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. CARs showed significant self-association in nanoclusters that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3). CARs also segregated more efficiently from the abundant membrane phosphatase CD45 in CAR-T cells forming such interfaces. CAR clustering and segregation from CD45 correlated with the effector functions of Ca++ influx and target cell killing. Our results shed new light on the nanoscale organization of CARs on the surfaces of CAR-Ts engaging on- and off-target cells, and its potential significance for CAR-Ts' efficacy and safety.

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. Blocking T-type Ca++-channels using Mibefradil reduced cortical actin tension in these cells and enhanced their membrane vibrations and dissipation of intracellular mechanical work to the cell surroundings. We also found increased vibrations of cell membranes in five different malignant cells lines derived from T cell leukemia, lung, prostate, bladder, and melanoma cancers, as compared to their corresponding benign cells. This was demonstrated by utilizing TIRF microscopy in single cells and dynamic laser speckles measurements in an in vitro model of multiple cells in a tissue. Our results show that cell membrane vibrations and dissipation of mechanical work are higher in malignant cells relative to benign cells. Accordingly, these properties may be used to detect and monitor cellular and tissue malignancies.

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. We demonstrate aberration-free high-resolution imaging of interfaces between live T cells and antigen-presenting cells, known as immune synapses, as an outstanding example. Imaging methods may include multiple diffraction-limited and super-resolution microscopy techniques (e.g., bright-field, confocal, STED, and dSTORM). Thus, our simple and widely compatible approach allows imaging with high- and super-resolution the intricate structure and molecular organization within a variety of cell-cell interfaces.

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. Upon EGF activation, a fraction of NRas and BRAF (dis)assembled synchronously at the PM in co-clusters. NRas and BRAF clusters associated with GPI-enriched domains, serving as possible nucleation sites for these clusters. NRas and BRAF association in mutual clusters was reduced by the NRas farnesylation inhibitor lonafarnib, yet enhanced by the BRAF inhibitor vemurafenib. Surprisingly, dispersed NRas molecules associated with the periphery of self-clusters of either Grb2 or NF1. Thus, NRas-mediated signaling, which is critical in health and disease, is regulated by dynamic interactions with functional clusters of BRAF or other related proteins at the PM.

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. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

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. NRas and BRAF resided in self-clusters that did not associate well in resting cells. In EGF-activated cells, NRas clusters became more diffused while overall protein levels at the PM increased; thus allowing enhanced association of NRas and BRAF and downstream signaling. In multiple melanoma cell lines, mutant NRas resided in more pronounced self-clusters relative to wild-type (WT) NRas yet associated more with the clustered and more abundant BRAF. In cells resistant to trametinib, a clinical MEK inhibitor (MEKi), a similar coclustering of NRas and BRAF was observed upon EGF activation. Strikingly, treatment of cells expressing mutant NRas with trametinib reversed the effect of mutant NRas expression by restoring the nonoverlapping self-clusters of NRas and BRAF and by reducing their PM levels and elevated pERK levels caused by mutant NRas. Our results indicate a new mechanism for signal regulation of NRas in melanoma through its nanoscale dynamic organization and a new mechanism for MEKi function in melanoma cells carrying NRas mutations but lacking MEK mutations. SIGNIFICANCE: Nanoscale dynamic organization of WT and mutant NRas relative to BRAF serves as a regulatory mechanism for NRas signaling and may be a viable therapeutic target for its sensitivity to MEKi.

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 α11.2 of the L-type voltage-gated Ca2+-channel Cav1.2, at positions G406R or G402S. Although both mutations cause cardiac arrhythmias, only Cav1.2G406R is associated with the autism-spectrum-disorder (ASD). We show that transcriptional activation by Cav1.2G406R and Cav1.2G402S is driven by membrane depolarization through the Ras/ERK/CREB pathway in a process called excitation-transcription (ET) coupling, as previously shown for wt Cav1.2. This process requires the presence of the intracellular ß-subunit of the channel. We found that only the autism-associated mutant Cav1.2G406R, as opposed to the non-autistic mutated channel Cav1.2G402S, exhibits a depolarization-independent CREB phosphorylation, and spontaneous transcription of cFos and MeCP2. A leftward voltage-shift typical of Cav1.2G406R activation, increases channel opening at subthreshold potentials, resulting in an enhanced channel activity, as opposed to a rightward shift in Cav1.2G402S. We suggest that the enhanced spontaneous Cav1.2G406R activity accounts for the increase in basal transcriptional activation. This uncontroled transcriptional activation may result in the manifestation of long-term dysregulations such as autism. Thus, gating changes provide a mechanistic framework for understanding the molecular events underlying the autistic phenomena caused by the G406R Timothy mutation. They might clarify whether a constitutive transcriptional activation accompanies other VGCC that exhibit a leftward voltage-shift of activation and are also associated with long-term cognitive disorders.

