Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated with oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the stability of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.

Probing phosphorylation events in biological membranes: The transducer function.

The extracellular environment is sensed by receptors in the plasma membrane. Some of these receptors initiate cytoplasmic signaling cascades involving phosphorylation: the addition of a phosphate group to a specific amino acid, such as tyrosine, in a protein. Receptor Tyrosine Kinases (RTKs) are one large class of membrane receptors that can directly initiate signaling cascades through their intracellular kinase domains, which both catalyze tyrosine phosphorylation and get phosphorylated. In the first step of signaling, the ligands stabilize phosphorylation-competent RTK dimers and oligomers, which leads to the phosphorylation of specific tyrosine residues in the activation loop of the kinases. Here we discuss quantitative measurements of tyrosine phosphorylation efficiencies for RTKs, described by the "transducer function". The transducer function links the phosphorylation (the response) and the binding of the activating ligand to the receptor (the stimulus). We overview a methodology that allows such measurements in direct response to ligand binding. We discuss experiments which demonstrate that EGF is a partial agonist, and that two tyrosines in the intracellular domain of EGFR, Y1068 and Y1173, are differentially phosphorylated in the EGF-bound EGFR dimers.

Investigations of membrane protein interactions in cells using fluorescence microscopy.

The interactions between proteins in membranes govern many cellular functions. Our ability to probe for such interactions has greatly evolved in recent years due to the introduction of new fluorescence techniques. As a result, we currently have a choice of methods that can be used to assess the spatial distribution of a membrane protein, its association state, and the thermodynamic stability of the oligomers in the native milieu. These biophysical measurements have revealed new insights into important biological processes in cellular membranes.

Quantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding.

Signaling bias is the ability of a receptor to differentially activate downstream signaling pathways in response to different ligands. Bias investigations have been hindered by inconsistent results in different cellular contexts. Here we introduce a methodology to identify and quantify bias in signal transduction across the plasma membrane without contributions from feedback loops and system bias. We apply the methodology to quantify phosphorylation efficiencies and determine absolute bias coefficients. We show that the signaling of epidermal growth factor receptor (EGFR) to EGF and TGFα is biased towards Y1068 and against Y1173 phosphorylation, but has no bias for epiregulin. We further show that the L834R mutation found in non-small-cell lung cancer induces signaling bias as it switches the preferences to Y1173 phosphorylation. The knowledge gained here challenges the current understanding of EGFR signaling in health and disease and opens avenues for the exploration of biased inhibitors as anti-cancer therapies.

Quantitative characterization of tetraspanin 8 homointeractions in the plasma membrane.

The spatial distribution of proteins in cell membranes is crucial for signal transduction, cell communication and membrane trafficking. Members of the Tetraspanin family organize functional protein clusters within the plasma membrane into so-called Tetraspanin-enriched microdomains (TEMs). Direct interactions between Tetraspanins are believed to be important for this organization. However, studies thus far have utilized mainly co-immunoprecipitation methods that cannot distinguish between direct and indirect, through common partners, interactions. Here we study Tetraspanin 8 homointeractions in living cells via quantitative fluorescence microscopy. We demonstrate that Tetraspanin 8 exists in a monomer-dimer equilibrium in the plasma membrane. Tetraspanin 8 dimerization is described by a high dissociation constant (Kd = 14 700 ± 1100 Tspan8/µm2), one of the highest dissociation constants measured for membrane proteins in live cells. We propose that this high dissociation constant, and thus the short lifetime of the Tetraspanin 8 dimer, is critical for Tetraspanin 8 functioning as a master regulator of cell signaling.

RECURRENT OPENING AND CLOSURE OF MACULAR HOLE AFTER PARS PLANA VITRECTOMY FOR RHEGMATOGENOUS RETINAL DETACHMENT.

PURPOSE: To report a case of full-thickness macular hole formation and closure twice after pars plana vitrectomy for macula-off rhegmatogenous retinal detachment. METHODS: Case report. RESULTS: A 45-year-old woman with a macula-off rhegmatogenous retinal detachment underwent uncomplicated pars plana vitrectomy with silicone oil tamponade, which was removed 3 months postoperatively. A full-thickness macular hole developed spontaneously 18 months after silicone oil removal, concurrently with the formation of epiretinal membrane and perifoveal cystoid changes. The hole was noted to close spontaneously within 2 months with normal foveal contour formation and improved vision, but then reopened for the second time after 10 months. A second spontaneous closure was observed within 2 months. CONCLUSION: Dynamic course of macular hole formation and resolution in a previously vitrectomized eye may justify a more conservative approach with observation for some time before surgical intervention is favored.

Electrospun Nanofibers With pH-Responsive Coatings for Control of Release Kinetics.

Functional and stimuli-responsive nanofibers with an enhanced surface area/volume ratio provide controlled and triggered drug release with higher efficacy. In this study, chemotherapeutic agent Rose Bengal (RB) (4,5,6,7-tetrachloro-2', 4',5',7'-tetraiodofluoresceindisodium)-loaded water-soluble polyvinyl alcohol (PVA) nanofibers were synthesized by using the electrospinning method. A thin layer of poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) p(4VP-co-EGDMA) was deposited on the RB-loaded nanofibers (PVA-RB) via initiated chemical vapor deposition (iCVD), coating the fiber surfaces to provide controllable solubility and pH response to the nanofibers. The uncoated and [p(4VP-co-EGDMA)-PVA] coated PVA-RB nanofiber mats were studied at different pH values to analyze their degradation and drug release profiles. The coated nanofibers demonstrated high stability at neutral and basic pH values for long incubation durations of 72 h, whereas the uncoated nanofibers dissolved in <2 h. The drug release studies showed that the RB release from coated PVA-RB nanofibers was higher at neutral and basic pH values, and proportional to the pH of the solution, whereas the degradation and RB release rates from the uncoated PVA-RB nanofibers were significantly higher and did not depend on the pH of environment. Further analysis of the release kinetics using the Peppas model showed that while polymer swelling and dissolution were the dominant mechanisms for the uncoated nanofibers, for the coated nanofibers, Fickian diffusion was the dominant release mechanism. The biocompatibility and therapeutic efficiency of the coated PVA-RB nanofibers against brain cancer was investigated on glioblastoma multiforme cancer cells (U87MG). The coated PVA nanofibers were observed to be highly biocompatible, and they significantly stimulated the ROS production in cells, increasing apoptosis. These promising results confirmed the therapeutic activity of the coated PVA-RB nanofibers on brain cancer cells, and encouraged their further evaluation as drug carrier structures in brain cancer treatment.

Multifunctional one-dimensional polymeric nanostructures for drug delivery and biosensor applications.

Advances in nanotechnology in the last decades have paved the way for significant achievements in diagnosis and treatment of various diseases. Different types of functional nanostructures have been explored and utilized as tools for addressing the challenges in detection or treatment of diseases. In particular, one-dimensional nanostructures hold great promise in theranostic applications due to their increased surface area-to-volume ratios, which allow better targeting, increased loading capacity and improved sensitivity to biomolecules. Stable polymeric nanostructures that are stimuli-responsive, biocompatible and biodegradable are especially preferred for bioapplications. In this review, different synthesis techniques of polymeric one-dimensional nanostructures are explored and functionalization methods of these nanostructures for specific applications are explained. Biosensing and drug delibiovery applications of these nanostructures are presented in detail.