The Fc receptor-cytoskeleton complex from human neutrophils

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

The Fc receptor-cytoskeleton complex from human neutrophils☆ Angelica K. Florentinus, Andy Jankowski, Veronika Petrenko, Peter Bowden, John G. Marshall ⁎ Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada

AR TIC LE I N FO

ABS TR ACT

Article history:

The Fc receptor complex and its associated phagocytic cytoskeleton machinery were

Received 7 April 2011

captured from the surface of live cells by IgG coated microbeads and identified by mass

Accepted 14 August 2011

spectrometry. The random and independently sampled intensity values of peptides were

Available online 3 September 2011

similar in the control and IgG samples. After log transformation, the parent and fragment intensity values showed a normal distribution where ≥99.9% of the data was well above

Keywords:

the background noise. Some proteins showed significant differences in intensity between

Fc receptor

the IgG and control samples by ANOVA followed by the Tukey–Kramer honestly

Actin

significant difference test. However many proteins were specific to the IgG beads or the

Myosin

control beads. The set of detected cytoskeleton proteins, binding proteins and enzymes

Tubulin

detected on the IgG beads were used to predict the network of actin-associated regulatory

Dynein

factors. Signaling factors/proteins such as PIK3, PLC, GTPases (such CDC42, Rho GAPs/GEFs),

Kinesin

annexins and inositol triphosphate receptors were all identified as being specific to the activated receptor complex by mass spectrometry. In addition, the tyrosine kinase Fak was detected with the IgG coated beads. Hence, an activated receptor cytoskeleton complex and its associated regulatory proteins were captured from the surface of live human primary leukocytes. © 2011 Elsevier B.V. All rights reserved.

Abbreviations: AEBSF, 4-(2-Aminoethyl) benzenesulfonyl fluoride; CID, Collision-induced dissociation; Da, Dalton; ESI, electro spray ionization; GAP, GTPase activating protein; GEF, guanine exchange factor; HPMI, HEPES Park Memorial Institute; LARC, live-cell affinity receptor chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PBS, phosphate buffered saline; PIK4, phosphatidyl inositol 4 phosphate kinase; PIK3, phosphatidylinositol 3 phosphate kinase; PI3P, phosphatidylinositol 3 phosphate phosphatase; NCBI, National Center for Biotechnology Information; PMA, phorbol ester; PNM, polymorphonuclear leukocytes a.k.a. neutrophil granulocytes; PMSF, phenylmethanesulfonyl fluoride; RCF, relative centrifugal force; RPMI, Rosswell Park Memorial Institute; S.E., standard error; Tris, tris(hydroxymethyl)aminomethane. ☆ Contributions: AKF tested the reagents, manually collated the data, examined the mass spectra, compiled the specific protein scores and created the protein interaction models; AJ performed the LARC experiment on the human neutrophils and performed the LC-ESI-MS/ MS; VP assisted with the collection, manual validation and compilation of the data; JGM contributed the neutrophils, assisted with the LARC experiment and the LC-ESI-MS/MS, supervised the work and wrote the paper. ⁎ Corresponding author at: Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, Ontario, Canada M5B 2K3. Tel.: +1 416 979 5000x4219. E-mail address: [email protected] (J.G. Marshall). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.08.011

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

1.

Introduction

Neutrophil granulocytes, or polymorphonuclear (PMN) leukocytes, are the most abundant white blood cells that fight infection by phagocytosis of foreign particles. White blood cells such as neutrophils retain the capacity to move throughout the body to attack immunoglobin (Ig)-opsonized microbes and have significant implications in disease [1]. Neutrophils have large amounts of vigorously active endo and exo peptidase activities that will rapidly and completely degrade the sample proteins after cell disruption. Neutrophils must be pre-treated with high concentrations of covalent modification reagents such as DFP and should be homogenized in ice cold buffer with AEBSF and PMSF to prevent proteolysis before the proteins are rapidly and completely digested with excess trypsin. Neutrophils should be rapidly isolated and used and cannot be isotopically labelled in vivo to completion [2]. The cocktail of competitive and covalent modification reagents required to preserve the samples interferes with the in vitro derivitization by isotopically labelled chemical tags. Neutrophils cannot be effectively treated with silencing RNA or transfected with mutant constructs. Protein interactions are fundamental to the functions of the cytoskeleton in cell polarity, mobility, signal transduction and phagocytosis. Although there are potentially thousands of cytoskeletal proteins encoded and expressed by the genome, only a certain subset of cytoskeleton proteins play a role in the IgG-Fc receptor mediated phagocytosis. However, the precise identity of these specific isoforms that are involved in white blood cells and immune reactions are presently unknown. Since the Fc receptor is not known to directly bind actin, the identity of the co-factor(s) that connect this receptor to the cytoskeleton are of great interest to many investigators. The identity of the factors that link the Fc receptor complex to the cytoskeleton and thus orchestrate phagocytosis remain to be determined [3]. Affinity chromatography from live cells was developed to capture the Fc receptor supramolecular complex from murine macrophages as a cell model [4]. A similar approach has been recently used to capture the Integrin receptor complexes from living cells where signaling components of the pathway were identified [5]. In this study, actin and tubulin cytoskeletal components were identified to specifically complex with the activated Fc receptor using LARC (Live-cell Affinity Receptor Chromatography) in human neutrophils. Polystyrene microbeads coated with IgG were used to capture the activated Fc receptor complex from the surface of live human neutrophils, where the same IgG coated beads mixed with crude extracts and background binding proteins to the hydrophobic support served as controls. Controlled LC-ESI-MS/MS and computation of protein interactions provided a list of specific isoforms of the cytoskeletal proteins that have been long suspected of playing important roles in regulating Fc mediated immunity.

1.1.

Mass spectral analysis of the cytoskeleton

There is little direct mass spectrometric data for the cytoskeleton proteins of the phagocytic receptors [4,6,7]. Remodeling of the cytoskeleton plays an important role in phagocytosis

451

and is regulated by a large variety of different proteins [8,9]. Once the actin filaments have been formed, myosin molecular motors can drive along the fibers with the hydrolysis of ATP to exert the necessary force that ensures the enclosure of the foreign particle by the plasma membrane [10–12]. The specific isoforms of proteins that participate in actin remodeling are central to understanding Fc-mediated phagocytosis in neutrophils [4,13–18]. The capture of the Fc receptor complex from the surface of live human neutrophils would therefore provide the specific isoforms of actin associated factors that govern the formation of the actin supramolecular complex during phagocytosis in neutrophils. The movement of the PMN cell towards these particles and their subsequent engulfment are mediated by the cytoskeleton. Clustering of activated Fc receptors at sites of contact with foreign particles stimulates cell signaling, local rearrangement of the actin cytoskeleton and remodeling of the membrane [19]. However, the exact factors that regulate remodeling of the actin cytoskeleton are not completely understood [8]. The random and independent sampling of parent ions from the IgG-ligand versus control beads permits the comparison of the resulting log normal distributions by ANOVA and Tukey–Kramer means testing to permit the quantitative calculation of the Fc receptor specific proteins.

1.2.

Actin

Actin plays a central role in the phagocytic function of the Fc receptor [20]. Stimulation of the Fc receptor results in the recruitment of the cytoskeleton to an activated subdomain of the plasma membrane where signaling events occur [19,21,22]. Actin is a highly abundant protein in animal cells of 42 kDa (24) that is found in two states: monomeric or globular actin (G-actin) and filamentous actin (F-actin). Actin polymerizes into long filaments in the cytoskeleton that are required to form the pseudopodia which reach out from cells to engulf foreign particles. Cytoskeleton filaments are responsible for cellular shape, tension and cell motility [23]. In addition, the tubulin-dynein network also exerts influence in the function of the Fc receptor [6].

1.3.

Fc receptor

The family of Fc receptors and their cognate ligands, the immunoglobulins, are part of the Ig superfamily. The ligand of Fc gamma RI is the fragment crystal (Fc) region of the immunoglobulin IgG, which triggers the phagocytic engulfment of foreign particles coated by antibodies. The Fc receptors have affinities for IgG that range from Kd values of E-6 to E-10 and can be directly captured from homogenates using the ligand IgG [4]. Phagocytosis involves the internalization of foreign particles by specialized cells and is a crucial component of the innate immune system, an ancient defense system against infection [9]. The cells of the adaptive immune system guide the innate immune system by the production of antibodies that are bound by the Fc receptor. Thus, the Fc receptor sits at a special location in the immune system that connects the adaptive and innate responses. In our model of Fc receptor mediated phagocytosis, the receptor becomes activated upon binding to foreign particles that are opsonized with gamma

452

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

globulin (IgG) [18]. The receptor associated enzyme, PIK3 has been detected by classical affinity chromatography from homogenates of RAW macrophages and by live-cell affinity chromatography against IgG [4]. However, the identity of the proteins that regulate the assembly and action of the cytoskeleton in response to ligation of the Fc receptor remain to be fully elucidated.

