Microneedle-mediated nanomedicine to enhance therapeutic and diagnostic efficacy.

Nanomedicine has been extensively explored for therapeutic and diagnostic applications in recent years, owing to its numerous advantages such as controlled release, targeted delivery, and efficient protection of encapsulated agents. Integration of microneedle technologies with nanomedicine has the potential to address current limitations in nanomedicine for drug delivery including relatively low therapeutic efficacy and poor patient compliance and enable theragnostic uses. In this Review, we first summarize representative types of nanomedicine and describe their broad applications.

Ligand bias underlies differential signaling of multiple FGFs via FGFR1.

The differential signaling of multiple FGF ligands through a single fibroblast growth factor (FGF) receptor (FGFR) plays an important role in embryonic development. Here, we use quantitative biophysical tools to uncover the mechanism behind differences in FGFR1c signaling in response to FGF4, FGF8, and FGF9, a process which is relevant for limb bud outgrowth. We find that FGF8 preferentially induces FRS2 phosphorylation and extracellular matrix loss, while FGF4 and FGF9 preferentially induce FGFR1c phosphorylation and cell growth arrest.

Tumor-on-chip modeling of organ-specific cancer and metastasis.

Every year, cancer claims millions of lives around the globe. Unfortunately, model systems that accurately mimic human oncology - a requirement for the development of more effective therapies for these patients - remain elusive. Tumor development is an organ-specific process that involves modification of existing tissue features, recruitment of other cell types, and eventual metastasis to distant organs.

Quantifying the Interaction between EGFR Dimers and Grb2 in Live Cells.

Adaptor proteins are a class of cytoplasmic proteins that bind to phosphorylated residues in receptor tyrosine kinases and trigger signaling cascades that control critically important cellular processes, such as cell survival, growth, differentiation, and motility. Here, we seek to characterize the interaction between epidermal growth factor receptor (EGFR) and the cytoplasmic adaptor protein growth factor receptor-bound protein 2 (Grb2) in a cellular context. To do so, we explore the utility of a highly biologically relevant model system, mammalian cells under reversible osmotic stress, and a recently introduced Förster resonance energy transfer microscopy method, fully quantified spectral imaging.

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors.

The activity of receptor tyrosine kinases (RTKs) is controlled through their lateral association in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared with homodimers, because of limitations in current experimental methods.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors.

Effect of thanatophoric dysplasia type I mutations on FGFR3 dimerization.

Thanatophoric dysplasia type I (TDI) is a lethal human skeletal growth disorder with a prevalence of 1 in 20,000 to 1 in 50,000 births. TDI is known to arise because of five different mutations, all involving the substitution of an amino acid with a cysteine in fibroblast growth factor receptor 3 (FGFR3). Cysteine mutations in receptor tyrosine kinases (RTKs) have been previously proposed to induce constitutive dimerization in the absence of ligand, leading to receptor overactivation.

Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes.

Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes.

Production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions.

Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation buffer containing chloride salts and no formaldehyde or DTT.