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

Journal Pre-proof Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions Dustin M.E Lillico, Joshua G. Pemberton, Rikus Niemand, James L. Stafford

PII:

S0898-6568(19)30239-6

DOI:

https://doi.org/10.1016/j.cellsig.2019.109443

Reference:

CLS 109443

To appear in: Received Date:

12 September 2019

Accepted Date:

14 October 2019

Please cite this article as: Lillico DME, Pemberton JG, Niemand R, Stafford JL, Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions, Cellular Signalling (2019), doi: https://doi.org/10.1016/j.cellsig.2019.109443

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

Dustin M.E Lillicoa, Joshua G. Pembertonb, Rikus Niemanda, and James L. Stafford a,*

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Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.

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Section on Molecular Signal Transduction, Program for Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

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Summary Sentence: Dynamic recruitment of Nck and pSyk drives distinct ITAM-dependent and -independent phagocytic modes.

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Running Title: Recruitment of key mediators of the F-actin cytoskeleton during LITR-mediated regulation of membrane dynamics.

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Correspondence: Dr. JL Stafford, Department of Biological Sciences, University of Alberta, CW319-A, Biological Sciences Building, 11455 Saskatchewan Drive, Edmonton, Alberta, Canada, T6G 2R3.

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Phone: 1 (780) 492-9258. Fax: 1 (780) 492-9234. E-mail: [email protected]

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Graphical Abstract

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 on the signalling activity of immunoregulatory receptor-types that initiate dynamic changes in plasma membrane architecture required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating

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phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel

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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

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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

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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

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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

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phagocytic processes.

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Abbreviations; Leukocyte immune-type receptors (IpLITRs); immunoreceptor tyrosine-based activation motif (ITAM); immunoreceptor tyrosine-based inhibitory motif (ITIM); rat basophilic leukemia-2H3 cells (RBL-2H3); filamentous (F); cytoplasmic tail (CYT); non-catalytic region of tyrosine kinase adaptor protein 1 (Nck); plasma membrane (PM); neural Wiskott-Aldrich Syndrome protein (N-WASp); WASp family verprolin-homologous protein-2 (WAVE2); src homology 2 domains (SH2); spleen tyrosine kinase (Syk); Rho-guanine nucleotide exchange factor (GEFs), actin-related protein 2/3 (Arp2/3); minimal essential media (MEM); αhemaglutinin (HA); green fluorescent protein (GFP); cell staining buffer (CSB), monoclonal antibodies (mAbs); pearson’s correlation coefficient (PCC); mean fluorescence intensities (MFIs); antibody staining buffer (ASB), p21-kinase (PAK); FcR-like protein 4 (FcRL4), Tlymphocyte specific tyrosine kinase (Lck); platelet endothelial cell adhesion molecule-1 (PEACAM-1); src-family kinases (SFK); macrophage receptor with collagenous structure (MARCO)

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Key words: comparative immunology, phagocytosis, filopodia, intracellular signaling, cytoskeleton, immunoregulatory receptors, innate immunity, fish

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 superfamily1,2. By using epitope-tagged

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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

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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

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molecular events that allow these receptors to actively regulate innate immune cell effector responses, including phagocytosis3–8.

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Of the known IpLITR variants, our previous work has characterized a classical immunoreceptor tyrosine-based activation motif (ITAM)-dependent phagocytic pathway

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controlled by the stimulatory IpLITR 2.6b/IpFcRγ-L chimera4,6,8,9, as well as a unique phagocytic mode that utilizes the immunoreceptor tyrosine-based inhibitory motif (ITIM)containing receptor IpLITR 1.1b4,7–9. We have also demonstrated that stable expression of

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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 protrusions8. 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

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1.1b-mediated phagocytic process, filopodia-like structures initiate target contact and then retract to secure captured targets to the cell surface8. 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 engulfment8. IpLITR 1.1b-mediated signaling also initiated secondary waves of F-actin polymerization events associated with the tethering and internalization of extracellular targets8. 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 dynamics8. 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 effectors7,10. Within its relatively long CYT, IpLITR 1.1b contains six tyrosine residues that can be

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reversibly phosphorylated to form transduction platforms for the control of effector cell responses7,10. Three of the tyrosine residues found in IpLITR 1.1b are located within the

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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

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located within inhibitory motifs at the distal CYT region1. 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

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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

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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

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motifs7. 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 complexes13. Unfortunately, the biochemical associations of Nck and Syk with IpLITR 1.1b do

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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 1.1bmediated 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

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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

of Nck and pSyk during two different phagocytic modes.

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MATERIALS AND METHODS

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1.1b-mediated membrane dynamics and provide new details regarding the variable recruitments

IpLITR-expressing RBL-2H3 cells

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Generation of RBL-2H3 cells stably expressing N-terminal hemagglutinin (HA)-tagged IpLITRs using the pDISPLAY expression vector has been described previously4. Briefly,

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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

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TS32.17 L1.1b sequence. Transfected RBL-2H3 cells were grown at 37°C and 5% CO2 in complete culture media (minimal essential media (MEM) supplemented with Earl’s balance salt solution (GE Healthcare; Baie d’Urfe, CAN)), 2 mM L-Glutamine (Life Technologies, Inc.; Burlington,CAN), 100 units/mL Penicillin (Life Technologies, Inc.), 100 μg/mL Streptomycin (Life Technologies, Inc.), 400 μg/mL G418 disulfate salt solution (Sigma-Aldrich; St.

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Louis,USA) and 10% heat inactivated fetal bovine serum (FBS; Sigma-Aldrich). Surface expression of IpLITRs was monitored by flow cytometry using an α-hemaglutinin (HA) monoclonal antibody (mAb; Cedarlane Laboratories Ltd; ON, CAN) as described previously4,6. Co-expression of IpLITRs and LifeAct-GFP in RBL-2H3 cells IpLITR-expressing RBL-2H3 cells were transfected with LifeAct-green fluorescent protein (GFP; a generous gift from Dr. Nicholas Touret, University of Alberta) by nucleofection

according to the manufacturers recommended protocol (Amaxa; Cell Line Nucleofector Kit T, RBL-2H3; Lonza, Cologne, Germany) as previously described8. In general, IpLITR-expressing cells were grown to confluence (~ 2.6x106 cells/well) in 6-well tissue culture plates (Fisher Scientific Company; Ottawa, CAN) then harvested using RBL-2H3 harvest buffer (1.5 mM EDTA, 135 mM NaCl, 5 mM KCl, 20 mM HEPES), washed with PBS, and then transfected with 100 μL of Cell Line Nucleofector Solution T (Amaxa) containing 5 μg of LifeAct-GFP plasmid. Samples were transferred into a nucleofection cuvette and transfected using the Nucleofector II Device (Amaxa) using the program X-001. Following transfection, cells were placed into pre-

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warmed selection media (complete MEM supplemented with 400 μg of G418) and allowed to grow to confluency. Cells were harvested and sorted based on their GFP expression (high FL-1

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intensities) using the FACSCanto II instrument (BD Bioscience; CA, USA). Sorted cells were then plated, grown to confluence, and then harvested to examine their IpLITR and LifeAct-GFP

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expression levels by flow cytometry as previously described4,6.