2.

Materials and methods

2.1.

Materials

the mixture was let stand at room temperature for 45 min. The supernatant was layered on two tubes of 10 ml of Ficoll– Hypaque solution and centrifuged for 20 min at 3000 RCF in a tabletop centrifuge before removing the supernatant, lymphocyte Band and Ficoll layers. Then, 36 ml of distilled water was added to lyse the red blood cells by gentle stirring for 20 s before adding 4 ml of 10× PBS. The neutrophils were collected by centrifugation for 5 min, re-suspended in HPMI and pelleted again. The cells were counted and re-suspended at 107cells/ml [2] before treatment with 2 mM diisopropylfluorphosphate for 15 min on ice before final collection and use.

2.4. Dextran T500 and Ficoll–Hypaque solution were obtained from Amersham/Pharmacia (Uppsala, Sweden). The microbeads were obtained from Bang's beads (Fishers IN, USA). The solvents were of optical grade and obtained from Caledon Laboratories (Georgetown ON, Canada). The 510 Meta-laser confocal microscope was obtained from Carl Zeiss (Jenna, Germany). The XP-100 ion trap was obtained from Thermo Fisher Scientific (Thermo Electron Corporation, Waltham MA, USA). The HPLC was from Agilent Technologies (Santa Clara CA, USA). Protein sequencing grade trypsin was obtained from Roche (Mississauga ON, Canada). The human IgG ligand and all other buffers, salts and reagents were obtained from Sigma– Aldrich (St. Louis MO, USA).

2.2.

Reagent testing

The capacity of the IgG coated microbeads to induce phagocytosis was confirmed using human U937 macrophages treated with PMA for three days to induce differentiation into adherent cells that show the phagocytic phenotype. The U937 cells were obtained from ATTC and cultured in RPMI with 5% fetal calf serum as previously described. Polystyrene (latex) microbeads (2 μm) were coated with a 1/50 dilution of IgG in PBS for 30 min before pelleting and washing the beads. The capacity to induce phagocytosis and the formation of actin driven pseudopods was tested on differentiated U937 cells, plated in 6-well dishes on 25 mm cover slips. The cells were washed three times with RPMI buffered with 20 mM HEPES and the reagent test was performed in HPMI. The cells were incubated with the IgG coated beads and fixed prior to completion of phagocytic engulfment. Cells without treatment, control beads or IgG coated beads were fixed with 4% paraformaldehyde in PBS for 30 min prior to quenching in glycine, followed by permeabilization with 0.1% Triton-X100 in PBS. The cells were stained for filamentous actin with Rhodamine phylloidin, as previously described [19]. The successful formation of the actin pseudopods and phagocytosis was imaged with a Zeiss 510 Meta-laser confocal microscope.

2.3.

Preparation of human neutrophils

Human neutrophils were isolated as previously described [2]. All solutions were kept at room temperature and contained within polypropylene tubes. About 100 ml of fresh human blood was collected by venipuncture, with 0.9 ml heparin (1000) added to the syringe. Nine ml of 4.5% dextran T500 in 0.9% saline (final dextran 0.7%) was added to the blood and

Live-cell affinity receptor chromatography (LARC)

Fc receptor complexes were isolated from the surface of live cells using the LARC approach and analyzed using LC-ESIMS/MS [4]. The Fc receptor complex was captured from the surface of live cells using IgG coated microbeads. Cells with microbeads bound via IgG to the activated Fc receptor were homogenized on ice. Initially, human neutrophils in HPMI were divided into two pools (treated and controls). One aliquot of fresh neutrophils was used for the capture of the Fc receptor complex from live cells. Microbeads bearing IgG, the ligand of the Fc receptor, were incubated with live neutrophils in suspension on ice for 30 min. The unbound beads were washed away by brief centrifugation in HPMI, prior to sampling the Fc receptor complex at 0, 2.5, 5, 7.5 and 10 min of warming. The beads from the various sampling times were combined. Another aliquot of fresh neutrophils was homogenized on ice along with the IgG coated beads to control for nonspecific binding in vitro. The cells and IgG coated beads were then homogenized twice through a French press in PBS plus 2 mM PMSF, 2 mM EDTA, 2 mM AEBSF and eukaryotic protease inhibitor cocktail. The results were examined with a microscope to ensure that most cells were broken. The IgG microbeads that were bound to the Fc receptor were collected over a sucrose gradient in an ultra centrifuge, as previously described [4]. The affinity receptor chromatography experiment from the live cells and the control were performed together using the same batch of cells, beads and buffers. The entire experiment was independently replicated on three different dates, separated by a few weeks with a fresh batch of cells, beads, buffers and other reagents on each occasion. The proteins that were released into the HPMI media over the course of the experiment were collected and after centrifugation of the activated cells were also analyzed and are termed background.

2.5.

LC/LC-ESI-MS/MS

The captured Fc receptor complexes and controls were analyzed by LC-ESI-MS/MS. Both the IgG coated LARC beads and the IgG coated control beads mixed with crude homogenates were subjected side-by-side to the same fractionation and analysis steps in three independent experiments. First, the proteins isolated on the beads were fractionated using a step gradient of NaCl in PBS (150 to 1000 mM NaCl). The proteins eluted from the beads in the various salt fractions were then digested with trypsin prior to LC-ESI-MS/MS. The proteins remaining on the beads were re-suspended in 80% organic

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

solvent, as previously described, and then digested with trypsin to yield a total of 15 fractions from one experiment [4]. The independent affinity receptor chromatography experiments, each with 15 sub-fractions, were analyzed separately after each experiment a few weeks apart. IgG coated control beads were added to neutrophils on ice at the time of homogenization to control for non-specific binding and were identically isolated, fractionated and analyzed alongside each receptor chromatography experiment. The tryptic peptides from the proteins were collected over C18 ZipTips and eluted in 2 μl of 65% acetonitrile 5% formic acid, before the eluted peptides were diluted to 20 μl in 0.02% acetic acid for injection. The peptides were resolved by C18 reverse phase chromatography over a 90 minute gradient from 5% to 65% acetonitrile at a flow rate of 2 μl per minute with a Vydac 15 cm, 300 μm ID, 5 μm particles with 300 Ǻ pore size. The peptides were separated with an Agilent 1100 series capillary pump via a metal needle to form an electrospray into a Decca XP-100 ion trap [4]. The randomly and independently sampled parent and fragment ion intensity values were recorded in Mascot generic format (mgf) files.

2.6.

Quantitative statistical analysis

For the purpose of a quantitative statistical analysis the peptide identifications provided by SEQUEST [24] from a federated human protein database assembled in 2009 were matched to the parent and fragment intensity data from the mgf files using Structured Query Language (SQL) and computed using the Statistical Analysis System (SAS) [25]. The parent and fragment ions were apparently randomly and independently sampled without replacement from the LC column over the course of the acetonitrile gradient. The parent and fragment intensity values were log transformed, fit to the normal distribution and tested using the log normal probability plot in SAS. The parent and fragment intensity values were treated as continuous variables. The peptide and protein sequences were treated as nominal variables. The nominal and continuous variables were declared using the SAS JMP graphical interface prior to calculation of ANOVA models. The tables and graphical results from the “model” and “fit y to x” SAS routines were converted to metafiles for inclusion in figures. The statistical analysis was calculated using SAS JMP on a 32 bit personal computer. The background noise in the LCESI mass spectrometer was about E3 in the MS mode and about E2 in the MS/MS mode and signals below these levels were omitted from the statistical analysis. Normal distribution analysis was used to demonstrate a high confidence in the completeness of the sampling of identified peptides.

2.7.

Protein identification

The correlation algorithm within SEQUEST was used to match MS/MS spectra to amino acid sequences from the stable human RefSeq NP (known proteins) database [26] from July 2004 that was updated and found to be in close agreement in 2007 as described by Zhu et al., [27]. The peptides were collected together under individual proteins to generate a total score with the BIOWORKS application, using the manufacturer's (Thermo Electron Corporation) software and recommended settings

453

[28,29]. The cytoskeleton-associated proteins listed in the table met the criteria of at least three independent peptides thus assuring near certain identification [29–31]. Proteins obtained by affinity capture of the Fc receptor complex from live cells were compared to those observed on the control beads to account for non-specific binding. Proteins previously implicated in actin or myosin biology and/or the proteins shown to interact with actin or myosin proteins in previous experiments were manually examined [4]. Proteins in the Fc receptor preparation with a significant score which also showed at least three different peptides assuring near certain identification, were tabulated alongside the corresponding control values that were detected [28,29,31]. The approach of building confidence in protein identification through replicated experiments and multiple peptides has been tested on viral cultures that are relatively pure and several lines of evidence indicate that proteins identified with 3 or more independent peptide sequences are highly reliable [29– 31]. The Bioworks scoring system accurately predicted important drug target proteins in RAW macrophages [4]. A previous statistical analysis by the Chi Square distribution and comparison to Cargile's random amino acid sequence tables both indicated that proteins with three or more peptides were reliable [32,33].

