Clathrin-mediated endocytosis regulates fMLP-mediated neutrophil polarization

Abstract

A cell's ability to establish polarization is one of the key steps in directional migration. Upon the addition of a chemoattractant, N-formylmethionyl-leucyl-phenylalanine (fMLP), neutrophils rapidly develop a front end marked by a wide and dense actin network which is a feature of cell polarization. Despite a general understanding of bi-directional crosstalk between endocytosis and polarization, it remains unclear how clathrin-mediated endocytosis (CME) induced by chemoattractant binding to formyl peptide receptor (FPR) affects neutrophil polarization. In this work, we characterized the spatial organization of FPR and clathrin-coated pits (CCPs), the functional unit of CME, with and without fMLP and found that fMLP induced different distributions of FPR and CCPs. We further found that cells had impaired polarization induced by fMLP when CME is inhibited by small molecule inhibitors. Under these conditions, pERK, pAkt308, and pAkt473 were all severely blocked or had altered dynamics. The spatial organization between actin and two major clathrin-mediated endocytic proteins, clathrin and β-arrestin, were distinct and supported clathrin and β-arrestin's functional roles in mediating neutrophil polarization. Together these results suggest that CME plays a pivotal role in a complex process such as cell polarization.

Clathrin Heavy Chain Knockdown Impacts CXCR4 Signaling and Post-translational Modification

“…While clathrin inhibition has not been previously associated with a change in ERK1/2 signaling, previous studies with CXCR4 have focused on transient pharmacological inhibition of CME (30 min timescale) (English et al, 2018), whereas in this study shRNAs were used to specifically knockdown CHC over the course of 3–5 days) and consequently provides additional evidence for cellular compensation. Additionally, this effect appears to be different in HL-60 derived neutrophils where inhibition of clathrin abrogated ERK1/2 signaling and polarization (Tan et al, 2018). This could be due to a strong dependence of signaling on the clathrin scaffold for a professional migratory cell as well as differences between pharmacological vs. genetic perturbations.…”

Induced Nanoscale Membrane Curvature Bypasses Clathrin’s Essential Endocytic Function

“…Clathrin-mediated endocytosis (CME), the best-studied form of extracellular cargo uptake, is a constitutive process that regulates a wide array of cellular functions including signaling, adhesion, and migration (Tan et al, 2018;Ezratty et al, 2009;Paul et al, 2015). To generate vesicles, adaptor proteins such as AP2 bind to cargoes, early curvature-generating proteins such as FCHo1/2, and the coat protein clathrin, marking a clathrin-coated pit (CCP) (Jackson et al, 2010;Henne et al, 2010).…”

Mechanical Regulation of Endocytosis: New Insights and Recent Advances

“…However, whether this redundancy acts as a fail-safe switch to sustain CME under different mechanical stimuli needs to be further studied. Mechanical factors like area of confinement, local membrane tension, and polarity of a cell may play a role in the selective initiation of CME at specific membrane sites [8,25,76,77] . Endocytic proteins like AP2, epsin 1, amphiphysin 1 show preferential recruitment to regions of pre-existing sub-micron curvature.…”