Examination of the constitutive co-localization of Nck and pSyk with IpLITRs

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RBL-2H3 cells (3x105) expressing IpLITR 2.6b/IpFcRγ-L or IpLITR 1.1b were grown on sterile 18mm diameter #1 ½ circular coverslips (Electron Microscopy Sciences; Hatfield,, USA)

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overnight in a 6-well tissue culture plate (Fisher Scientific Company, Ottawa, ON, CAN) at 37°C and 5% CO2. The following day, cells were washed twice with pre-warmed 37°C PBS, fixed with 4% PFA, and then washed again with antibody staining buffer (ASB; 0.5% sodium azide;

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Sigma-Aldrich, 1% BSA in PBS). Coverslips were transferred with the cell side down onto a section of parafilm containing 50 μL of 5 μg/mL mouse αHA in ASB and incubated at 4°C for 30 min. Cells were then washed twice with ASB and then placed onto another section of parafilm containing 50 µl of 20 μg/mL goat α-mouse Alexa-647 (Molecular Probes) for 30 min

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at 4°C. Cells were then washed with ASB and incubated with cell permeabilization buffer (Biolegend; CA, USA) for 15min at room temperature. Following permeabilization, cells were washed with cell staining buffer (CSB; Biolegend) and then placed cell-side down onto parafilm containing either 1:100 (v/v) α-phospho (p)Syk) or 1:50 (v/v) α-Nck rabbit mAbs (Cell Signaling Technology; Danvers, MA, USA) in CSB for 30 min at room temperature. After staining with the α- pSyk or α-Nck mAbs, samples were again washed with CSB, and the coverslips were transferred cell side down onto parafilm containing 20 μg/mL goat α-rabbit Alex-488 (Molecular Probes; Eugene, OR, USA) for 30 min at room temperature. For positive controls, IpLITR 1.1b-

expressing RBL-2H3 cells were incubated with an αHA mAb followed by incubations with two goat α-mouse secondary antibodies; one conjugated to Alexa-657 and subsequently a second antibody conjugated to Alexa-488. These two antibodies were used to stain surface IpLITRs with two distinct fluorophores that both bind the primary αHA mAb. Finally, cells were washed with CSB and the coverslip was placed onto a microscope slide containing a drop of Prolong® Gold antifade (Molecular Probes) mounting media. Images were acquired using a Zeiss LSM 710 laser scanning confocal microscope

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(Objective 60x, 1.3 oil plan-Apochromat; Munich, Germany) located at the Cross Cancer Institute Microscopy Facility (Faulty of Medicine & Dentistry; University of Alberta). Image

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analysis was performed using Zen software (2011; Carl Zeiss; Oberkochen, Germany) or Imaris 9.2.1. For co-localization assessments, individual cells were isolated from z-stack series and

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regions of interest on each cell were designated using Zen (cell surface PM regions vs. filopodialike structures). Z-stacks for each region of the cell were then examined using the Imaris 9.2.1 co-localization analysis tool for the entire 3D volume of the selected region and the calculation

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of co-localization within these regions was determined using the Pearson’s Correlation Coefficient (PCC). PCC values predict the covariance of two independent signals for all

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individual pixels within two separate image channels14,15. This approach also removes background signal offset within a given image and therefore the covariance, or co-localization, of two signals measured is independent of signal intensity14,15. PCC values <0.5 are considered

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inconclusive and indicate that the overlap of two analyzed signals is no greater than that due to random chance. In comparison, PCC values >0.5 indicate that the overlap of two analyzed signals is not due to random chance and the higher the PCC value is above 0.5, the greater the probability that the co-localization is occurring. This analysis was performed on three individual cells for each cell-type tested and three regions of interest were used for each cell (total of 9

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regions). Statistical significance between cell-types for a given intracellular protein was performed using a student t-test with a two-tailed paired analysis. Visualization of Nck and pSyk recruitment during IpLITR-triggered phagocytosis To examine the recruitment of Nck and pSyk during IpLITR-mediate phagocytosis, Nterminal HA-tagged IpLITRs were specifically activated using 4.5 µm non-fluorescent carboxylate microspheres (Polysciences, Warrington, PA, USA) pre-coated with Protein A (from

Staphylococcuc aureus; Sigma-Aldrich) and opsonized with mouse αHA mAb as previously described4,6,7. Briefly, IpLITR-expressing RBL-2H3 cells (3x105) were grown on sterile 18 mm diameter #1 ½ circular coverslips placed in a 6-well tissue culture plate. Coverslips were washed with PBS and then 1mL of phagocytosis buffer (1% BSA in OptiMem (Thermofisher, Inc.) containing 9x105 αHA coated beads was added prior to centrifugation of the plate at 1400xg for 1 min. The cell and bead mixture was incubated for 30 min at 37°C; a time-point that was previously determined to allow for visualization of the various stages of IpLITR-mediated phagocytosis8. After incubation, cells were washed with PBS and then fixed using 4% PFA at

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37°C for 10 min. Fixed samples were then stained with a goat α-mouse secondary antibody to detect the αHA mAb on the surface of the target beads, as previously described16. This procedure

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allows for accurate discrimination of the relative association of the microbead with the cell

surface as areas of the bead that are intimately associated with the PM exclude the antibody

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staining17. Briefly, coverslips were placed cell-side down onto a section of parafilm containing 2 μg/mL of goat α-mouse Alexa-647 (Molecular Probes) for 30 min at 4°C. After staining of the

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beads, coverslips were washed with ASB and incubated with cell permeabilization buffer (Biolegend) for 15 min at room temperature. Permeabilized cells were then washed with CSB