2.8.

Generation of protein interaction models

The previous literature on actin and cytoskeleton assembly and regulation was used as a starting point to identify proteins that might form a complex with actin [9]. Actinassociated proteins and related isoforms or types of proteins that were specific to the IgG coated beads were considered for the model of protein interactions. Multiple filtering criteria were used to select proteins for inclusion into the working model. Proteins from the captured Fc receptor complex from human neutrophils observed by LC-MS/MS were compared to the existing literature and a protein–protein interaction model was generated using iHOP [34]. The computer software programs STRING [35], Cytoscape [36] and Osprey [37] were also used to create a protein–protein interaction model, wherein additional proteins that might participate in the cytoskeleton were predicted. In some instances, BLAST analysis [38] was used to search for homologs of the proteins previously implicated by protein–protein interaction experiments in the receptor specific MS/MS correlation results. Proteins similar to those predicted by the computer model were subsequently confirmed by manual examination and addition of the MS/MS data for inclusion in the model. The iterative process of prediction from the literature and protein–protein interaction data, along with confirmation by manual examination of the LCESI-MS/MS data, resulted in the tables and protein network diagrams shown.

3.

Results

3.1.

Experimental workflow

The workflow of the LARC beads bound to live cells and the control beads incubated with crude homogenates or experimental medium to account for non-specific binding over the

454

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

course of the cellular incubation and homogenization are shown in Fig. 1. In brief, the IgG coated beads were bound to cells on ice for 30 min prior to washing away the unbound beads and warming over the course of receptor activation. The receptor activation was quenched with ice cold buffer before homogenizing the cells in an ice cold French press prior to isolating the beads over sucrose gradients. Control beads were added to the cells on ice immediately prior to homogenization and then treated identically.

3.2.

Confirmation of reagents

The sufficiency of the IgG coated beads to trigger the Fc receptor was confirmed in the U937 human macrophage system differentiated from a monocytic cell line by treatment with PMA. The binding of IgG coated microbeads to human phagocytes resulted in the formation of a supermolecular complex of actin at the site of receptor activation and pseudopodia formation as illustrated in the human cell model (Fig. 2). Human

Fig. 1 – Flow chart of the Live-cell Affinity Receptor Chromatography (LARC) of the Fc receptor by ligand coated microbeads from the surface of live human neutrophils compared to non-specific binding from crude homogenates with the same IgG coated beads as a control.

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

A

#720-722 NL: 3.02E6

B

Fig. 2 – The formation of the actin supramolecular complex at the site of Fc receptor activation by IgG coated microbeads in human cells. Panels: [A], laser confocal micrograph of IgG coated microbead binding to the Fc receptor on human U937 cells stained for actin with Rhodamine phylloidin. The arrow shows the location of an IgG coated microbead; [B], spectrum of beta-actin collected from the IgG coated microbeads used to capture the Fc receptor complex from the surface of live cells. The peak label ° indicates a loss of water. The peak label * indicates a loss of NH3. The peptide HQGVMVGMGQKDSYVGDEAQSK was identified from beta-actin with M + H 2352.5911 and a z value of 3 + .

IgG bound to microbeads were used to capture the activated Fc receptor and its associated cytoskeleton from the surface of live human phagocytic cells. The formation of the actin complex at the site of receptor crosslinking and activation by the ligand-coated beads can be easily visualized by staining with Rhodamine phylloidin (Fig. 2). Beads without ligands displayed little binding to the cells (not shown).

3.3.

Quantification of actin, myosin and associated proteins

The MS/MS spectra were fit to peptides and proteins using a well-established function [24]. A small subset of the total data set containing 1,162,425 parent and fragment ion intensity

455

values were collected alongside their corresponding peptide and protein sequences in SQL for subsequent statistical analysis with SAS [25]. The grand mean of transformed parent ion intensity (IgG and control) was E6.835 with a standard deviation of E0.611 counts. At a column loading of 1–5 μg very few of the parent ions sampled and identified had mean intensity values of less than E3 (1000 counts). Thus, where peptides were only detected in IgG samples, only a small fraction of these might have been randomly missed in the control at intensity values of greater than one thousand counts (E3). The average intensity values for the 908 control peptides was E7.31 (S.E. 0.019) and for the 2908 IgG peptides was E6.77 (S.E. 0.011) counts. The parent and fragment intensity data was found to approximate a normal distribution after log transformation (Fig. 3). The observed parent and fragment intensity values ranged from E7 to as little as E2 and average intensity values for many proteins were typically E5 to E3 counts. Complete ANOVA models were created at the level of treatment, protein and peptides for parent ion signals of greater or equal to E3 (1000) counts from actin, myosin or associated proteins that showed significant effects [p(x) ≤0.0001]. On average, the 59 actin-associated proteins from the control samples showed slightly higher mean intensity values of E4.25 (S.E. 0.00134) compared to a mean of E4.04 (S.E., 0.00088) in the 200 or so proteins in the IgG-ligand samples. There were some statistically significant differences between the 22 proteins shared by the control versus IgG sample with some proteins more intense in the control and others elevated in the IgG samples (see Supplemental data Tables 1 and 2). ANOVA followed by the Tukey–Kramer Honestly Significance Difference (HSD) was used to compare the statistical differences between proteins found in both IgG and control: A subset of 237 actin and myosins and their potential regulatory GTPases or binding proteins are shown along with the number of ions observed, the protein mean intensity values of peptides and fragments, the standard deviation and confidence intervals as well as the comparison of all means by Tukey–Kramer HSD (Supplemental data Tables 1 and 2).

3.4.

Summary of LC-ESI-MS/MS

The cytoskeleton and associated proteins in this study met the minimum criteria of at least a minimum score of 2400 and showed three independent peptides. The data was manually inspected and confirmed showing similar trends (Supplemental data Table 3). Selected results of LC-ESI-MS/MS experiments based on manual examination showing many different representative types of protein are summarized in Table 1. In some cases proteins were specifically detected with signal intensity values of ≥E3 in either the IgG or control samples. Actin is a major cellular protein that, like all proteins, binds chromatography supports non-specifically to some degree, even after ultracentrifugation in sucrose. The use of control affinity chromatography beads incubated with crude extracts or the experimental media may assist in the showing specific isoforms that while found on IgG beads are also well detected from incubating homogenates with porous chromatographic supports. Hence, the same IgG coated beads were homogenized with cells on ice [4] in order to account for the non-specific interactions of proteins with the microbeads. Many proteins from homogenates were observed to apparently bind non-specifically to the IgG beads and

456

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

A

Table 1 – Summary of the cytoskeletal proteins detected by LC-ESI-MS/MS. The IgG beads ligated to live cells (LARC) and the same beads incubated with crude extracts as a control were replicated side by side in three independent experiments and the results were combined for correlation analysis with SEQUEST Bioworks. Actin-associated proteins were detected in both IgG LARC and control beads. About 159 actin-associated proteins were exclusively detected in the LARC beads or exceeded the control by at least one confidence interval and were therefore subjected to further analysis (see Table 2). A total of 161 proteins were detected in the background from the experimental media that presumable resulted from cell lysis and degranulation over the course of the experiment.

log parent and fragment ion intensity

9 8 7 6 5 4 3

Experimental Specific Score >=2400 Difference >=2400

2

log normal probability

B

0.99 0.9 0.5 0.1 0.01 0.001 1e-5 2

3

4

5

6

7

8 9 10

log parent and fragment ion intensity Fig. 3 – The normal distribution of parent and fragment ion intensity values from the LC-ESI-MS/MS of control and IgG coated micro-beads together. Panels: A, the fitted log normal distribution log transformed ion intensity values; B, the log normal diagnostic plot. The normality of 1,162, 424 parent and fragment ions intensity values is shown. Note the parent ion intensity distribution alone had a grand average of E6.835 and a standard deviation of 0.611 and so ≥99.9% of the sampled and identified peptides in this study were well above the threshold of E3 counts in the background noise. However the fragments may have counts as low as E2. Fragment counts of less than E2 appeared to be noise and were omitted.

are presented alongside as the control. The background proteins released into the experimental isolation medium from the neutrophils that activate and degranulate during the experiments were also determined. Background proteins included actin related protein 2/3 complex subunit 4 isoform b, a protein similar to actin-binding protein anillin, the Wiskott–Aldrich syndrome protein, and a protein similar to Ataxin-10. The protein isoforms detected in the background samples differed from the isoforms detected in the activated Fc receptor samples (LARC). In addition, profilin 1 and kinesin light chain 1A were also similarly

Live cell

Crude homogenate

Background

349 117 245 234

243 11 56 20

8 6 3 1

detected in the background samples, both of which are different from the isoforms detected in the affinity experiments. In the background samples ~161 proteins, including specific isoforms of abundant actin related proteins, were observed to be released into the experimental medium. In contrast, approximately 349 proteins were observed with the IgG-Fc receptor complex isolated from live cells (LARC) and about 243 proteins were detected on the control IgG beads incubated with crude extracts (IAC sample). About 245 protein captured from live cells attained significant scores greater than 2400 (p ≤ 0.05) with at least three independent peptides thereby assuring near certain identification in the Fc receptor complex [29–33]. About 117 of the cytoskeleton proteins observed (33.5%) were specific to the Fc receptor and 11 were specific to the control IAC samples (Table 1).

