Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy.

Rapid, high-resolution volumetric imaging without moving heavy objectives or disturbing delicate samples remains challenging. Pupil-matched remote focusing offers a promising solution for high NA systems, but the fluorescence signal's incoherent and unpolarized nature complicates its application. Thus, remote focusing is mainly used in the illumination arm with polarized laser light to improve optical coupling. Here, we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light, lets them pass through the remote focusing module separately, and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks with an axial range of 70 µm and camera-limited acquisition speed. Owing to its general nature, we believe this technique will find its application in many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging, such as confocal, 2-photon, and light sheet variants.

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy.

The ability to image at high speeds is necessary for biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging. However, owing to the incoherent and unpolarized nature of the fluorescence signal, manipulating this emission light through remote focusing is challenging. Therefore, remote focusing has been primarily limited to the illumination arm, using polarized laser light to facilitate coupling in and out of the remote focusing optics. Here, we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light, lets them pass through the remote focusing module separately, and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks with an axial range of 70 µm and camera-limited acquisition speed. Owing to its general nature, we believe this technique will find its application in many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging, such as confocal, 2-photon, and light sheet variants.

Heterochiral modifications enhance robustness and function of DNA in living human cells.

Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactions in situ via a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror-image, ʟ-DNA nucleotides to produce heterochiral "gapmers". We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ-nucleic acid protection of oligonucleotides and DNA circuitry for applications in vivo.

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy.

The ability to image at high speeds is necessary in biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging. However, owing to the incoherent and unpolarized nature of the fluorescence signal, manipulating this emission light through remote focusing is challenging. Therefore, remote focusing has been primarily limited to the illumination arm, using polarized laser light for facilitating coupling in and out of the remote focusing optics. Here we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light and lets them pass through the remote focusing module separately and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks, with an axial range of 70 µm and camera limited acquisition speed. Owing to its general nature, we believe this technique will find its application to many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging such as confocal, 2-photon, and light sheet variants.

Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcεRI Aggregation and Structural Changes.

Immunoreceptor tyrosine-based activation motif (ITAM)-containing Fc receptors are critical components of the innate and adaptive immune systems. FcεRI mediates the allergic response via crosslinking of IgE-bound receptors by multivalent antigens. Yet, the underlying molecular mechanisms that govern the response of FcεRI to specific antigens remain poorly understood. We compared responses induced by two antigens with distinct geometries, high valency DNP-BSA and trivalent DF3, and found unique secretion and receptor phosphorylation profiles that are due to differential recruitment of Lyn and SHIP1. To understand how these two antigens can cause such markedly different outcomes, we used direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging combined with Bayesian Grouping of Localizations (BaGoL) analysis to compare the nanoscale characteristics of FcεRI aggregates. DF3 aggregates were found to be smaller and more densely packed than DNP-BSA aggregates. Using lifetime-based Förster resonance energy transfer (FRET) measurements, we discovered that FcεRI subunits undergo structural rearrangements upon crosslinking with either antigen, and in response to interaction with monovalent antigen presented on a supported lipid bilayer. The extent of conformational change is positively correlated with signaling efficiency. Finally, we provide evidence for forces in optimizing FcεRI signaling, such that immobilizing DF3 on a rigid surface promoted degranulation while increasing DNP-BSA flexibility lowered degranulation. These results provide a link between the physical attributes of allergens, including size, shape, valency, and flexibility, and FcεRI signaling strength. Thus, the antigen modulates mast cell outcomes by creating unique aggregate geometries that tune FcεRI conformation, phosphorylation and signaling partner recruitment.

Application of Biophysical Techniques to Cellular and Molecular Oncology.

Dysregulated cellular processes drive malignant transformation, tumor progression, and metastasis, and affect responses to therapies [...].

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) with important roles in many cellular processes as well as cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. However, it is not clear how these dimers relate to higher-order EGFR oligomers detected at the cell surface. We used single-particle tracking (SPT) and Förster resonance energy transfer (FRET) imaging to examine how each domain within EGFR contributes to receptor dimerization and the rate of its diffusion in the cell membrane. We show that the EGFR extracellular region is sufficient to drive receptor dimerization, but that the EGF-induced EGFR slow-down seen by SPT requires formation of higher order oligomers, mediated in part by the intracellular tyrosine kinase domain - but only when in its active conformation. Our data thus provide important insight into higher-order EGFR interactions required for EGF signaling.

High-precision estimation of emitter positions using Bayesian grouping of localizations.

Single-molecule localization microscopy super-resolution methods rely on stochastic blinking/binding events, which often occur multiple times from each emitter over the course of data acquisition. Typically, the blinking/binding events from each emitter are treated as independent events, without an attempt to assign them to a particular emitter. Here, we describe a Bayesian method of inferring the positions of the tagged molecules by exploring the possible grouping and combination of localizations from multiple blinking/binding events. The results are position estimates of the tagged molecules that have improved localization precision and facilitate nanoscale structural insights. The Bayesian framework uses the localization precisions to learn the statistical distribution of the number of blinking/binding events per emitter and infer the number and position of emitters. We demonstrate the method on a range of synthetic data with various emitter densities, DNA origami constructs and biological structures using DNA-PAINT and dSTORM data. We show that under some experimental conditions it is possible to achieve sub-nanometer precision.