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and placed onto parafilm containing either 1:100 (v/v) α-pSyk or 1:50 (v/v) α-Nck rabbit mAbs (Cell Signaling Technology) in CSB for 30 min at room temperature and subsequently washed with CSB before staining with 20 μg/mL of goat α-rabbit Alexa-550 secondary antibody in CSB

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for 30 min at room temperature. Lastly, coverslips were washed with CSB and mounted onto microscope slides containing a small droplet of Prolong® Gold antifade mounting media that was allowed to solidify overnight at room temperature. Imaging was performed as previously described6,8,9. Individual cellular events were then isolated from captured z-stacks and the mean fluorescence intensities (MFIs) for bead staining (blue) as well as for the intracellular Nck and

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pSyk (red) were calculated along a user defined analysis line using representative z-stack series for each signal tested using ImageJ 1.51. Calculated MFIs for each channel were converted into an MFI histogram representing the changes in signal intensity across the region of the cell for each z-stack image18,19. RESULTS 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 RBL2H3 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 activation and greatly enhances its catalytic activity20. In each staining experiment, three PM regions from three representative cells were analyzed (see

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analysis regions i-ix, dashed boxes; Fig. 1A, right panels). Each z-stack series was analyzed using the PCC value to predict the probability of co-localization between IpLITRs and pSyk

within the indicated region of interest. PCC values <0.5 indicate that the pixel localization of two

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fluorescent channels examined are below the probability threshold for co-localization,14,15

whereas PCC values of >0.5 suggest that the two signals co-localize within the same pixel

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space. As shown in Figure 1A, parental RBL-2H3 cells do not show an IpLITR signal (red), as observed in the αHA staining (left panels), but pSyk (green) was readily detected in these cells

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(Fig. 1A, middle panels). The calculated PCC values in regions i-ix for parental RBL-2H3 cells ranged from 0.09 to 0.33 (Fig. 1D); thereby defining the baseline probability of signal co-

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localization in un-transfected cells. When stable IpLITR 2.6b/IpFcRγ-L-expressing cells were examined for IpLITR (red) and pSyk (green) staining (Fig. 1B, left and middle panels respectively), based on the calculated PCC values (<0.5) in the analysis regions i-ix (right

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panels), significant levels of signal co-localization was not observed (Fig. 1D). Similar results were also obtained using IpLITR 1.1b-expressing cells, which showed no significant colocalization (PCC<0.5) between IpLITR 1.1b and pSyk in all areas of the PM examined (Fig. 1C; regions i-ix); including within extended filopodia-like structures (see Fig. 1D; regions iii, v, vi, viii, and ix).

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Nck staining was readily detected in parental RBL-2H3 cells (Fig. 2A, middle panels),

however, no significant co-localization values (PCC <0.5) were calculated (Fig. 2A, regions i-ix, left panels, and Fig. 2D). Furthermore, as observed for pSyk, no apparent co-localization occurred in resting RBL-2H3 cells expressing IpLITR 2.6b/IpFcRγ-L (Fig. 2B and Fig. 2D). In contrast, IpLITR 1.1b-expressing cells (Fig. 2C and Fig. 2D) showed a significant increase in PCC values calculated across multiple regions of the PM (ii (0.84), iii (0.83), v (0.90), vi (0.88), viii (0.83), and ix (0.74)). Interestingly, these regions all contained filopodia-like structures;

unlike regions i, iv, and vii, which are regions of the cell surface devoid of cellular protrusions (Fig. 2C, left panels). Quantitative analysis of the co-localization data is shown in Figure 3. In addition, positive controls for the PCC-derived co-localization values are provided in Supplementary Figure 1. The positive control was generated by incubating IpLITR 1.1bexpressing cells with an αHA mouse mAb and then subsequently staining the cells with two different goat α-mouse IgG secondary antibodies in succession; one conjugated to Alexa-488 probe, while the other being conjugated with an Alexa-647 probe. Since both of these secondary antibodies selectively bind mouse IgG, these probes stained IpLITR 1.1b on the cells surface by

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binding to the αHA mouse mAb. This control experiment shows consistently high PCC values, ranging from 0.7-0.94, providing further evidence that this confocal imaging analysis was

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effective for measuring co-localization of IpLITR 1.1b and Nck in constitutively-generated

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membrane protrusions.

Recruitment of activated pSyk during IpLITR-triggered phagocytosis

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To specifically examine the recruitment of pSyk at the sites of IpLITR activation, αHA mAb opsonized beads were incubated with IpLITR-expressing RBL-2H3 cells that were

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subsequently stained for intracellular pSyk using α-rabbit mAbs conjugated with Cy3 (red). These cells were co-stained with α-mouse IgG pAbs conjugated with Cy5 (blue) to detect the surface exposed areas of the target beads. Phagocytosed beads are protected by the PM and thus

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are not accessible to antibody staining. In comparison, any exposed surfaces of the beads, in areas not enclosed by the PM, are stained blue. A series of representative examples of the pSyk staining pattern during the IpLITR 2.6b/IpFcRγ-L-mediated phagocytic process is shown in Figure 4. In each representative image (Fig. 4A-4D), confocal z-stacks (top panel), 3D reconstructions (middle panel), and staining intensity histograms (bottom panel) complete with

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MFI for pSyk (red) as well as the extracellular exposed areas of the selected beads (blue) are shown. Arrowed lines seen in the middle and bottom panels indicate the areas of the images that were selected for MFI analyses, with the direction of the arrow correlating with the distance in µm (starting at 0 µm) of the x-axis reported for each of the histogram plots (bottom panels). In Figure 4A, an extracellular bead is identified with an asterisk (*), which appears to be loosely associated with the surface of an IpLITR 2.6b/IpFcRγ-L-expressing cell (stained green using the LifeAct-GFP probe). Staining for the bead (blue ring) is clearly observed around the

exposed circumference of the target. This is also indicated by the relatively high MFI values calculated in the blue channel at the bead location (~40,000; Fig. 4A, blue histogram line between 1.55 μm and 6.20 μm). No significant pSyk localization is observed at the interface between the bead and the cell surface (Fig. 4A, z-stack images and 3D renders), which also correlates with the relatively low MFI values in the red channel calculated along the lined arrow (~10, 000 MFI; Fig. 4A, middle panel and red histogram line in the bottom panel). Shown in Figure 4B, is a partially engulfed bead (*), with reduced staining intensity (blue) at the bead-PM

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interface. At the contact site between the bead and the cell surface, pSyk staining is clearly visible as a thin red line outlining the area of the bead that is in close contact with the PM and also where IpLITR 2.6b/IpFcRγ-L receptors are being specifically activated by the αHA mAb

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(Fig. 4B, bead (*) in z-stack images and 3D renders). The calculated MFI for the bead (blue

histogram line) and pSyk (red histogram line) are displayed in the bottom panel of Figure 4B.

