Two independent but synchronized Gβγ subunit–controlled pathways are essential for trailing-edge retraction during macrophage migration

Abstract

Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (G)-coupled receptors (GPCRs), G protein βγ (Gβγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα pathway. However, it is not clear how the activation of G-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the G pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gβγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cβ and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the G-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.

Statins Perturb GβγSignaling and Cell Behavior in a GγSubtype Dependent Manner

“…We have demonstrated that farnesylated Gg types, including Gg9, attenuate Gbg-effectors PLCb and phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) activations, whereas geranylgeranylated Gg types enhance their signaling (Senarath et al, 2018). We also showed that endogenously expressed Gg3 in RAW cells are required for Gi pathway activation-mediated PIP3 production and PLCb activation during cell migration (Siripurapu et al, 2017;Senarath et al, 2018). In migratory cells, lamellipodia formation at the leading edge is driven by PIP3, produced as a result of Gbg-mediated PI3K activation (Kölsch et al, 2008).…”

“…RAW 264.7 cells incubated with either 10 mM fluvastatin, 20 mM atorvastatin, or 10 mM lovastatin for 12 hours were pretreated with the small-molecule calcium indicator fluo-4 AM (2.28 mM). Calcium imaging was performed as described previously (Siripurapu et al, 2017). After baseline, fluo-4 fluorescence was captured, endogenous c5a receptors were activated with 20 mM c5a, and fluo4 imaging was continued.…”

Gγ identity dictates efficacy of Gβγ signaling and macrophage migration

“…The Gγ dependent differential PIP3 generation in HeLa cells hints at a plausible mechanism of how Gβγ effectors are recruited to the PM and activated by PM bound fraction of HiAf-Gβγ. We recently showed that Gβγ controls PLCβ activation, induces Ca 2+ mobilization, governing the trailing edge retraction during RAW cell migration (18). Similar to PI3Kγ, PLCβ1 and PLCβ2 are also cytosolic (41,42).…”

“…Altered cell motilities are implicated in pathological processes such as immune deficiencies, lack of wound healing, tissue repair and cancer metastasis (14)(15)(16)(17). We have recently shown that Gβγ is a key regulator of inhibitory G protein (Gi) -coupled GPCR activation induced macrophage migration (18). In addition to PI3K-PIP3 signaling at the leading edge, we demonstrated that Gβγ mediated activation of PLCβ pathway is essential for macrophage migration.…”

“…Recently, we demonstrated that Gicoupled GPCR activation induced RAW cell migration requires an increase in cytosolic calcium (Ca 2+ ) which is governed by Gβγ mediated activation of PLCβ to induce trailing edge retraction (18). Thus, we examined if PLCβ activity in RAW cells is also controlled in a Gγ subtype dependent manner, in the same way it controlled PI3Kγ activation.…”