3.5. Activated Fc receptor complex associated cytoskeletal proteins in human neutrophils The federated library of ~135,000 human proteins contained at least 162 Fc or Ig superfamily related receptor proteins plus 1642 known cytoskeleton associated proteins (Table 2). Thus a specific subset of only 160 of the 1642 cytoskeleton proteins detected in genomic experiments were observed to associate with the Fc receptor complex from human neutrophils (Table 2). About 234 proteins from live cells attained a score greater by at least one significance interval from the controls. In many cases, one or a few isoforms of a protein family were specifically associated with the Fc receptor, whereas other isoforms were only detected in the crude homogenate bead controls. Specific isoforms of actin, myosin, cofilins, profilins, WASP/Scar, ARP2/3, FERM proteins ezrin, moesin and radixin, cortactin, HS1, LIM kinase and others such as tubulin, dynein and kinesin were observed with the captured Fc receptors.

3.6.

Fc receptor

The Fc receptor itself, may be recovered by affinity chromatography from crude homogenates by affixing its cognate

457

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

Table 2 – Genetically encoded versus experimentally detected cytoskeletal proteins. The count of distinct cytoskeleton proteins specific to the Fc receptor complex compared to cytoskeleton proteins listed in the human federated library of expressed proteins from cDNA sequences and predicted genomic sequences. The cytoskeleton proteins specific to the Fc receptor complex from LARC and not prominent in the IAC or background sample as shown. Protein category

Descriptor

Ig superfamily Fc & immunoglobulin receptor receptor Actin and associated Actin Actinin Actin binding protein Actin capping Gelsolin Actin nucleation Wiskott–Aldrich Syndrome Actin related protein WASP interacting protein Actin bundling Ataxin Fascin Nucleotide dissociation Filament dis-/assembly Cofilin Profilin Thymosin Lim protein Annexin Annexin Myosin Myosin Tropomyosin Myosin light chain Myosin light chain kinase Spectrin Desmin Nebulin Titin Nebulette Vimentin Desmoyokin or AHNAK Microtubule network Tubulin & associated Dynein Kinesin Kinectin Dynactin Regulatory proteins HS1 Cortactin Filamin Coronin Villin Advillin Supervillin

NR human Fc federated receptor library complex 162

9

84 32 54

10 2 3

17

1

15

1

15 3

7 1

66 18

3 4

Table 2 (continued) Protein category

Descriptor

NR human Fc federated receptor library complex

Paxillin Contactin FERM & band 4.1 Fak

11 47 56 6

1 2 7 1

SWI/SNF BRG related Sum

43 1642

9 160

Nuclear proteins

ligand IgG to microbeads [4]. As expected, Fc receptor related molecules were captured by either LARC or IAC including members of the FcγRI, II, III, and N variants (Table 3). The polymeric immunoglobulin receptor was detected in the experimental medium controls. BLAST analysis also confirmed the presence of novel proteins that show homology with Fc receptors [38]. Given the amplification provided by signal activation pathways, there are stochiometrically more molecules of actin and downstream effectors than activated Fc receptors detected in the analysis. The manual confirmation of the proteins with three independent peptides is shown for each protein in the Supplemental data.

3.7. Live-cell affinity receptor chromatography (LARC) from human neutrophils 3 7 24 30

1 2 2 1

30

19

282 48 13 14

29 1 2 1

80 14 16 22 13 7 12

1 1 1 1 2 1

157

5

111 165 6 25

1 22 1 1

2 8 34 30 15 1 6

1 3 4 2 1 1 1

3.7.1.

Actin

To avoid the confounding effect of background protein binding, the results of the cytoskeleton proteins that achieved significant detection in the LARC specific fraction are presented alongside controls (controls= background + beads incubated with crude extracts). After these precautions actin or actin-like Table 3 – The Fc receptor proteins captured by the cognate ligand of commercial Human Immunoglobulin bound to polystyrene microbeads used for Live-cell Affinity Receptor Chromatography (LARC) versus Immuno affinity Chromatography (IAC) alongside background binding proteins from the experimental medium. The polymeric Immunoglobulin receptor was only observed in the background sample and the Fc receptors were only observed in the LARC and IAC samples. The number of peptides from different splice variants and isoforms that were observed grouped under each receptor class shown. The receptor types shown attained a score of 2400 and/or at least three independent peptides thereby confirming near certain identification.

Fc RgI Fc RgII Fc RgIIIb Fc like 3 Fc Ra Fc Re Fc Rn Fc alpha/mu FCRLc2

LARC

IAC

Background

9 4 7 2 3 4 7 3 3

5 1 2 2 6 0 1 0 0

0 0 0 0 0 0 0 0 0

458

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

Table 4 – Actin cytoskeleton proteins specifically associated with the Fc receptor complex captured from IgG-coated microbeads (LARC) compared to control microbeads incubated with crude extracts or background proteins released into the experimental buffers. The protein names provided are from the NCBI RefSeq NP annotation fields. Proteins with 3 peptides and Bioworks score ≥2400 are shown. Control refers to the non-specific background plus the presumed background binding of non-IgG-receptor proteins to the IAC beads. No score is shown where no protein was detected. Protein category

LARC

Actins and associated Actin, beta Beta-actin Similar to beta-actin Actin Chain A Actin, skeletal muscle Actin-like 7A Anillin, actin binding protein Similar to astrotactin 2 A Chain Actin Microfilament and actin filament cross-linker protein isoform b Neural tissue-specific F-actin binding protein I Actin Chain A Trabeculin, alpha actin binding protein ABP620 Similar to actin 3 Similar to human alpha-actinin 3 Alpha-actinin

69719 2426332 494238 30351 68706 6094 3666 2635 147043 13966 2543 30351 2928 33485 41559 3548

Actin nucleation Wiskott–Aldrich syndrome protein family member 2 WASPIP protein WAVE-associated Rac GTPase activating protein Actin-related protein 2; ARP2 Actin related protein 2/3 complex subunit 1B ARP3 actin-related protein 3 homolog Actin related protein 2/3 complex subunit 5 Actin-related protein 8; ARP8 Actin-related protein 8; ARP8 Similar to actin-related protein 3-beta

3626 4503 4566 3690 2797 3835 104585 12889 4807 3326

Actin capping Gelsolin (amyloidosis, Finnish type)

3035

Actin bundling Ataxin 1 Ataxin 2 Ataxin-7protein Fascin 1 Fascin 2; retinal fascin Fascin 3; fascin Neurofascin precursor

4330 9266 5126 841 2239 1018 1121

Nucleotide dissociation and filament assembly/disassembly Cofilin 1 Platelet profilin Profilin 1 Thymosin beta-4 Similar to thymosin beta-like protein LIM actin-binding protein 1

14028 1617 775 45809 1258 5095

Annexins Annexin I; lipocortin I Similar to annexin A2 Annexin III Annexin VI isoform 1 Annexin XI Annexin XI

646514 4055 2715 8242 3258 3422

Myosin motors Myosin IB Myosin ID Myosin motors

4592 2793

Controls

883698 162941 4787 65500 145 237 69948 5392 4787 34982 16267

409 745 125 172 645

Gene symbol

Accession no

ACTB ACTB ACTB GSN ATRB ACTL7A ANLN ASTN2 GSN MACF1 PPP1R9A GSN MACF1

14250401 28336 42655680 7546413 71611 5729720 8923832 27714675 7245526 33188443 13431727 7546413 11137615 18595749 969079 28723

ORF1 ACTN1

WASF2 WIPF1 SRGAP3 ACTR2 ARPC1B ACTR3 ARPC5 ACTR8 ACTR8

13431974 12804123 24369934 5031571 5031601 5031573 5031593 27923738 39812115 28503542