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Here, the exposed region of the bead has a relatively large MFI value (~40,000), whereas the staining intensity from the bead is reduced to <10,000 MFI at the target-PM interface (Fig. 4B,

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blue histogram line). Conversely, the decrease in the MFI from the bead (blue) is accompanied by an increased intensity of pSyk staining at the same location (Fig. 5B, red histogram line;

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~38,000 MFI between 6.20 and 6.98 μm). Figure 4C (top and middle panels) shows a phagocytosed bead (*) that is completely surrounded with pSyk staining (red) but devoid of any stain from the bead (blue). The levels of pSyk (red histogram line) are shown as two distinct

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peaks in Figure 4C (>20, 000 MFI and >30,000 MFI). At these same locations, no staining from the bead is evident (blue histogram line). Finally, shown in Figure 4D, is another phagocytosed target (*) that is devoid of both staining for the bead and pSyk. The lack of staining is clearly shown in the confocal images as well as on the MFI histogram plot (Fig. 4D). Representative examples of IpLITR 1.1b-mediated recruitment of pSyk during

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extracellular target capture and engulfment are shown in Figure 5. In Figure 5A, two individual beads (* and ) are each attached to distinct F-actin rich PM structures (green). At each of the interfaces between membrane extensions and the targets, pSyk staining (red) is increased, but an overall decreased level of staining for the bead (blue) is observed at these sites of contact. Using the arrowed line to calculate the MFIs for both the bead and pSyk, each bead shows an accumulation of pSyk at each membrane-bead interaction zone resulting in four distinct histogram peaks (Fig. 5A, histogram). These same positions also have relatively low staining

intensities measured for the bead. Figure 5B shows two beads (* and ) captured within what appear to be a continuous F-actin rich membranous extension (green) that are clearly visible in the 3D render of the confocal z-stack. However, unlike the two beads described in Figure 5A, one of these beads () is completely surrounded by the membrane (green) while a second bead (*) appears to be only partially encircled. At the membrane-target interfaces, reduced staining is evident for bead (*), with an increase in staining for pSyk at the same site. For the other target (), no staining is observed for the bead but it is contained within a distinct ring of staining for

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activated pSyk (Fig. 5B, see z-stack images and 3D renders). The MFI profiles for these images, calculated along the arrowed line, are shown in Figure 5B (bottom panel); which support that staining for pSyk is specifically increased at the interface between the bead and the surface of

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this IpLITR 1.1b-expressing cell. Shown in Figure 5C, a bead (*) is found attached to the cell surface (green) in what appears to be a phagocytic cup-like structure. As expected, the side of the

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bead that contacts the IpLITR 1.1b-expressing cell is devoid of blue staining, however, this interface contains a distinct ring of pSyk (red; Fig. 5C). The profile of the MFI histogram for this

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target-cell interaction (Fig. 5C, bottom panel) shows that the bead surface opposite the PM also shows increased staining with an α-mouse IgG pAb (~45,000 MFI at 2.33-3.10 μm; blue

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histogram line), yet at this same site, the staining is relatively low (i.e. ~10,000 MFI) when using the α-pSyk rabbit mAb (Fig. 5C, red histogram line). Conversely, at the cell-bead interface, pSyk staining was increased (~50,000 MFI; red histogram line) while the staining observed for the

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bead remained low (~3000 MFI; blue histogram line). A similar phenotype is also observed in Figure 5D, showing multiple targets attached to the surface with underlying staining for pSyk at the interfaces of the bead and cell. In this figure, two beads of particular interest, indicated by the symbols (*) and (), were used to create MFI intensity histograms (Fig. 5D, bottom panel). Lastly, as shown in Figure 5E, we observe the recruitment of pSyk to the membrane area

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surrounding a bead that has undergone complete engulfment by an IpLITR 1.1b-expressing RBL-2H3 cell. This is evident from a lack of extracellular staining of any exposed portions of the bead, combined with the increase in localization of pSyk at that the same position (Fig. 5E). Specifically, the MFI calculated across the bead (Fig. 5E, lined arrow) shows a relatively large amount of pSyk staining at the basal portion of the internalized bead (~45,000 MFI; red histogram line). These values gradually diminish as the arrow approaches the portion of the bead located at the cell surface, but no extracellular (blue histogram line) staining was observed.

Overall, these results show that IpLITR 1.1b-expressing RBL-2H3 cells recruit pSyk at the PMtarget interfaces during the active capture of extracellular targets by filopodia as well as throughout the subsequent engulfment process. Analysis of Nck recruitment patterns during IpLITR-triggered phagocytosis To examine the dynamics of Nck recruitment during IpLITR-mediated phagocytosis, a similar imaging-based phagocytic assay was performed as described above but using an α-Nck rabbit mAb. Extracellular regions of the target beads were once again identified using α-mouse

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IgG to detect the αHA mAb coated on the beads in addition to visualizing fluorescently-labelled F-actin. The first representative example of Nck recruitment during IpLITR 2.6b/IpFcRγ-L-

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mediated phagocytosis is shown in Figure 6, where three different beads (*, , ) were selected for analysis (Fig. 6A). The first bead (*) is attached to the cell surface (green) in a phagocytic

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cup-like structure and Nck (red) is observed at the base of the bead at the cell interface (Fig. 6A). Most of this bead is stained blue, indicating that it is captured but only partially engulfed by the

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cell, with distinct accumulations of Nck observed at the base of the bead-membrane interface. The MFI analysis histograms for the surface staining of the exposed bead (blue), F-actin (green line), and Nck (red line) are all shown in the MFI histogram (Figure 6B, top panel). The second

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bead () analyzed appears to have been completely internalized by the cell, as indicated by the absence of any surface staining for the bead (blue; Fig. 6A) and is surrounded by a bright ring of Nck (Fig. 6A, 6B). Lastly, a third bead (, Fig. 6A), which has also been completely engulfed

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by the IpLITR 2.6b/IpFcRγ-L-expressing RBL-2H3 cell, again shows no surface staining for the bead, but there is a relatively high overall intensity of Nck surrounding the internalized target (Fig. 6B). Additional representative examples of Nck recruitment during the IpLITR 2.6b/IpFcRγ-L-mediated phagocytic process are provided in Supplementary Figures 2 and 3.

