RESUMO
Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-ß aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Magnetismo/métodos , Fagocitose/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação da Expressão Gênica , Estudos de Associação Genética/métodos , Genoma Humano , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos , Células RAW 264.7 , Transdução de Sinais/genética , Células U937RESUMO
Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore, including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon fluorescence microscopy biopsy analysis after a single administration.
