SOFI) enable imaging biological samples well beyond the diffraction-limit of light. SOFI imaging is typically faster, yet has lower resolution than SMLM. Since the same (or similar) data format is acquired for both methods, their algorithms could presumably be combined synergistically for reconstruction and improvement of overall imaging performance.

Gp41 dynamically interacts with the TCR in the immune synapse and promotes early T cell activation.

The HIV-1 glycoprotein gp41 critically mediates CD4 T-cell infection by HIV-1 during viral entry, assembly, and release. Although multiple immune-regulatory activities of gp41 have been reported, the underlying mechanisms of these activities remain poorly understood. Here we employed multi-colour single molecule localization microscopy (SMLM) to resolve interactions of gp41 proteins with cellular proteins at the plasma membrane (PM) of fixed and live CD4 T-cells with resolution of ~20-30 nm.

Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation.

T cells have a central function in mounting immune responses. However, mechanisms of their early activation by cognate antigens remain incompletely understood. Here we use live-cell multi-colour single-molecule localization microscopy to study the dynamic separation between TCRs and CD45 glycoprotein phosphatases in early cell contacts under TCR-activating and non-activating conditions.

The L-type Voltage-Gated Calcium Channel co-localizes with Syntaxin 1A in nano-clusters at the plasma membrane.

The secretory signal elicited by membrane depolarization traverses from the Ca-bound α1.2 pore-forming subunit of the L-type Ca-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism.

Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane.

The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy.

Development of nanoscale structure in LAT-based signaling complexes.

The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). These multi-molecular complexes are dynamic structures that activate highly regulated signaling pathways. Previously, we have demonstrated nanoscale structure in LAT-based complexes where the adapter SLP-76 (also known as LCP2) localizes to the periphery of LAT clusters.

Hierarchical nanostructure and synergy of multimolecular signalling complexes.

Signalling complexes are dynamic, multimolecular structures and sites for intracellular signal transduction. Although they play a crucial role in cellular activation, current research techniques fail to resolve their structure in intact cells. Here we present a multicolour, photoactivated localization microscopy approach for imaging multiple types of single molecules in fixed and live cells and statistical tools to determine the nanoscale organization, topology and synergy of molecular interactions in signalling complexes downstream of the T-cell antigen receptor.

Architecture and Characteristics of Bacterial Nanotubes.

Bacteria display an array of contact-dependent interaction systems that have evolved to facilitate direct cell-to-cell communication. We have previously identified a mode of bacterial communication mediated by nanotubes bridging neighboring cells. Here, we elucidate nanotube architecture, dynamics, and molecular components.

Mechanisms of localized activation of the T cell antigen receptor inside clusters.

The T cell antigen receptor (TCR) has been shown to cluster both before and upon engagement with cognate antigens. However, the effect of TCR clustering on its activation remains poorly understood. Here, we used two-color photo-activated localization microscopy (PALM) to visualize individual molecules of TCR and ZAP-70, as a marker of TCR activation and phosphorylation, at the plasma membrane of uniformly activated T cells.