Biology subjects

Lidke, D.

Publications and source records attributed to Lidke, D..

3 recordsLinked to original sources

bioRxiv2023

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 Forster 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.

biophysics↗

bioRxiv2021

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

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. 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 both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the I 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. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activity state. 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 G13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and G13 binding to the cytoplasmic tail of an integrins {beta} subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.

cell biology↗

bioRxiv2017

Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics

Crosslinking of IgE-bound Fc{varepsilon}RI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the Fc{varepsilon}RI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after Fc{varepsilon}RI activation. We found that Syk colocalizes transiently to Fc{varepsilon}RI and that Syk-Fc{varepsilon}RI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-Fc{varepsilon}RI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the Fc{varepsilon}RI signaling pathway occurs at the level of Syk-Fc{varepsilon}RI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.\n\nSummarySchwartz et al. use single molecule imaging to quantify the transient nature of Fc{varepsilon}RI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

cell biology↗