GSN

4504165

ATXN1 ATXN2 ATXN7 FSCN1 FSCN2 FSCN3 NFASC

4506793 4506795 3192950 13623415 6912626 9966791 38372284

CFL1 PFN1 PFN1 TMSB4X TMSB4Y ABLIM1

5031635 3891601 30584265 136580 38086762 21284383

ANXA1 ANXA2 ANXA3 ANXA6 ANXA11 ANXA11

4502101 12314197 4826643 4502109 4557317 4557317

MYO1B MYO1D

37549339 41150753

2218

151 1518 1119 935

120367 99790

1878

340898 20877 6071

459

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

Table 4 (continued) Protein category

LARC

Controls

Accession no

MYO1E MYO3A MYO5A MYO5C MYO6 MYO5B MYO9A MYO9B MYO9B MYO7A MYO10 MYO15A MYH2 MYH4 MYH7 MYH8 MYH13 MYH14 MYO18B MYH6 MYH2 MYL6 MYO1F MYH9 MYH11 MYH9 MYO1A TPM3 MRLC2 MYL6 MYLK

4826844 7958618 10835119 9055284 13431720 39932736 5902012 4758750 33356170 11360310 7108753 22547229 8923940 11024712 4557773 4505301 11321579 33563340 20338989 27764861 13431716 33620739 1924940 12667788 13124879 625305 3337398 22748619 15809016 127148 16950611

134798 22748619 19698555 5030431 37848 115527120 55769541 5453758

Myosin IE; myosin-IC Class III myosin 17 Myosin VA (heavy polypeptide 12, myoxin) Myosin VC; myosin 5 C Myosin VI Myosin Vb (Myosin 5B) Myosin IXA Myosin IXB Myosin IXB Myosin VIIa, long form Myosin X Myosin XV; unconventional myosin-15 Myosin, heavy polypeptide 2, skeletal muscle Myosin, heavy polypeptide 4, skeletal muscle Myosin, heavy polypeptide 7, cardiac muscle Myosin, heavy polypeptide 8, skeletal muscle Myosin, heavy polypeptide 13, skeletal muscle Myosin, heavy polypeptide 14; myosin heavy chain 14 Myosin heavy chain 1 isoform b Myosin heavy chain 6; myosin heavy chain, cardiac muscle Myosin heavy chain, skeletal muscle 2 Myosin light chain, alkali, nonmuscle Myosin-IF Myosin, heavy polypeptide 9, non-muscle Smooth muscle myosin heavy chain 11 Myosin heavy chain nonmuscle form A Brush border myosin-I Tropomyosin 3 Myosin regulatory light chain MRCL2 Myosin light chain alkali, smooth-muscle isoform Myosin light chain kinase isoform 1

2223 5989 14827 4199 1310 2602 13451 13312 5512 33809 5250 10834 3652 6560 6980 7842 19720 4572 10046 2419 5571 29522 12396 3079 20953 9019 3780 6647 9698 28535 4710

2473 415 22638 22773 3702 2244 1555 23987 521

Proteins associated with muscle contractions Spectrin beta chain, erythrocyte Tropomyosin 3 Desmin Vimentin factor IIIC, polypeptide 1 Vimentin N-terminal fragment Nebulin Desmoyokin – human 11 X-linked isoform b Nebulette; actin-binding Z-disk protein

5325 6647 9182 367188 10163 27453 15482 5207

7191 2244 559 17034 9402 5546 7188 1202

SPTB TPM3 DES VIM

2313 7537 6933 4660 2465 4640 13523 15228

2306 1014 4263 2000 650 1889 4390 8719 2254 27318 344 356 443 2145 1210 920 1554 1688 276 1351

HCLS1 CTTNBP2 CTTNBP2 SHANK2 CTTN FLNA FLNC FLNB FLNB CORO1A

Candidate regulatory proteins Hematopoietic cell-specific Lyn substrate 1 Cortactin binding protein 2 Similar to cortactin binding protein 2 SH3 and multiple ankyrin repeat domains 2 Isoform 1 Src substrate cortactin Filamin 1 Filamin C, gamma Filamin B, beta Similar to filamin B, beta Coronin, actin binding protein, 1A Similar to coronin, actin-binding protein, 2A Similar to advillin Supervillin isoform 2 Similar to villin-like Paxillin gamma Contactin 1 isoform 1 precursor; glycoprotein gP135 Contactin 6; neural adhesion molecule Erythrocyte membrane protein 4.1 N Erythrocyte membrane protein band 4.1 like 4B Similar to erythrocyte membrane protein band 4.1-like 2 Erythrocyte membrane protein band 4.1 like 4B

42815 3369 5860 7593 4111 4509 10601 8102 10909 2813 2656 2813

1535 247 1656 4277 4715

Gene symbol

1275 419 2574 797 198 440 1379 2740 438 3176 210

NEB AHNAK NEBL

AVIL SVIL VILL PXN CNTN1 CNTN6 CNTN1 EPB41L4B EPB41L2 EPB41L4B

4885405 16975496 27709074 19743794 2498954 4503745 4557597 4503747 27674097 5902134 27714525 13278708 11496982 119584910 1912057 28373117 7657361 16356663 21361812 21961573 21361812 (continued on next page)

460

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

Table 4 (continued) Protein category

LARC

Controls

Gene symbol

Accession no

3257 3259 17325 997

EPB41L2 EZR MSN RDX PTK2

11544608 119717 14625824 27486345 27886593

TUBA4A TBCD TUBGCP5 TUBB1 TUBGCP2 PLEKHA8

32015 41350333 24308378 2119276 5729840 32449733

KIF1C KIF3B KIF5A KIF5B KIF5C KIF11 KIF13A KIF14 KIF20A KIF21A KIF21B KIF23 KIF26A

KIF17 KLC3 KIF2A

42661074 4758646 4826808 4758648 4758650 13699824 27730235 7661878 5032013 38348350 41112866 6754472 37546015 27370633 12054032 22060166 9910266 13878563 21594340

KIF1A KIFC3 KTN1

19924175 34098691 33620775

SMARCC1 SMARCA5 SMARCA1 ARID1B ARID1A ARID1B PPRC1 KIF4A KIF4A

21237802 21071058 21071044 22047962 28467001 11320942 40068462 40807452 9255863 13959694

PKM2

4505839

Candidate regulatory proteins 4.1 G protein Ezrin (Cytovillin) (Villin 2) Moesin/anaplastic lymphoma kinase fusion Similar to radixin Focal adhesion kinase 1, protein tyrosine kinase 2

3957 4511 982 2840 6993

Tubulins Alpha-tubulin Beta-tubulin cofactor D Tubulin, gamma complex associated protein 5 Beta-tubulin – human Tubulin, gamma complex associated protein 2 FAPP2 proteinpolypeptide 11; dynein

3939 2918 2464 50470 2815 3348

50 510 296 770 465 117

3141 3480 12544 3922 2952 3200 3675 6654 3014 3358 578 2993 8538 4405 7355 2636 4045 3371 2433

277 599 1298 2004 1257 1014 248 1207 1412

Kinesins Kinesin family member 1C Kinesin family member 3B; kinesin family protein 3B Kinesin family member 5A; spastic paraplegia 10 Kinesin family member 5B; kinesin 1 (110–120 kD) Kinesin family member 5C; heavy chain, neuron-specific Kinesin family member 11 Similar to kinesin family member 13A Kinesin family member 14 Kinesin family member 20A; RAB6 interacting Similar to kinesin family member 21A; N-5 kinesin Kinesin family member 21B Kinesin family member 23 isoform 2; mitotic Kinesin-like 1 Kinesin family member 26A Similar to kinesin-like protein at 64D Kinesin-13A2 Similar to kinesin-related protein 3A Kinesin-like 7; kinesin-like protein 2 Probable kinesin light chain 3 Kinesin heavy chain member 2 Kinesin heavy axonal transport of synaptic vesicles Member 1A Kinesin-like protein Kinectin 1; CG-1 antigen

3421 2527 3798

3559 165 208 718 2030 569 1679 993

479 1217

Nuclear proteins SWI/SNF SWI/SNF SWI/SNF similar to SWI/SNF BRG1-binding protein ELD BRG1-Associated Factor BRG1-binding protein isoform 3 PGC-1 related co-activator BRG Chromokinesin Chromosome-associated kinesin

6144 7251 5118 3428 3428 4275 3156 2596 2938 2629

709 1950 1051 332 741 350 1328 632

Example of non specific proteins Pyruvate kinase, muscle; Pyruvate kinase-3

23708

74438

or functionally related proteins such as actinin were found to be specifically present at the Fc receptor complex (Table 4). The actin isoforms in the live sample compared to the homogenate control sample were consistent with the formation of the actin network in response to activation of the Fc receptor by the IgG coated microbeads. Excellent spectra and significant scores for beta-actin isoforms were observed to be specific to IgG beads (Fig. 2 & Table 4). Spectra for actin-like proteins similar to

675

KIF13A

beta-actin are shown in the Supplemental tables and figures. Hence, actin and related molecules were found to be specifically associated with the activated Fc receptor complex.

3.7.2.

