Elsevier

Cellular Signalling

Volume 66, February 2020, 109443
Cellular Signalling

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions

https://doi.org/10.1016/j.cellsig.2019.109443Get rights and content

Abstract

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.

Introduction

Phagocytosis is an essential component of innate immunity in multicellular organisms. To better understand the cellular control of phagocytosis, our research has focused on characterizing the phagocytic modes initiated by the diverse family of channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs). These fish innate immune proteins share basic structural, as well as distant phylogenetic relationships, with several immunoregulatory proteins within the mammalian immunoglobulin superfamily [1,2]. By using epitope-tagged receptor constructs that allow for the expression of IpLITRs in mammalian cell lines, we have taken advantage of commercially available antibodies to efficiently engage these receptors even though their endogenous ligands are not known. This strategy has helped define the basic cellular mechanisms that control the immunoregulatory actions of IpLITRs and has delineated the molecular events that allow these receptors to actively regulate innate immune cell effector responses, including phagocytosis [[3], [4], [5], [6], [7], [8]].

Of the known IpLITR variants, our previous work has characterized a classical immunoreceptor tyrosine-based activation motif (ITAM)-dependent phagocytic pathway controlled by the stimulatory IpLITR 2.6b/IpFcRγ-L chimera [4,6,8,9], as well as a unique phagocytic mode that utilizes the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor IpLITR 1.1b [4,[7], [8], [9]]. We have also demonstrated that stable expression of IpLITR 1.1b, but not IpLITR 2.6b/IpFcRγ-L, specifically triggers transfected rat basophilic leukemia (RBL)-2H3 cells to actively generate filamentous (F)-actin-dense cellular protrusions [8]. Active deployment of filopodia by macrophages is a well-known phenomenon, however, relatively little is known about the immunoregulatory receptor-types that participate in the control of filopodial dynamics within innate immune cells. During the early stages of the IpLITR 1.1b-mediated phagocytic process, filopodia-like structures initiate target contact and then retract to secure captured targets to the cell surface [8]. This mode of active target-capture is followed by the formation of phagocytic cup-like structures at the membrane-target interface, which occasionally resulted in target engulfment [8]. IpLITR 1.1b-mediated signaling also initiated secondary waves of F-actin polymerization events associated with the tethering and internalization of extracellular targets[8]. Based on these observations, we hypothesized that constitutive preassembly of IpLITR 1.1b with intracellular effectors allows for the selective modulation of the cytoskeletal machinery to drive dynamic membrane remodeling events prior to the formation of stable receptor-ligand interactions that further reinforce filopodia dynamics [8]. Additionally, it is likely that the target acquisition and engulfment pathways facilitated by IpLITR 1.1b require the differential participation of unique membrane proximal and distal regions of its cytoplasmic tail (CYT) for the recruitment and activation of distinct intracellular effectors [7,10].

Within its relatively long CYT, IpLITR 1.1b contains six tyrosine residues that can be reversibly phosphorylated to form transduction platforms for the control of effector cell responses [7,10]. Three of the tyrosine residues found in IpLITR 1.1b are located within the membrane proximal region of its CYT and they are not embedded within any conventional inhibitory or stimulatory signaling motifs; whereas, two of the three remaining tyrosines are located within inhibitory motifs at the distal CYT region [1]. Although the recruitment of signaling molecules to the CYT region of IpLITR 1.1b has yet to be investigated directly, our previous biochemical work has shown that IpLITR 1.1b binds the non-catalytic region of tyrosine kinase adaptor protein 1 (Nck) [7]; a protein that assembles regulators of actin polymerization at the plasma membrane (PM), including neural Wiskott-Aldrich Syndrome protein (N-WASp) or WASp family verprolin-homologous protein-2 (WAVE2) [11,12]. We also identified spleen tyrosine kinase (Syk) as a potential signaling effector that could be targeted to the CYT region of IpLITR 1.1b by interacting with phosphorylated tyrosines, which are positioned in two tandem ITIM motifs [7]. When recruited to IpLITR 1.1b, we predicted that Syk would then bind and activate intracellular Rho-guanine nucleotide exchange factor (GEFs) that may directly regulate one of the many known Rho GTPases that are responsible for controlling rapid actin-driven membrane remodeling through activation of the actin-related protein 2/3 (Arp2/3) nucleator and WAVE2 complexes [13]. Unfortunately, the biochemical associations of Nck and Syk with IpLITR 1.1b do not confirm if these molecules are required for IpLITR 1.1b-mediated responses, however, these findings provide the framework for exploring a new mode of ITAM-independent phagocytosis facilitated by IpLITR 1.1b.