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Collectively, these imaging studies demonstrate that although Nck is present throughout the cells, staining for Nck is specifically enriched at bead interfaces at the PM during the immobilization and engulfment stages of IpLITR 2.6b/IpFcRγ-L-induced phagocytosis. Due to the dynamic membrane structures that are formed by IpLITR 1.1b-expressing

cells6,8, both individual bead-cell contacts, as well multiple contact sites along these structures were analyzed using F-actin (green), Nck (red), and staining for the extracellular bead surface (blue) to visualize these complex cell-target interactions. As shown in Figure 7, three individual

targets (*, , ) are observed at a stage where they are all captured by an elongated F-actin enriched (green) extension of the PM (Fig. 7A). While portions of these immobilized beads remain exposed (blue staining), a reduction in bead staining is observed at the sites where the intensity of the staining for Nck was the highest (Fig. 7A,B). All beads captured along this filopodia-like structure were also individually analyzed for the relative accumulation of Nck (Fig. 7C). Collectively, this analysis suggests that Nck is specifically enriched at the interface between the filopodia and the surface of any captured beads (red histogram lines; Figure 7C,D). Another representative example of the dynamics of Nck recruitments during filopodia-mediated

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target capture is shown in Figure 8. In this example, two beads (* and ) make contact with an F-actin and Nck-rich membrane extension (Fig. 8A). Although large portions of these beads are

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not surrounded by the PM (blue stain), discrete pockets of Nck staining are observed at the

membrane-target interfaces. When quantified along the arrowed line (Fig. 8B), three clear peaks

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of Nck are observed at the target contacts, with a broader F-actin- and Nck-rich region preceding the contact site of the first bead (*; Fig. 8B). Additional examples of representative Nck

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recruitment patterns during IpLITR 1.1b-triggered target capture and engulfment are provided in

DISCUSSION

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Supplementary Figures 4-6.

Our recent work has shown that the expression of IpLITR 1.1b, but not IpLITR

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2.6b/IpFcRγ-L, constitutively stimulates the formation of filopodia in stably transfected RBL2H3 cells6,8,9. 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 detailed kinetic and pharmacological profiling for the IpLITR

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2.6b/IpFcRγ-L- and IpLITR 1.1b-mediated phagocytic processes revealing the unique aspects of the atypical target capture and engulfment phenotype of the latter.6,8,9 We proposed that these responses are likely due to the unique domain structure and signaling potential associated with the CYT of IpLITR 1.1b, which allows for considerable diversity in the integrated dynamics of cytoskeletal and membrane remodelling events associated with expression of this IpLITR variant4,6,8,9. Building from these findings, the goal of the present study was to investigate the localization of two key transduction effectors, Nck and pSyk, both of which are proposed to be

associated with the control of F-actin polymerization, during constitutive IpLITR 1.1b-induced formation of filopodia and IpLITR-triggered phagocytic events. First, we examined if Nck and/or pSyk endogenously co-localized with IpLITRs in transfected RBL-2H3 cells. These experiments were performed by fluorescently co-labeling surface expressed N-terminal epitope tagged IpLITRs with either intracellular Nck or pSyk. Using confocal imaging, we assessed the probability that IpLITRs interacted with Nck and/or pSyk within the PM of un-stimulated cells. Co-localization was measured using PCC, which has been successfully used previously to examine the co-localization of Nck with the cytoskeletal structural protein paxillin, specifically

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within membrane protrusions18. In our study, we show that that Nck endogenously co-localizes with IpLITR 1.1b within filopodia structures, but not at other locations of the PM that do not

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possess these F-actin rich extensions. In comparison, PCC values determined for signals derived from IpLITR 1.1b and pSyk were below the threshold for co-localization in IpLITR 1.1b-

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generated filopodia structures as well as in parental RBL-2H3 cells. Interestingly, in IpLITR 2.6b/IpFcRγ-L-expressing cells, both Nck and pSyk did not colocalize with this receptor (i.e.

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PCC <0.5); suggesting that this classical stimulatory IpLITR receptor subtype does not endogenously recruit these signaling effectors.

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In a variety of cell types, Nck functions as an important regulator of filopodia formation by serving as an adaptor protein that organizes signaling complexes at the PM18,19,21–26. For example, in fibroblasts, Nck endogenously associates with p21-kinase (PAK) and, upon

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stimulation with growth factors, this constitutive complex is transported to the membrane to initiate F-actin polymerization events27. PAK has also been identified as a regulatory factor for filopodia formation, and the constitutive interaction of PAK with Nck, followed by the subsequent engagement of Nck to IpLITR 1.1b, may be a possible mechanism for facilitating IpLITR 1.1b-induced filopodia formation. Nck also constitutively binds to Vav1 in resting T

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cells 19. This interaction requires the proline-rich region in Vav1 and causes significantly altered patterns of F-actin polymerization19. Consequently, IpLITR 1.1b could also facilitate localized filopodia formation without prior receptor activation through Nck recruitment of Vav1, which would be important for the activation of small GTPases that can facilitate the activation of Factin nucleation effectors such as WAVE228. Interestingly, previous biochemical studies indicate that Vav1 associates with the CYT of IpLITR 1.1b7. One of the likely requirements for an endogenous association between Nck and IpLITR 1.1b would be tyrosine phosphorylation of the

CYT. As such, our co-localization data presented here suggest that IpLITR 1.1b is basally phosphorylated to allow for the recruitment of Nck, but we have not been able to detect phosphorylated IpLITR 1.1b in unstimulated RBL-2H3 cells (data not shown). However, recent examinations of the human FcR-like protein 4 (FcRL4) support the idea that immunoregulatory receptors are basally phosphorylated without prior activation and that this ligand-independent phosphorylation is important for regulating immune cell activities29. Specifically, the CYT region of FcRL4 is endogenously phosphorylated at defined ITIMs in order to facilitate the

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binding and activation of intracellular phosphatases29. Based on these findings, ligandindependent phosphorylation of IpLITR 1.1b could facilitate constitutive associations with Nck and potentially allow for the endogenous induction of filopodia. Overall, the co-localization of