Actin nucleation

The Wiskott–Aldrich Syndrome protein itself (WASP) was detected in the background and is the product of a gene that, when mutated, gives rise to immunodeficiency and bleeding

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

[39,40]. WASPs are nucleation promoting factors that may transport actin monomers and regulate the activity of the ARP2/3 complex. The ARP2/3 complex in turn connects ATP-bound actin monomers to the barbed ends of the actin filaments. The process of nucleating new actin filaments is tightly regulated by the actin-related proteins (ARP) that mimic the growing “barbed” or “plus” end surface of actin filaments, thus permitting the formation of a new growing actin complex. The Wiskott–Aldrich protein isoforms 1, 2 and 3 related proteins or homologues and binding factors were all found to be specifically associated with the activated Fc receptor complex (Table 4). Many of the regulatory factors associated with actin were significantly detected in the activated Fc receptor complex, but not in the control beads, which is consistent with the polymerization of actin under the direction of the Fc receptor complex (Table 3). ARP proteins 2, 3, 3-beta and 8 (among others) were also specific to the Fc receptor complex along with novel homologues and other related proteins (Table 4). Hence, many of the factors that regulate actin nucleation and filament extension/polymerization were associated with the activated Fc receptor. Representative spectra for ARP proteins are shown in the Supplemental tables and figures.

3.7.3.

Actin capping

The actin filaments grow and push the membrane forward until the barbed end is capped by proteins such as gelsolin. Proteins that might act to cleave or cap actin were observed to be specifically associated with the Fc receptor complex. The gelsolin protein attained a significant score in the IgG-Fc complex (Table 4). Other specific isoforms of gelsolin-related molecules, such as the F actin capping protein Cap Z and the gelsolin related proteins, were detected in the activated Fc fraction but did not always meet the required score or showed three independent peptides (criteria for inclusion in Table 1). Reasonably convincing spectra for gelsolin A are shown in the Supplemental tables and figures.

461

light chain kinase were all shown to specifically associate/ bind to the activated Fc receptor complex. Many MS/MS spectra for multiple isoforms of myosins were recorded as shown in the Supplemental tables and figures.

3.7.6. Nucleotide dissociation and filament assembly and disassembly Actin monomers are maintained in two discrete pools within the cell. One pool of Mg-ATP-actin may readily polymerize at the barbed or plus end of the actin filament. Another pool of actin monomers may be bound to profilin or sequestered by thymosin β4. The Mg-ATP-actin monomers that are polymerized into F-actin retain ATP bound to a cleft in the actin monomer. ATP is not required for polymerization of actin, but its presence is required to avoid disassembly by the actin depolymerizing factor cofilin. At some period after binding, distal from the elongating barbed ends, the ATP is hydrolyzed to ADP near the pointed ends of the elongating actin filaments. Proteins such as cofilin bind to the filaments, depolymerize the pointed end and cofilin catalyzes the exchange of ADP for ATP [20]. Loss of the γ phosphate and the binding of cofilin initiates the depolymerization. The monomers of actin that are released by cofilin can then be sequestered by thymosin β4. Alternatively, cofilin may sever filaments there by exposing the barbed ends to initiate elongation of F-actin. Cofilin and thymosin β4 were both observed to be specifically associated with the cytoskeleton of the Fc receptor. LIM protein that may regulate the return of monomers to actin filaments was also observed to specifically associate/bind to the activated Fc receptor (Table 4). In contrast, profilin was strongly associated with the control beads incubated in crude extract.

3.7.7.

Proteins commonly associated with muscle contraction

Protein interaction experiments from the literature and data collated, may suggest a role for ataxins and fascins, which are required for bundling and crosslinking the actin fibers and which may function at or near the cell surface. The presence of ataxins, but not fascins, was observed with the Fc receptor complex (Table 4).

Based on studies of the actin-myosin motor complexes from muscle, it has been shown that a large number of these proteins interact with actin, including actinin, desmin, desmoyokin, vimentin, nebulin, paramyosin, spectrin and nebulette actin binding protein. In our experiments we also observed these same proteins binding to the activated Fc receptor (Table 4). While some muscle actin complex components, such as tropomyosin 3 and beta-tropomyosin, were detected, others such as troponins were notably absent from the activated Fc receptor complex.

3.7.5. Myosins, myosin light chain kinase and myosin binding partners and regulatory factors

3.7.8. Proteins that may connect the Fc receptor to the cytoskeleton

The association of myosin with the actin filament gives the “arrowhead” outline of the barbed end where monomer addition occurs. Myosin proteins bind actin and hydrolyze ATP to exert motor force. Many different isoforms of myosin light and heavy chains and their regulatory proteins were observed, including myosin VA heavy polypeptide 12, myosin VC, Vb, IX A, B, C and variants, as well as VIIa, X, XV and unconventional myosin-15, heavy polypeptide 2, 4, 7, 8, 9, 13 and 14. Myosin heavy chains, including myosin heavy chain 1 isoform b 6, cardiac muscle alpha isoform, skeletal muscle adult 2, smooth muscle isoform 11 and non-muscle form A were all detected (Table 4). Brush border myosin-I polypeptide 1 was also observed. In addition, myosin regulatory light chain, non-muscle myosin alkali light chains and myosin

The Fc receptor complex directs the function of a large actin supramolecular complex. It has been suggested that the receptor, its associated cytoskeletal proteins, and the surrounding plasma membrane subdomain, are all required to form the active receptor complex [19]. The Fc receptor itself may be intimately associated with the actin cytoskeleton by virtue of intermediate actin binding proteins. Hence, proteins that interact with actin might serve to bridge or connect the activated Fc receptors to the cytoskeleton. The anchoring of actin to the plasma membrane may be partly regulated by Band 4.1, ezrin, moesin and radixin, together known as the FERM proteins (Table 4). Other membrane proteins that may play a role in regulating the actin cytoskeleton include coronin the Src substrate cortactin, or their binding proteins [41–47]. All of these proteins were

3.7.4.

Actin bundling and crosslinking proteins

462

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

observed in the Fc receptor complex. Although, the related molecule HS1 also scored high, it was also observed in the crude homogenate control, perhaps indicating a weak association with the Fc receptor. Lastly, villin, advillin, supervillin and other filamin family members were also observed to be associated with the activated Fc receptor (Table 4), which is consistent with their proposed function of connecting receptors to the cytoskeleton [48–52].

3.7.9.

Tubulin, dynein, kinesin and dynactin

Members of the tubulin-dynein cytoskeleton system were also observed to strongly associate/bind to the Fc receptor. For example, beta-tubulin attained a score of 50,469 in the receptor beads, as compared to 769 in the control beads. Alpha-tubulin, beta-tubulin cofactor, tubulin gamma complex associated 2, dynactin and dynein were also observed to specifically associate with the activated Fc receptor complex (Table 4). In addition, many kinesin isoforms were also found to be specific to the Fc receptor complex. Sample spectra for peptides belonging to beta-tubulin, beta-tubulin cofactor D and dynein are shown in the Supplemental tables and figures.

3.7.10. BRG1 and SWI/SNF Proteins that were formerly shown to be critical for the regulation of transcription and gene expression are now known to mediate the connection of nuclear machinery to the actin cytoskeleton. It is now understood that not only the movement of the chromosomes but also other aspects of nucleic acid metabolism – including chromatin remodeling, DNA unwinding and transcription itself – may be dependent in part on nuclear actin to generate motor force [53,54]. Some proteins that interact with actin in the nucleus, such as BRG1 and SWI/SNF proteins, were also shown to specifically associate with the Fc receptor complex isolated from early phagosomes (Table 4).

3.8.

Protein interaction network modeling

Proteins specific to the IgG-Fc receptor complex, and known to associate with the actin-myosin network were analyzed using information hyperlinked over proteins (iHOP). In addition, the mathematical network modeling packages Cytoscape, Osprey and STRING were also employed. The generally established members of the actin network (Table 2) were also used as the starting point for an exploration of the specific MS/MS data guided by the previously noted literature and databases. The results of the network model generated by STRING as shown in Fig. 4 are from the protein interactions summarized in Supplementary data Table 3. Further detected cytoskeleton interactions are listed or illustrated in the Supplementary materials. Uncharacterized isoforms or homologues of actin-myosin proteins were obtained from the primary LC-ESI-MS/MS data. The central nodes in these predicted protein-interacting networks showed very high levels of agreement between these different models indicating that these proteins may be important targets in receptor function (Fig. 4).

3.8.1.