Based on the working hypothesis that Nck and Syk are key players during IpLITR 1.1b-mediated regulation of cytoskeletal reorganization and membrane dynamics, the primary focus of this study was to directly examine the recruitment of Nck and phosphorylated (p)Syk during the IpLITR 1.1b-stimulated phagocytic process. First, we examined whether Nck and pSyk are present in constitutively generated filopodia-like structures in the absence of extracellular targets. Then, using a microbead-based phagocytosis assay, PM-target interactions were examined to assess the dynamics of Nck and pSyk localization during the induction of IpLITR-mediated phagocytosis. Our results show that Nck, but not pSyk, constitutively associates with IpLITR 1.1b and specifically accumulates within filopodia-like structures. Furthermore, during the distinct phases of IpLITR 1.1b target-binding, immobilization, and engulfment, both Nck and pSyk were found to dynamically accumulate at the membrane-microbead interfaces. Overall, these results provide the first evidence for unique molecular events that are required for IpLITR 1.1b-mediated membrane dynamics and provide new details regarding the variable recruitments of Nck and pSyk during two different phagocytic modes.

Section snippets

IpLITR-expressing RBL-2H3 cells

Generation of RBL-2H3 cells stably expressing N-terminal hemagglutinin (HA)-tagged IpLITRs using the pDISPLAY expression vector has been described previously [4]. Briefly, pDISPLAY IpLITR 2.6b/FcRγ-L is a chimeric receptor which contains two extracellular Ig-like domains (GenBank Accession: ABI23577) fused with the ITAM-containing signaling adaptor IpFcRγ-L [3]. pDISPLAY IpLITR 1.1b (GenBank Accession: ABI16050) contains the full length TS32.17 L1.1b sequence. Transfected RBL-2H3 cells were

Nck, but not phosphorylated-Syk, co-localizes with IpLITR 1.1B in constitutively generated filopodia-like structures

To examine the localization of activated pSyk with IpLITR proteins, parental RBL-2H3 cells (Fig. 1A) as well as IpLITR 2.6b/IpFcRγ-L- (Fig. 1B) and IpLITR 1.1b-expressing RBL-2H3 cells (Fig. 1C) were co-stained for confocal imaging with an αHA mouse mAb, to detect IpLITR expression, and an α-rabbit mAb specific for pSyk phosphorylated at Y525 and Y526. Phosphorylation of Syk at two tyrosines, Y525 and Y526, which are located within the activation loop of kinase domain, is required for Syk

Discussion

Our recent work has shown that the expression of IpLITR 1.1b, but not IpLITR 2.6b/IpFcRγ-L, constitutively stimulates the formation of filopodia in stably transfected RBL-2H3 cells [6,8,9]. IpLITR 1.1b also promotes secondary waves of F-actin polymerization during the initial tethering and subsequent internalization of immobilized extracellular targets; dynamic events that are believed to be distinct from those that drive constitutive filopodia formation [8]. Furthermore, we have performed

Declaration of Competing Interest

There are no conflicting funding, employment, or personal financial interests regarding this submission.

Acknowledgments

This work was supported by grants from; the Natural Sciences and Engineering Council of Canada (NSERC; grant# RGPIN-2012-341209) awarded to James Stafford; graduate teaching assistantship awarded by the Department of Biological Sciences to Dustin Lillico; an NSERC PDF and Banting Fellowship awarded to Joshua Pemberton.

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