Docking of Syk to FcεRI is enhanced by Lyn but limited in duration by SHIP1.

The high-affinity immunoglobulin E (IgE) receptor, FcεRI, is the primary immune receptor found on mast cells and basophils. Signal initiation is classically attributed to phosphorylation of FcεRI ß- and γ-subunits by the Src family kinase (SFK) Lyn, followed by the recruitment and activation of the tyrosine kinase Syk. FcεRI signaling is tuned by the balance between Syk-driven positive signaling and the engagement of inhibitory molecules, including SHIP1. Here, we investigate the mechanistic contributions of Lyn, Syk, and SHIP1 to the formation of the FcεRI signalosome. Using Lyn-deficient RBL-2H3 mast cells, we found that another SFK can weakly monophosphorylate the γ-subunit, yet Syk still binds the incompletely phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). Once recruited, Syk further enhances γ-phosphorylation to propagate signaling. In contrast, the loss of SHIP1 recruitment indicates that Lyn is required for phosphorylation of the ß-subunit. We demonstrate two noncanonical Syk binding modes, trans γ-bridging and direct ß-binding, that can support signaling when SHIP1 is absent. Using single particle tracking, we reveal a novel role of SHIP1 in regulating Syk activity, where the presence of SHIP1 in the signaling complex acts to increase the Syk:receptor off-rate. These data suggest that the composition and dynamics of the signalosome modulate immunoreceptor signaling activities.

Logic-gated antibody pairs that selectively act on cells co-expressing two antigens.

The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect®, an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

EGFR transactivates RON to drive oncogenic crosstalk.

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation - arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

Integrin activation is an essential component of SARS-CoV-2 infection.

SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's ß subunit, we demonstrate that talin-mediated signaling is essential for productive infection.

γ9δ2T cell diversity and the receptor interface with tumor cells.

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

High-speed single molecule imaging datasets of membrane proteins in rat basophilic leukemia cells.

A high-speed fluorescence microscope operating at a 490 Hz frame rate was used to image two different membrane proteins- the high-affinity IgE receptor FcɛRI, a transmembrane protein, and an outer-leaflet GPI-anchored protein. The IgE receptor was imaged via IgE labeled with Janelia Fluor 646 and the GPI-anchored protein was imaged using a GPI-GFP fusion protein and an ATTO 647 N labeled anti-GFP nanobody. Data was collected for both proteins in untreated cells and cells that had actin stabilized by phalloidin. This dataset can be used for development and testing of single-particle tracking methods on experimental data and to explore the hypothesis that the actin cytoskeleton may affect the movement of membrane proteins.

RTK signaling requires C3ar1/C5ar1 and IL-6R joint signaling to repress dominant PTEN, SOCS1/3 and PHLPP restraint.

How receptor tyrosine kinase (RTK) growth signaling is controlled physiologically is incompletely understood. We have previously provided evidence that the survival and mitotic activities of vascular endothelial cell growth factor receptor-2 (VEGFR2) signaling are dependent on C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 joint signaling in a physically interactive platform. Herein, we document that the platelet derived and epidermal growth factor receptors (PDGFR and EGFR) are regulated by the same interconnection and clarify the mechanism underlying the dependence. We show that the joint signaling is required to overcome dominant restraint on RTK function by the combined repression of tonically activated PHLPP, SOCS1/SOCS3, and CK2/Fyn dependent PTEN. Signaling studies showed that augmented PI-3KÉ£ activation is the process that overcomes the multilevel growth restraint. Live-cell flow cytometry and single-particle tracking indicated that blockade of C3ar1/C5ar1 or IL-6R signaling suppresses RTK growth factor binding and RTK complex formation. C3ar1/C5ar1 blockade abrogated growth signaling of four additional RTKs. Active relief of dominant growth repression via joint C3ar1/C5ar1 and IL-6R joint signaling thus enables RTK mitotic/survival signaling.

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

Differential epidermal growth factor receptor (EGFR) phosphorylation is thought to couple receptor activation to distinct signaling pathways. However, the molecular mechanisms responsible for biased signaling are unresolved due to a lack of insight into the phosphorylation patterns of full-length EGFR. We extended a single-molecule pull-down technique previously used to study protein-protein interactions to allow for robust measurement of receptor phosphorylation. We found that EGFR is predominantly phosphorylated at multiple sites, yet phosphorylation at specific tyrosines is variable and only a subset of receptors share phosphorylation at the same site, even with saturating ligand concentrations. We found distinct populations of receptors as soon as 1 min after ligand stimulation, indicating early diversification of function. To understand this heterogeneity, we developed a mathematical model. The model predicted that variations in phosphorylation are dependent on the abundances of signaling partners, while phosphorylation levels are dependent on dimer lifetimes. The predictions were confirmed in studies of cell lines with different expression levels of signaling partners, and in experiments comparing low- and high-affinity ligands and oncogenic EGFR mutants. These results reveal how ligand-regulated receptor dimerization dynamics and adaptor protein concentrations play critical roles in EGFR signaling.

Characterization of the Signaling Modalities of Prostaglandin E2 Receptors EP2 and EP4 Reveals Crosstalk and a Role for Microtubules.

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

Combinatorial diversity of Syk recruitment driven by its multivalent engagement with FcεRIγ.

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αßγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk-FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk's bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.