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. We show that early contacts are sufficient for robust TCR triggering and ZAP-70 recruitment. With cell spreading, TCR activation and ZAP-70 recruitment increase and shift to the edges of the growing tight contacts. CD45 segregates from TCR at tight contacts and is enriched at high local curvature membrane. Surprisingly, cortical actin and LFA localized at contact regions of intermediate tightness. Our results show in molecular detail the roles of early and tight T cell contacts in T cell activation, as both sensing and decision-making entities.

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. The simulation allows for membrane molecules, embedded at intercellular contacts, to diffuse and interact, while capturing the topography and energetics of the plasma membranes of the interface. We provide a detailed example related to pattern formation in the early IS. Using simulation predictions and three-color single molecule localization microscopy (SMLM), we detected the intricate mutual patterning of T cell antigen receptors (TCRs), integrins and glycoproteins in early T cell contacts with stimulating coverslips. The simulation further captures the dynamics of the patterning under the experimental conditions and at the IS with antigen presenting cells (APCs). Thus, we provide a generic tool for simulating realistic cell-cell interfaces, which can be used for critical hypothesis testing and experimental design in an iterative manner.

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. For that, we first defined a measure of the acquired-SNR for each method. This measure was ∼x10 to x100 higher for SOFI as compared to SMLM, indicating faster recognition and acquisition of features by SOFI. This measure also allowed fluorophore-specific optimization of SOFI reconstruction over its time-window and time-lag. We show that SOFI-assisted SMLM imaging can improve image reconstruction by rejecting common sources of background (e.g. out-of-focus emission and auto-fluorescence), especially under low signal-to-noise ratio conditions, by efficient optical sectioning and by shortening image reconstruction time. The performance and utility of our approach was evaluated by realistic simulations and by SOFI-assisted SMLM imaging of the plasma membrane of activated fixed and live T-cells (in isolation or in conjugation to antigen presenting cells). Our approach enhances SMLM performance under demanding imaging conditions and could set an example for synergizing additional imaging techniques.

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. We observed that gp41 clusters dynamically associated with the T cell antigen receptor (TCR) at the immune synapse upon TCR stimulation. This interaction, confirmed by FRET, depended on the virus clone, was reduced by the gp41 ectodomain in tight contacts, and was completely abrogated by mutation of the gp41 transmembrane domain. Strikingly, gp41 preferentially colocalized with phosphorylated TCRs at the PM of activated T-cells and promoted TCR phosphorylation. Gp41 expression also resulted in enhanced CD69 upregulation, and in massive cell death after 24-48 hrs. Our results shed new light on HIV-1 assembly mechanisms at the PM of host T-cells and its impact on TCR stimulation.

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. Using atomic force microscopy, we identify these cell contacts with engaged microvilli and characterize their morphology, rigidity and dynamics. Physical modelling and simulations of the imaged cell interfaces quantitatively capture the TCR-CD45 separation. Surprisingly, TCR phosphorylation negatively correlates with TCR-CD45 separation. These data support a refined kinetic-segregation model. First, kinetic-segregation occurs within seconds from TCR activation in engaged microvilli. Second, TCRs should be segregated, yet not removed too far, from CD45 for their optimal and localized activation within clusters. Our combined imaging and computational approach prove an important tool in the study of dynamic protein organization in cell interfaces.

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 Ca2+-bound α11.2 pore-forming subunit of the L-type Ca2+-channel (Cav1.2) to syntaxin 1 A (Sx1A) via an intra-membrane signaling mechanism. Here, we report the use of two-color Photo-Activated-Localization-Microscopy (PALM) to determine the relation between Cav1.2 and Sx1A in single-molecule detail. We observed nanoscale co-clusters of PAmCherry-tagged Sx1A and Dronpa-tagged α11.2 at a ~1:1 ratio. PAmCherry-tagged Sx1AC145A, or PAmCherry-tagged Sx2, an inactive Cav1.2 modulator, in which Cys145 is a Ser residue, showed no co-clustering. These results are  consistent with the crucial role of the single cytosolic Sx1ACys145 in clustering with Cav1.2. Cav1.2 and the functionally inactive transmembrane-domain double mutant Sx1AC271V/C272V engendered clusters with a ~2:1 ratio. A higher extent of co-clustering, which coincides with compromised depolarization-evoked transmitter-release, was observed also by oxidation of Sx1ACys271 and Cys272. Our super-resolution-imaging results set the stage for studying co-clustering of the channel with other exocytotic proteins at a single-molecule level.

Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner­both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.

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. Imaging and second-order statistics revealed that ZAP-70 recruitment and TCR activation localized inside TCR clusters. Live cell PALM imaging showed that the extent of localized TCR activation decreased, yet remained significant, with cell spreading. Using dynamic modeling and Monte-Carlo simulations we evaluated possible mechanisms of localized TCR activation. Our simulations indicate that localized TCR activation is the result of long-range cooperative interactions between activated TCRs, or localized activation by Lck and Fyn. Our results demonstrate the role of molecular clustering in cell signaling and activation, and are relevant to studying a wide range of multi-molecular complexes. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Fifteen years of APC/cyclosome: a short and impressive biography.

The APC/C (anaphase-promoting complex/cyclosome) discovered exactly 15 years ago by Avram Heshko and Marc Kirschner is by far the most complex ubiquitin ligase discovered so far. The APC/C is composed of roughly a dozen subunits and measures a massive 1.5 MDa. This huge complex, as well as its multiple modes of regulation, boasts impressive evolutionary conservation. One of its most puzzling features is its split personality: regulation of mitotic exit events on the one hand, and its ongoing activity during G(1)-phase, G(0)-phase and in terminally differentiated cells. The present short review is intended to provide a basic description of our current understanding of the APC/C, focusing on recent findings concerning its role in G(1)-phase and in differentiated cells.

APC/CCdh1 specific degradation of Hsl1 and Clb2 is required for proper stress responses of S. cerevisiae.

Cdh1 activates the Anaphase Promoting Complex/Cyclosome (APC/C(Cdh1)) throughout G(1) to degrade key cell cycle proteins. Cdh1 is not essential for cell proliferation, in spite of the fact that overexpression of some its degradation substrates is highly toxic. We report here that cdh1Delta cells are sensitive to stresses that activate the CWI (Cell Wall Integrity) and Hog1 MAP kinase pathways. Stresses did not activate APC/C(Cdh1) and cellular sensitivity was thus clearly due to constitutively elevated substrate levels. To explore the contribution of stabilization of individual APC/C(Cdh1) substrates to stress sensitivity, we generated cell lines expressing stabilized substrate mutants under their endogenous promoters. Cells expressing stabilized Hsl1 were sensitive to caffeine and failed to activate the Slt2 pathway. Cells expressing partially stable Clb2 were particularly sensitive to different stresses, possibly due to reduced Sic1 levels. Cells expressing stabilized Cdc5 were much less stress sensitive. Interestingly sensitivity of cdh1Delta cells does not seem to be restricted to G(1) but is manifested also during S and G(2) when the APC/C(Cdh1) is inactive anyway. We thus hypothesize that a role of G(1) specific APC/C(Cdh1) activity is to reset substrate levels to enables appropriate regulation of substrate accumulation in the subsequent phases of the cell cycle.

A cellular screening assay to test the ability of PKR to induce cell death in mammalian cells.

Long double-stranded RNA (>30 bp), usually expressed in cells infected with RNA viruses, triggers antiviral responses that induce apoptosis of the infected cells. PKR can be selectively activated in glioblastoma cells by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in these cells. Harnessing PKR for the selective killing of cancer cells is potentially a powerful strategy for treating cancer, but we were unable to induce apoptosis by this approach in a T cell lymphoma. We therefore established a cellular screening assay to test the ability of PKR to induce death in cell lines, especially those originating from human cancers. This "PKR killing screen" is based on the infection of cells with an adenoviral vector encoding GyrB-PKR, followed by coumermycin treatment. Cancers represented by cell lines in which PKR activation leads to cell death are good candidates for the dsRNA killing approach, using antisense to RNA molecules specifically expressed in these cells. The PKR killing screen may also serve as a tool for exploring PKR signaling and other related pathways, by identifying new cases in which PKR signaling is inhibited or impaired.