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Nck and IpLITR 1.1b within constitutively-generated filopodia-like structures supports previous biochemical studies from our lab that showed specific recruitment of Nck to the proximal CYT

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region of IpLITR 1.1b7. This interaction likely occurs at a predicted Nck-binding consensus motif located at Y433,7,11 strongly suggesting that interactions between IpLITR 1.1b and Nck are

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required for the constitutive generation of dynamic F-actin containing filopodia. The second objective of this study was to examine the selective recruitment of Nck and

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active pSyk to sites of receptor activation during the IpLITR-triggered phagocytic process. For these experiments, IpLITR staining was not measured directly, but these receptors were selectively engaged using microbeads opsonized with αHA mAbs. Consequently, the interface

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between target beads and the PM represented sites of IpLITR clustering and were used as a proxy for examining IpLITR-induced signaling events17. A similar approach has recently been used to track the recruitment and activation of intracellular signaling effectors during immune receptor activation. For example, the localization of the T-lymphocyte specific tyrosine kinase (Lck) was determined by measuring MFI values along a cross sectional line during CD8+ T cell

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stimulation19. In addition, as mentioned above, the localization of Nck and paxillin has been monitored at the leading edge of membrane protrusions by measuring the intensities of fluorescently-labelled Nck and paxillin18. As expected, IpLITR 2.6b/IpFcRγ-L-expressing cells recruited both Nck and pSyk at the interface between extracellular targets and the PM. The pattern of recruitment for these molecules were consistent with the known phases of the classic ITAM-triggered phagocytic

process30,31. For example, beads that are loosely associated with the membrane showed little Nck or pSyk staining at the target-cell interface. However, as phagocytosis of the target progressed, a loss of surface-exposed staining for the targeted bead directly correlated with increased recruitment of both Nck and activated pSyk; a response expected to occur during activation of the IpLITR 2.6b/IpFcRγ-L signalling complex. Subsequently, as indicated by the total loss of surface staining for the engulfed bead, both Nck and pSyk were progressively reduced from the membrane interface; likely indicating that sustained IpLITR 2.6b/IpFcRγ-L-mediated signaling events were no longer required following F-actin mediated engulfment of an extracellular target.

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The recruitment patterns observed for Nck and pSyk during IpLITR 2.6b/IpFcRγ-L-mediated phagocytic process closely correlate with those observed for other ITAM-induced phagocytic

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responses11,32,33.

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Interestingly, while IpLITR 1.1b-expressing cells specifically recruited Nck and pSyk to interaction sites between immobilized beads and the PM, these effectors were also observed at points of contact with beads positioned at the leading edge or on the surface of extended

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filopodia-like structures. This suggests that Nck and pSyk are likely important for the initial capture as well as engulfment phases of IpLITR 1.1b-mediated phagocytosis. If IpLITR 1.1b can

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endogenously associate with Nck, then it is not surprising that recruitment of this adaptor protein can be reinforced to drive the extended phagocytic process. The observed recruitment of pSyk to IpLITR 1.1b during microbead capture and engulfment is an interesting observation that directly

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supports our working hypothesis, whereby the tandem SH2 domains of pSyk can be accommodated by phosphorylated tryrosines located within the two tandem ITIMs of IpLITR 1.1b6–8. A similar mechanism has been demonstrated for the ITIM-containing receptor platelet endothelial cell adhesion molecule-1 (PEACAM-1), which has two ITIMs that are situated 22 amino acids apart; a distance that was shown to allow for binding of pSyk34. Interestingly, 22

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amino acids also separate the tyrosine residues located within the tandem ITIMs (Y477 and Y499) of IpLITR 1.1b and therefore could act as a possible docking site for activated pSyk. Similarly, the inhibitory platelet receptor G6B-b was also shown to bind Syk to tandem ITIMs within its CYT region35. Overall, these results provide further evidence in support of the hypothesis that Nck and pSyk are two key regulators of the IpLITR 1.1b-stimulated phagocytic process.

Nck and Syk are major regulatory proteins that involved in F-actin polymerization events within a wide variety of cellular process, including phagocytosis36–39. The role of Nck as a transduction scaffold requires one SH2 and three SH3 binding domains, which drive a diversity of protein-protein interactions. As a result of being able to drive multivalent signalling responses, Nck has been shown to be involved in phagocytic activities initiated by FcRs, carcinoembryonic antigen related cell adhesion molecule (CEACAM)3, and CEACAM411,27,40. Unlike the scaffolding role of Nck, Syk-associated phagocytic responses rely on its enzymatic activity as a non-receptor tyrosine kinase to initiate downstream signaling events that typically lead to F-actin

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polymerization37,38. Structurally, Syk contains two SH2 domains, which it uses to bind

phosphorylated tyrosines that are normally present within the ITAMs of many phagocytic

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receptor-types37,41. Syk also contains a functional tyrosine kinase domain that has been shown to phosphorylate a variety of intracellular target proteins41. In general, tyrosine residues embedded

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within the ITAMs of phagocytic receptor are phosphorylated via Src-family kinases (SFKs) during the initial stages of receptor activation that immediately follow ligand-binding. This

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initial event then induces both the binding as well as the phosphorylation and activation of Syk33,42. Specifically, intermolecular interactions found within the linker region between the two

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SH2 domains of Syk, as well as within its kinase domain, stabilize Syk in an inactive state43. However, following ITAM binding, the auto-inhibited conformation of Syk is released following phosphorylation of tyrosine residues within the activation loop; including Y525 and Y526 20,43,44.

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Activated Syk then induces the phosphorylation of downstream signaling molecules which leads to the formation of signaling complexes at the sites of activated receptors45 that can promote induction of the F-actin polymerization machinery27,33,45,46. Due to its vital role in driving F-actin polymerization, activation of Syk is requisite for many receptor-induced phagocytic responses; including for classical phagocytic receptors such as FcR and Dectin-133,34,37,47, as well as during

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responses initiated by atypical phagocytic receptors like macrophage receptor with collagenous structure (MARCO), Jedi-1, CD44, and CEACAM311,40,48–50. Overall, the data presented in this study provides new evidence regarding the initial

signaling events that occur during immunoregulatory receptor-mediated phagocytosis and insights into the potential mechanism(s) associated with IpLITR 1.1b-induced cellular responses. Our co-localization data clearly show interactions of the ITIM-containing receptor IpLITR 1.1b with intracellular effector proteins that are instrumental for regulating F-actin polymerization

during distinct phases of phagocytosis. Specifically, this data suggests that IpLITR 1.1b endogenously co-localizes with Nck during the constitutive formation of filopodia-like extensions of the PM. Furthermore, we show that Nck and pSyk are recruited by activated IpLITR 1.1b receptors during activated phagocytosis, thus providing the first evidence that an ITIM-containing immunoregulatory receptor can activate signalling networks associated with Nck and pSyk to induce extracellular target binding, immobilization, and engulfment. Based on our previous studies, it is likely that Nck binds to IpLITR 1.1b at a consensus motif located within the proximal CYT region, whereas pSyk could interact with tandem ITIMs located within