Predicted actin regulators of the Fc receptor complex

Upon binding of the Fc receptor to its ligand (IgG), the production and metabolism of PIP2 and PIP3 at the plasma membrane by

kinases, phophatases and phospholipases are thought to function in actin regulation [19,21,22]. Our protein interaction analysis indicates that annexins, phosphatidylinositol kinases and inositol triphosphate receptors are all central binding factors in the Fc receptor pathway. Receptor associated kinases and phosphatases act on proteins or lipids as well as GTPase pathways and play a role in regulating the formation of new actin nucleation sites at the plasma membrane [23]. The enzymes that regulate levels of PI(4,5)P2 – including phosphatidylinositol kinases and phosphatases, protein tyrosine and serine kinases and GTPase proteins, such as Rac and CDC42 – are all predicted by this and previous studies to interact with the actin cytoskeleton. The phosphorylation of membrane lipids apparently regulate the activation of GTPases [14] that in turn lead to the formation of actin filaments [55]. The production of PIP2 may also regulate the function of WASP/Scar, a protein previously shown to be required for the formation of the phagocytic cup [55]. Some of the predicted protein interactors that are known to be regulatory factors, and were found to be directly detectable in IgG beads by mass spectrometry (see Supplementary data tables). Many of the proteins specifically detected in the IgG-FcR complex captured from live cells have been previously shown to interact with actin-associated proteins. Specific isoforms of phosphatidylinositol 3 kinases (PIK3), the inositol1,4,5-receptor and other regulatory proteins, such as the spleen tyrosine kinase (Syk) that were previously predicted to interact with the IgG-FcR complex were confirmed by mass spectrometry in the affinity capture from both live cell and homogenate samples. The detection of PIK3, SYK and IPTR from the IAC fractions may indicates that these proteins are directly or indirectly associated with the Fc receptor itself. PIK3C2A is known to interact with myosins and has been observed in Fc receptor affinity fractions, using IgG coated microbeads in vitro with crude homogenates and more complex live cell samples. Similarly, inositoltriphosphate receptors (IPTR) were observed in LARC fractions and are known to directly interact with actins, filamins, cofilin, ataxins and myosins, as well as other cytoskeletal proteins. Therefore, ITPRs may serve as key connectors between the PIK3, the Fc receptor and the cytoskeleton. Likewise, specific isoforms and homologues from the monomeric GTPases CDC42 and Rac pathway, including their GAP and GEF proteins (e.g. ARFGAP), were calculated to play a role in actin regulation, based on the existing literature and protein interaction experiments (Fig. 4). The Rho GTPase activating protein 1 (RhoGAP), which is believed to activate CDC42 and the WAVE-associated Rac GTPase activating protein, was also found to be significantly associated with the Fc receptor complex. The protein–protein interaction studies indicated that regulatory proteins such as nexins, rapamycin-associated proteins, FAT tumor suppressor and the VEGF inducible protein are all specifically associated with the activated IgG-Fcγ-R complex. (Fig. 4). Moreover, the protein tyrosine kinase Fak was also observed to be specifically associated with the cytoskeleton of the Fc receptor supramolecular complex. All together, these studies have identified specific isoforms of the known regulatory proteins, as well as novel proteins not previously thought to play a role in phagocytosis, to be intimately associated with the activated IgG-FcγR supercomplex (Fig. 5). The presence of proteins associated with growth factor pathways was in

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

463

Fig. 4 – The protein–protein interaction network of the cytoskeleton specifically associated with the Fc receptor supramolecular complex of human neutrophils as illustrated with STRING. See supplemental data for details. Evidence of protein–protein interactions based on: Green: Neighborhood, Red: Gene fusion, Dark blue: Co-occurrence, Black: Co-expression, Pink: Experiments, Light blue: Databases, Yellow: Text mining; Purple, Homology. The 68 proteins in the network are detailed in supplemental Table 2.

agreement with silencing RNA screens from the phagocytic cells of Drosophila melanogaster [56,57].

4.

Discussion

4.1. Quantitative Analysis of the Fc-receptor versus the non-specific binding control The protein descriptor fields from the federated human protein library indicate that thousands of actin and actinassociated proteins are expressed in various human tissues and cells. However, this study shows that a specific subset of the known cytoskeleton-proteins are associated with the

activated Fc receptor from live cells and thus may be involved in FcR mediated phagocytosis. The observation that ≥99.9% of the log normal probability distribution was clearly above the cut off intensity value of the background noise indicates that there is a low probability that proteins only observed in the IgG sample were missed in the controls because of sampling error. The presence of only a subset of cytoskeleton proteins or isoforms that were specific to the Fc receptor complex indicates that the experiment was selective and did not merely detect all actin-associated proteins. The data seems to indicate that only one or a few isoforms of the many types of actin/myosin regulating proteins are expressed in neutrophils and associated with the active Fc receptor. Moreover, many new regulatory proteins were identified in this study that

464

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

Fig. 5 – Schematic diagram of the actin interaction network of the Fc receptor complex. The potential spatial arrangement of actin-associated proteins from Tables 1, 2 and 3. Candidate proteins for forging the physical connection(s) by which the Fc receptor directs the actions of the actin supramolecular complex are shown with the purple circle. Many other, as yet unknown, factors may be involved.

may be involved in phagocytosis, indicating that wellcontrolled LC-ESI-MS/MS experiments may be a selective and useful tool to identify proteins involved in regulating activated receptor complexes.

4.2. Sensitivity and specificity of LC-ESI-MS/MS for the actin supramolecular complex The limit of sensitivity of LC-ESI-MS/MS from complex mixtures has been estimated to be in the nanogram per ml range [58]. Actin and myosin are relatively abundant cellular proteins and are expected to be found in at least microgram quantities in cellular homogenates. Thus, LC-ESI-MS/MS should be sensitive enough to detect actin-myosin network components associated with the Fc receptor complex. Calculations regarding the specificity of LC-ESI-MS/MS confirm that mass spectrometry of peptides yield reliable identification of many proteins from mammalian samples [25,59]. The multiple peptide correlations observed to each of the proteins in this study indicate that the probability of false positive identification is low [29,31–33]. Since many of the key actin and myosin proteins were specifically associated with the ligand coated microbeads bound to the live cells it appears that controlled LC-ESI-MS/MS has the analytical sensitivity to identify the actin interaction protein network [4]. The strong detection of some actins, which rapidly dissociate from the internalized phagosome [21], is consistent with the capture of the activated Fc receptor at the cell surface.

4.3.

Agreement with other lines of evidence

The IgG coated beads seem to have faithfully captured the phagocytic cytoskeleton in agreement with previous biochemical and genetic experiments. It appears that the results of the

LARC approach are strongly biased towards the known Fc receptor complex and its associated regulatory and effector proteins [9]: Many proteins or isoforms – such as PI3K, Inositol triphosphate receptors, specific myosins, kinesins and RhoGEFs and GAPs, which were previously supposed to interact with the Fc receptor and actin cytoskeleton – were specifically identified with the Fc receptor in this study. The expression of the Fc receptor is known to be sufficient to drive the formation of a functional phagosome in 293 T adipocytes [3]. Thus, the Fc receptor itself seems to have sufficient structural features to interact, either directly or indirectly, with the actin cytoskeleton. Since the members of the Fc receptor family are not well known to be high affinity binding partners of the cytoskeleton, it seems likely that specific receptor-binding proteins might serve to connect the Fc receptor to the actin-myosin network in order to direct the forces involved in phagocytosis. The observation that elements of the actin cytoskeleton specifically associated with the activated FcR indicated that these proteins may be contiguously connected to the receptor itself from within or without the cell. The Fc receptor on the cell surface is grounded to the actin cytoskeleton by connections that are not fully elucidated. Villin, supervillin, annexins [60,61], FERM proteins [62] or perhaps other membrane and cytoskeleton-associated proteins – such as coronin [41–46], or cortactin repeat [63–65] – are all possible connector proteins that physically link the activated receptor complex to the actin-myosin machinery. The observation that filamin might be a key connection from a cell surface receptor to the cytoskeleton indicates that it may also be one of the cadre of structural regulatory proteins that serve to connect the Fc receptor to the cytoskeleton [49,50,52]. Alternatively, other isoforms of filamin might play a role in regulating the expression of Fc receptors on the cell surface and their recycling to the interior of the cell [48].

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

The analysis revealed an extended and detailed network of cytoskeleton-interacting proteins that could regulate phagocytosis, including the inositoltriphosphate receptors, filamins, paxillins and kinases [50,66–69] and a number of myosin and kinesin motors, which may function in the phagocytic engulfment of IgG coated particles by human neutrophils. The numerous molecular motor isoforms, which independently interact with the receptor complex and phospholipid kinases, or their products, may indicate that differential regulation of motor proteins controls the movement of the membrane in more than one dimension during the engulfment of the target particle. Tabulation of the protein-interaction model revealed protein isoforms at important network nodes. For example, microtubules may regulate PIK3 activity and its recruitment to the activated Fc receptor [70]. Analysis of the actin-interacting proteins indicated an even wider network of specific proteins and isoforms, which included receptor associated lipid kinases such as PIK3, annexins and inosotol triphosphate receptors, that may function together to regulate the cytoskeleton [60,61,71–74]. At the membrane PIK3 may also regulate monomeric G proteins, such as CDC42, that in turn regulate actin polymerization [14]. Proteins, such as guanine nucleotide hydrolases (GTPases), their activator (GAP) and exchange factors (GEF) might regulate the actin cytoskeleton. Manual inspection of the MS/MS data indicates that Rac, CDC42 GTPase, Rho GTPase activating protein 1 and the WAVEassociated Rac GTPase activating protein may function in this capacity [14,15,75–84]. Specific isoforms of actins, WASP, severing and capping proteins, assembly and disassembly proteins and their regulatory proteins were all observed. Moreover, there is evidence that protein tyrosine kinases, such as FAK, may also functionally connect the binding partners of the Fc family of receptors to the cytoskeleton [68,83].

