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 (GPCRs) 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. 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.

Directional reorientation of migrating neutrophils is limited by suppression of receptor input signaling at the cell rear through myosin II activity

“…Simply put, a cell that overreacted when the stimulation commenced would also overreact when it stopped, emerging as a strong responder. This combination of positive and negative regulation of Cdc42 downstream of receptors was also documented in cell-wide optogenetic stimulation experiments and may reflect temporal processing of input signals 14 .…”

“…We generated a neutrophil-like HL60 cell line stably expressing parapinopsin (a light-sensitive G αi -family GPCR) and a tdTomato/tdKatushka2 Cdc42 fluorescence resonance energy transfer (FRET) biosensor, spectrally compatible with the reversible parapinopsin stimulation. This system allows direct recording of downstream Cdc42 activity in cells whose migration is guided by parapinopsin 14 . We confined cells to migrate inside 1D straight microfluidic channels in response to spatial serum gradients (Fig.…”

“…A subtle point here is that although Cdc42 is a primary regulator of cell direction and formation of protrusive fronts in neutrophil-like cells 4 , there may be other components that could change faster. Nevertheless, evidence of temporal signal processing has also been shown in neutrophil-like cells when confined to migrate in a 2D plane under agarose 14 , in primary neutrophils upon fast switching of chemoattractant gradients 35 , and in myeloid cells whose persistent migration to certain intermediate chemokines involves temporal sensing 36 .…”

WASP integrates substrate topology and cell polarity to guide neutrophil migration

“…The Cdc42 PLB-895 knockout line was generated as described in (Bell et al, 2021). Briefly, PLB-895 cells were electroporated with a Cas9-sgRNA ribonucleoprotein (RNP) complex.…”

“…Two clonal lines were generated for WASP from separate CRISPR assemblies (guide 1: 5' CCAATGGGAGGAAGGCCCGG 3' and guide 2: 5' GCTGAACCGCTGGTGCTCCT 3') and one clonal line was generated for FBP17 (5' ACGAAATGAATGATTACGCA 3'). The Cdc42 PLB-895 knockout line was generated as described in (Bell et al, 2021). Briefly, PLB-895 cells were electroporated with a Cas9-sgRNA ribonucleoprotein (RNP) complex.…”