Rincon, E. | bioRxiv Science

bioRxiv2021

Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing

During chemotaxis, neutrophils use cell surface G-Protein Coupled Receptors (GPCRs) to detect chemoattractant gradients1-4. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities1, 5-8. Positive feedback could promote rapid signal spreading9, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell1. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing10. To directly measure information flow from receptors to Cdc42, we paired zebrafish parapinopsina, an optogenetic GPCR that allows reversible ON/OFF receptor control with a spectrally compatible red/far red Cdc42 FRET biosensor. Using this new toolkit, we show that positive and negative signals downstream of G-proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.

cell biology↗

bioRxiv2017

A map of gene expression in neutrophil-like cell lines

BackgroundHuman neutrophils are central players in innate immunity, a major component of inflammatory responses, and a leading model for cell motility and chemotaxis. However, primary neutrophils are remarkably short-lived, limiting their experimental usefulness in the laboratory. Thus, human myeloid cell lines have been established and characterized for their ability to undergo neutrophil-like differentiation in vitro. The HL-60 cell line and its PLB-985 sub-line are commonly used as a model for human neutrophil behavior, but how closely gene expression in differentiated cells resembles that of primary neutrophils has remained unclear.\n\nResultsIn the present study, we compared the effectiveness of differentiation protocols and used RNA sequencing (RNA-seq) to compare the transcriptomes of HL-60 and PLB-985 cells with published data for human and mouse primary neutrophils. Among commonly used differentiation protocols for neutrophil like cell lines, addition of dimethyl sulfoxide (DMSO) gave the best combination of cell viability and expression of markers for differentiation. However, combining DMSO with the serum-free-supplement Nutridoma resulted in an increased chemotactic response and cell surface expression of the neutrophil markers FPR1 and CD11b without a cost in viability. RNA-seq analysis of HL-60 and PLB-985 cells before and after differentiation showed that differentiation broadly increases the similarity in gene expression between the cell lines and primary neutrophils. Furthermore, the gene expression pattern of the differentiated cell lines correlated slightly better with that of human neutrophils than the mouse neutrophil pattern did. Finally, we created a publicly available gene expression database that is searchable by gene name and by protein domain content, where users can compare gene expression in HL-60, PLB-985 and primary human and mouse neutrophils.\n\nConclusionsOur study verifies that a DMSO-based differentiation protocol for HL-60 and PLB-985 cell lines gives superior differentiation and cell viability relative to other common protocols, and indicates that addition of Nutridoma may be preferable for studies of chemotaxis. Our neutrophil gene expression database will be a valuable tool to identify similarities and differences in gene expression between the cell lines and primary neutrophils, to compare expression levels for genes of interest, and to improve the design of tools for genetic perturbations.

genomics↗