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the distal region of the CYT7. Interestingly, these data also raise the possibility that rather than simply being basic platforms for the recruitment and assembly of signaling platforms, the

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structure of the CYT region itself may contribute to subsequent downstream signaling events. For instance, basal phosphorylation of the membrane proximal CYT region of IpLITR 1.1b may

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result from its proximity to the transmembrane segment; allowing it to remain accessible to membrane-associated signalling effectors that facilitate the endogenous association with Nck.

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However, in this resting state, the tandem ITIMs may be shielded from basal phosphorylation due to a closed conformation of the distal CYT region that is presented only after receptor

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engagement. Therefore, conformational dynamics within the CYT may also help to explain the differential recruitment of Nck and Syk to the resting and stimulated IpLITR 1.1b, respectively. In support of this, other studies examining both ligand- and phosphorylation-dependent

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conformational changes in immunoregulatory receptors have shown that the structure of the CYT directly influences signaling outcomes. Specifically, after binding immobilized fibrinogen, conformational changes in the CYT region of the IIb3 integrin receptor exposes binding sites for downstream signaling molecules and this presentation of the CYT represents an important regulatory mechanism for the assembly of cytoskeletal proteins at the PM51. Distinct changes in

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the conformation of the CYT are also associated with tyrosine phosphorylation, which can change the local geometries of the intracellular signaling segments of immunoregulatory receptors52,53. These phosphorylation-induced changes in molecular dynamics are in part due to structural changes mediated by local hydrogen bonding and electrostatic potential52, as well as altered binding interactions with membrane phospholipids53. Collectively, reversible and differential phosphorylation of CYT regions has a major effect on the conformation and

signaling potential of various immunoregulatory receptor-types, which likely contributes to the context-specific signalling observed in IpLITRs. Our detailed examinations of IpLITR 1.1b-mediated signalling and its unique ability to promote F-actin polymerization events reveals that functional plasticity within immunoregulatory signaling pathways emerged very early in vertebrate evolution. As such, IpLITR 1.1b-mediated formation of filopodia and the ability to actively interact with extracellular targets represents a unique model for further understanding how F-actin dynamics

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contribute to the integrated control of innate cellular responses. This model will serve to broaden our understanding of how other vertebrate immunoregulatory receptor-types recruit intracellular

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signaling components to regulate effector responses such as phagocytosis. Additionally, results from these studies clearly demonstrate how the unique composition of tyrosine residues within

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the CYT regions of immune receptor-types can form discrete signaling cassettes to facilitate functional plasticity within immunoregulatory receptor families. The fact that IpLITR 1.1b, an ITIM-containing and known inhibitory receptor, can constitutively network with components of

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the F-actin polymerization machinery, as well as recruit new effectors during the active capture

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immunoregulatory receptor.

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and engulfment of extracellular targets, highlights the immunoregulatory versatility of this

Conflict of 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

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Canada (NSERC; grant# RGPIN-2012-341209) awarded to James Stafford; graduate teaching

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PDF and Banting Fellowship awarded to Joshua Pemberton.

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assistantship awarded by the Department of Biological Sciences to Dustin Lillico; an NSERC

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Figure Legends

FIGURE 1. Examination of the constitutive co-localization of pSyk with IpLITRs in RBL2H3 cells. Parental RBL-2H3 cells (A) IpLITR 2.6b/IpFcRγ-L-expressing RBL-2H3 cells (B) and IpLITR 1.1b-expressing RBL-2H3 cells (C) were plated (2x105 cells) onto a coverslip, grown overnight, then fixed with 4% PFA at 37°C. Visualization of IpLITRs was performed using an αHA mAb and a secondary goat α-mouse Alexa-647 (red) antibody. Cells were then permeabilized and stained with a rabbit α-pSyk mAb that was visualized using a secondary goat

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α-rabbit Alexa-488 (green) to examine the relative localization of pSyk in relation to IpLITRs. Z-

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stack images were obtained at 63x magnification using a Zeiss LSM 710 laser scanning confocal microscope. Co-localization analysis was performed on representative z-stack images using Imaris 9.2.1 software at three distinct regions of the membrane (dashed box; i-ix) for three

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individual cells. PCC values were then calculated for all cell-types at each region examined (i-ix)

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(D).

FIGURE 2. Examination of the constitutive co-localization of Nck with IpLITRs in RBL2H3 cells. Parental RBL-2H3 cells (A) IpLITR 2.6b/IpFcRγ-L-expressing RBL-2H3 cells (B)

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and IpLITR 1.1b-expressing RBL-2H3 cells (C) were plated (2x105 cells) onto a coverslip, grown overnight, then fixed with 4% PFA at 37°C. Visualization of IpLITRs was performed using an αHA mAb and a secondary goat α-mouse Alexa-647 (red) antibody. Cells were then

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permeabilized and stained with a rabbit α-Nck mAb that was visualized using a secondary goat α-rabbit Alexa-488 (green) to examine the relative localization of pNck in relation to IpLITRs. Z-stack images were obtained at 63x magnification using a Zeiss LSM 710 laser scanning confocal microscope. Co-localization analysis was performed on representative z-stack images

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using Imaris 9.2.1 software at three distinct regions of the membrane (dashed box; i-ix) for three individual cells. PCC values were then calculated for all cell-types at each region examined (i-ix) (D).

