Biology subjects

Yan, P.

Publications and source records attributed to Yan, P..

3 recordsLinked to original sources

bioRxiv2019

Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis.

Regulation of the actin cytoskeleton is critical for normal glomerular podocyte structure and function. Altered regulation of the podocyte cytoskeleton can lead to proteinuria, reduced kidney filtration function and focal segmental glomerulosclerosis (FSGS). Mutations in inverted formin 2 (INF2), a member of the formin family of actin regulatory proteins, are the most common cause of autosomal dominant FSGS. INF2 is a multi-domain protein regulated by interaction between its N-terminal Diaphanous Inhibitory Domain (DID) and its C-terminal Diaphanous Auto-regulatory Domain (DAD). Although many aspects of the INF2 DID-DAD interaction are understood, it remains unclear why disease-causing mutations are restricted to the DID and how these mutations cause human disease. Here we report a proteolytic cleavage in INF2 that liberates the INF2 N-terminal DID to function independently of the INF2 C-terminal fragment containing the DAD domain. N-terminal DID region epitopes are differentially localized to podocyte foot process structures in normal glomeruli. This N-terminal fragment localization is lost in INF2-mediated FSGS, whereas INF2 C-terminal fragment epitopes localize to the podocyte cell body in both normal and disease conditions. INF2 cleavage is mediated by cathepsin proteases. In cultured podocytes, the wild-type INF2 N-terminal fragment localizes to membrane regions and promotes cell spreading, while these functions are impaired in a disease-associated INF2 mutant R218Q in the DID. These features are dependent on INF2-cleavage, with accompanying interaction of INF2 N-fragment with mDIA1. Our data suggest a unique cellular function of the DID dependent on INF2 cleavage and help explain the altered localization of FSGS-associated INF2 mutant polypeptides.

pathology↗

bioRxiv2019

Ultra-fast fit-free analysis of complex fluorescence lifetime imaging via deep learning

Fluorescence lifetime imaging (FLI) provides unique quantitative information in biomedical and molecular biology studies, but relies on complex data fitting techniques to derive the quantities of interest. Herein, we propose a novel fit-free approach in FLI image formation that is based on Deep Learning (DL) to quantify complex fluorescence decays simultaneously over a whole image and at ultra-fast speeds. Our deep neural network (DNN), named FLI-Net, is designed and model-based trained to provide all lifetime-based parameters that are typically employed in the field. We demonstrate the accuracy and generalizability of FLI-Net by performing quantitative microscopic and preclinical experimental lifetime-based studies across the visible and NIR spectra, as well as across the two main data acquisition technologies. Our results demonstrate that FLI-Net is well suited to quantify complex fluorescence lifetimes, accurately, in real time in cells and intact animals without any parameter settings. Hence, it paves the way to reproducible and quantitative lifetime studies at unprecedented speeds, for improved dissemination and impact of FLI in many important biomedical applications, especially in clinical settings.

bioengineering↗

bioRxiv2018

Tethered Bichromophoric Fluorophore Quencher Voltage Sensitive Dyes

Voltage sensitive dyes (VSDs) are used for in vitro drug screening and for in vivo imaging of patterns of electrical activity. However, wide application of this technology is limited by poor sensitivity. A promising approach uses a 2-component system consisting of charged membrane permeable quenchers together with fluorophores labeling one side of the membrane; this produces voltage-dependent fluorescence quenching. However, to achieve good sensitivity, the quencher compound must be used at high concentrations, which can perturb the membrane capacitance or have other pharmacological effects. By developing tethered bichromophoric fluorophore quencher (TBFQ) dyes, where the fluorophore and quencher are covalently connected by a long hydrophobic chain, the concentration required is minimized, and the sensitivity is maximized. A series of 13 TBFQ dyes based on the AminoNaphthylEthenylPyridinium (ANEP) fluorophore and the dipicrylamine anion (DPA) quencher have been synthesized and tested in an artificial lipid bilayer apparatus. The best one from the screening, TBFQ1, shows a 2.5 fold change in fluorescence per 100mV change in membrane potential, and the response kinetics is in 10-20 ms range. This sensitivity is an order of magnitude better than commonly used fluorescent voltage sensors. The design principles for TBFQ VSDs described here can be readily extended to other spectral regions and promise to greatly enhance our ability to monitor electrical activity in cells and tissues.

biophysics↗