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

Abstract

During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. 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 processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this 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 migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

“…We were also interested in whether the knockdown cells with altered mTORC1 signaling activity maintained their sensitivity to fMLP as a chemotactic agent, and explored this further in our FLCN knockdown line. Here we used photo-activation of caged fMLP to generate spatial gradients of the small chemotactic peptide 30 , using a standard assay where migratory cells are sandwiched between a BSA-coated coverslip and an agarose overlay to minimize requirements for integrin-based adhesion during migration 60 . While FLCN knockdown cells migrated with similar speeds as our control sgRNA line in this context (Fig.…”

Direct activation of the Ras-Akt network mediates polarity and organizes protrusions in human neutrophil migration

“…Although Ras activation-mediated cytoskeletal changes and motility occurred in absence of chemoattractant, we wanted to ensure that these phenotypes were caused solely by recruiting active Ras on the membrane and not basal G-protein coupled receptor signaling 86 . To test this, we inhibited heterotrimeric G-protein activity in neutrophils using a combination of Gα i and Gβγ inhibitors, pertussis toxin (PTX) and gallein, respectively (Figure S3A and B) 18, 47, 83, 87–90 . Dual inhibitor treatment prevented the typical uniform chemoattractant-induced burst of F-actin polymerization all around the cell periphery and subsequent polarized migration (Figure S3B).…”

“…Stable expression lines in HL-60 cells were generated by a combination of 3 rd -generation lentiviral- and PiggyBac™ transposon-integration based approaches 18, 132 . Virus was prepared in HEK293T cells grown to ∼80% confluency in 10 cm cell culture dish (Greiner Bio-One; 664160).…”

Noise Signal Recognition and Noise Reduction Algorithm of Ships

“…Considering the discreteness of the above functions, we can get the following by recurrence criterion [13][14][15].…”