FIGURE 3. PCC quantification values for the constitutive co-localization of pSyk and Nck with IpLITRs. Mean ±SEM PCC values for each analyzed image shown in Figures 1 and 2 are displayed. No statistical significance (ns) in PCC values was calculated for IpLITRs and pSyk in resting cells. Statistically significant values were calculated for IpLITRs with Nck when

comparing resting RBL-2H3 cells with IpLITR 2.6b/IpFcRγ-L-expressing cells (*). When compared with resting RBL-2H3 cells and IpLITR 2.6b/IpFcRγ-L-expressing cells, IpLITR 1.1b PCC values were significantly higher (**). Statistical analysis was performed using a student ttest with a two-tail paired variance and significance was determined by p≤0.05. FIGURE 4. Recruitment of pSyk during IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis. Phagocytosis assays were performed by incubating IpLITR 2.6b/IpFcRγ-L-expressing cells (1x105) stably transfected with LifeAct-GFP (green) with 4.5-µm non-fluorescent polystyrene

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beads (3x105) pre-opsonized with αHA mAb for 30 min at 37°C. Cells were fixed with 4% PFA at 37°C and the exposed regions of the opsonized beads were stained using the goat α-mouse

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Alexa-647. Cells were then permeabilized and stained for intracellular pSyk using a rabbit αpSyk mAb visualized using a goat α-rabbit Alexa-488. Z-stack images (top panel) were captured

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at 63x using a Zeiss LSM 710 laser scanning confocal microscope. Target beads of interest are indicated by an asterisk (*). Beads (blue), pSyk (red) and F-actin staining (green) colors were

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displayed using the Zen 2009 imaging software. Z-stack images (top panel) were converted into 3D renders (middle panel) to better visualize the spatial association of pSyk staining at the

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various bead-cell interfaces. Qualitative analysis of bead and pSyk staining intensities was performed using ImageJ 1.51 software (bottom panel) by determining the mean fluorescence intensities (MFI; y-axis) of both bead staining (blue line) and pSyk staining (red line) across the

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dash lined arrow visible in the left image of the middle panel and at the top of the MFI histogram in the bottom panel. The x-axis=distance in μm across the indicated line. Figure 4 A-D shows representative images displaying the temporal stages of the IpLITR 2.6b/IpFcRγ-L-activated phagocytic process.

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FIGURE 5. Recruitment of pSyk during IpLITR 1.1b-mediated phagocytosis. Phagocytosis assays were performed by incubating IpLITR 1.1b-expressing cells (1x105) stably transfected with LifeAct-GFP (green) with 4.5-µm non-fluorescent polystyrene beads (3x105) pre-opsonized with αHA mAb for 30 min at 37°C. Cells were fixed with 4% PFA at 37°C and the exposed regions of the opsonized beads were stained using the goat α-mouse Alexa-647. Cells were then permeabilized and stained for intracellular pSyk using a rabbit α-pSyk mAb visualized using a goat α-rabbit Alexa-488. Z-stack images (top panel) were captured at 63x using a Zeiss LSM 710 laser scanning confocal microscope. Target beads of interest are

indicated (*, ). Beads (blue), pSyk (red) and F-actin staining (green) colors were displayed using the Zen 2009 imaging software. Z-stack images (top panel) were converted into 3D renders (middle panel) to better visualize the spatial association of pSyk staining at the various bead-cell interfaces. Qualitative analysis of bead and pSyk staining intensities was performed using ImageJ 1.51 software (bottom panel) by determining the mean fluorescence intensities (MFI; yaxis) of both bead staining (blue line) and pSyk staining (red line) across the dash lined arrow visible in the left image of the middle panel and at the top of the MFI histogram in the bottom

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panel. The x-axis=distance in μm across the indicated line. Figure 5 A-E are representative images displaying the temporal stages of the IpLITR 1.1b-activated phagocytic process.

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FIGURE 6. Recruitment of Nck during IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis. Phagocytosis assays were performed by incubating IpLITR 2.6b/IpFcRγ-L-expressing cells

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(1x105) stably transfected with LifeAct-GFP (green) with 4.5-µm non-fluorescent polystyrene beads (3x105) pre-opsonized with αHA mAb for 30 min at 37°C. Cells were fixed with 4% PFA

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at 37°C and the exposed regions of the opsonized beads were stained using the goat α-mouse Alexa-647. Cells were then permeabilized and stained for intracellular Nck using a rabbit α-Nck

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mAb visualized using a secondary goat α-rabbit Alexa-550. Z-stack images (A) were obtained at a magnification of 63x using a Zeiss LSM 710 laser scanning confocal microscope, with beads of interested indicated (*, , ). Beads (blue), Nck (red) and F-actin staining (green) were viewed

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using the imaging software Zen 2009. Representative z-stacks for individual cells (A; top panels) were converted into 3D renders (A; middle panel) to better visualize the spatial association of Nck recruitments at the various bead-cell interfaces. Individual beads are again indicated (*, , ) in (B) where qualitative analysis was performed using ImageJ 1.51 software on the representative z-stack images (from A; top panels) by measuring the mean fluorescence

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intensities (MFI; y-axis) for both bead staining (blue line) and Nck staining (red line) across the lined arrow visible in the left images and at the top of the MFI histogram on the right side. The x-axis on the histograms=distance in μm across the indicated lines in the images.

FIGURE 7. Recruitment of Nck during IpLITR 1.1b-mediated phagocytosis. Phagocytosis assays were performed by incubating IpLITR 1.1b-expressing cells (1x105) stably transfected with LifeAct-GFP (green) with 4.5-µm non-fluorescent polystyrene beads (3x105) pre-opsonized with αHA mAb for 30 min at 37°C. Cells were fixed with 4% PFA at 37°C and the exposed regions of the opsonized beads were stained using the goat α-mouse Alexa-647. Cells were then permeabilized and stained for intracellular Nck using a rabbit α-Nck mAb visualized using a secondary goat α-rabbit Alexa-550. Z-stack images (A and D; top panels)

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were obtained at a magnification of 63x using a Zeiss LSM 710 laser scanning confocal microscope, and beads of interested are indicated (*, , ). Beads (blue), Nck (red) and F-actin

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staining (green) were viewed using the imaging software Zen 2009. Representative z-stack

images (A and D; top panels) were transformed into 3D renders (A and D; bottom panels) to

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better visualize the spatial association of Nck recruitments at the various bead-cell interfaces. Individual beads are again indicated (*, , ) in (B and E) where qualitative analysis was performed using ImageJ 1.51 software on the representative z-stack images (from A and D; top

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panels) by measuring the mean fluorescence intensities (MFI; y-axis) for both bead staining (blue line) and Nck staining (red line) across the lined arrow visible in the 3D rendered images

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and at the top of the MFI histogram (B and E). The x-axis on the histograms=distance in μm across the indicated lines in the images. Images and MFI intensity histograms are also provided

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for the individually analyzed beads (C and F).

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