465

ANOVA and Tukey–Kramer means testing of peptides or proteins indicated that there were many proteins specific to the Fc receptor complex and not the controls for non-specific background binding. The direct physical capture of the phagocytic protein complex from live cells was in agreement with previous biochemical and genetic studies and the predicted protein interaction calculations. Capture of an activated receptor complex from the surface of live cells permitted the illumination of receptorassociated cytoskeleton proteins as well as the regulatory factors from live human primary cells. The large number of the various myosin, dynein and kinesin motors specifically associated with the Fc receptor seems to indicate that the remodeling of the membrane and cytoskeleton with the generation of force in the pseudopods requires the concerted regulation of both the actin and microtubule network. Moreover, the abundant kinesins and their receptor kinectin are perhaps consistent with the delivery of membrane vesicles to the plasma membrane at the site of phagocytosis [85–87]. The data supports the proposal that inositol phosphorylation and reception may play a central role in the regulation of the cytoskeleton in phagocytosis [6,70–73]. Supplementary materials related to this article can be found online at doi:10.1016/j.jprot.2011.08.011.

Acknowledgment This research was supported with a Discovery grant from the Natural Science and Engineering Research Council (NSERC) to JGM.

REFERENCES 4.4.

Sufficiency of live-cell affinity receptor chromatography

The advent of LC-ESI-MS presents the possibility of many new strategies for detecting novel protein interactions. However, an experimental strategy that was unable to satisfactorily handle the interactions of actin could hardly be expected to work on the more challenging interactions of lower abundance molecules. The data shown here illustrates many of the advantages of capturing receptor complexes intact after assembly in the living cell – prior to cellular disruption and collection. Actin, along with its regulatory mechanisms, polymerization machinery and the protein complexes that exert force at the cellular level, were found to be specifically associated with the activated Fc receptor complex at IgG-coated microbeads on live cells in agreement with several other lines of evidence. The specific capture of the cytoskeleton from the live cells indicates that it is possible to capture an activated receptor supramolecular complex from the surface of live human primary cells.

5.

Conclusion

The presence of a small subset of human cytoskeleton proteins that were specifically observed in the phagocytic receptor complex provided the identity of the protein isoforms that may function in particle engulfment by human neutrophils. Quantitative analysis of the log-normal intensity distribution followed by

[1] Tsuboi N, Asano K, Lauterbach M, Mayadas TN. Human neutrophil Fcgamma receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases. Immunity 2008;28:833–46. [2] Marshall J, Krump E, Lindsay T, Downey G, Ford DA, Zhu P, et al. Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils. J Immunol 2000;164:2084–91. [3] Giodini A, Rahner C, Cresswell P. Receptor-mediated phagocytosis elicits cross-presentation in nonprofessional antigen-presenting cells. Proc Natl Acad Sci USA 2009;106:3324–9. [4] Jankowski A, Zhu P, Marshall JG. Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography. Anal Biochem 2008;380:235–48. [5] Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, et al. Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Sci Signal 2009;2(ra51). [6] Patel PC, Fisher KH, Yang EC, Deane CM, Harrison RE. Proteomic analysis of microtubule-associated proteins during macrophage activation. Mol Cell Proteomics 2009;8: 2500–14. [7] Xu P, Crawford M, Way M, Godovac-Zimmermann J, Segal AW, Radulovic M. Subproteome analysis of the neutrophil cytoskeleton. Proteomics 2009;9:2037–49. [8] Kodama A, Lechler T, Fuchs E. Coordinating cytoskeletal tracks to polarize cellular movements. J Cell Biol 2004;167:203–7. [9] Greenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Opin Immunol 2002;14:136–45.

466

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

[10] Araki N, Hatae T, Furukawa A, Swanson JA. Phosphoinositide-3-kinase-independent contractile activities associated with Fcgamma-receptor-mediated phagocytosis and macropinocytosis in macrophages. J Cell Sci 2003;116:247–57. [11] Swanson JA, Johnson MT, Beningo K, Post P, Mooseker M, Araki N. A contractile activity that closes phagosomes in macrophages. J Cell Sci 1999;112(Pt 3):307–16. [12] Harris E, Cardelli J. RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion. J Cell Sci 2002;115:3703–13. [13] Rogers LD, Foster LJ. The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum. Proc Natl Acad Sci USA 2007;104:18520–5. [14] Beemiller P, Zhang Y, Mohan S, Levinsohn E, Gaeta I, Hoppe AD, et al. A Cdc42 activation cycle coordinated by PI 3-kinase during Fc receptor-mediated phagocytosis. Mol Biol Cell 2010;21:470–80. [15] Hoppe AD, Swanson JA. Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol Biol Cell 2004;15:3509–19. [16] Castellano F, Chavrier P, Caron E. Actin dynamics during phagocytosis. Semin Immunol 2001;13:347–55. [17] Knaus UG, Heyworth PG, Kinsella BT, Curnutte JT, Bokoch GM. Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase. J Biol Chem 1992;267:23575–82. [18] Swanson JA, Hoppe AD. The coordination of signaling during Fc receptor-mediated phagocytosis. J Leukoc Biol 2004;76:1093–103. [19] Marshall JG, Booth JW, Stambolic V, Mak T, Balla T, Schreiber AD, et al. Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis. J Cell Biol 2001;153: 1369–80. [20] Tse SM, Furuya W, Gold E, Schreiber AD, Sandvig K, Inman RD, et al. Differential role of actin, clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis. J Biol Chem 2003;278:3331–8. [21] Corbett-Nelson EF, Mason D, Marshall JG, Collette Y, Grinstein S. Signaling-dependent immobilization of acylated proteins in the inner monolayer of the plasma membrane. J Cell Biol 2006;174: 255–65. [22] Botelho RJ, Teruel M, Dierckman R, Anderson R, Wells A, York JD, et al. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J Cell Biol 2000;151:1353–68. [23] Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003;112:453–65. [24] Lopez-Ferrer D, Martinez-Bartolome S, Villar M, Campillos M, Martin-Maroto F, Vazquez J. Statistical model for large-scale peptide identification in databases from tandem mass spectra using SEQUEST. Anal Chem 2004;76:6853–60. [25] Bowden P, Beavis R, Marshall J. Tandem mass spectrometry of human tryptic blood peptides calculated by a statistical algorithm and captured by a relational database with exploration by a general statistical analysis system. J Proteomics 2009;73:103–11. [26] Maglott DR, Katz KS, Sicotte H, Pruitt KD. NCBI's LocusLink and RefSeq. Nucleic Acids Res 2000;28:126–8. [27] Zhu P, Bowden P, Pendrak V, Thiele H, Zhang D, Siu M, et al. Comparison of protein expression lists from mass spectrometry of human blood fluids using exact peptide sequences versus BLAST. Clin Proteomics 2007;2:185–203. [28] Guzzetta AW, Thakur RA, Mylchreest IC. A robust micro-electrospray ionization technique for highthroughput liquid chromatography/mass spectrometry proteomics using a sanded metal needle as an emitter. Rapid Commun Mass Spectrom 2002;16:2067–72.

[29] Moore RE, Young MK, Lee TD. Qscore: an algorithm for evaluating SEQUEST database search results. J Am Soc Mass Spectrom 2002;13:378–86. [30] Chelius D, Huhmer AF, Shieh CH, Lehmberg E, Traina JA, Slattery TK, et al. Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods. J Proteome Res 2002;1:501–13. [31] Cargile BJ, Bundy JL, Stephenson Jr JL. Potential for false positive identifications from large databases through tandem mass spectrometry. J Proteome Res 2004;3:1082–5. [32] Zhu P, Bowden P, Tucholska M, Marshall JG. Chi-square comparison of tryptic peptide-to-protein distributions of tandem mass spectrometry from blood with those of random expectation. Anal Biochem 2011;409:189–94. [33] Zhu P, Bowden P, Tucholska M, Zhang D, Marshall JG. Peptide-to-protein distribution versus a competition for significance to estimate error rate in blood protein identification. Anal Biochem 2011;411:241–53. [34] Hoffmann R, Valencia A. Implementing the iHOP concept for navigation of biomedical literature. Bioinformatics 2005;21 (Suppl 2):ii252–8. [35] von Mering C, Jensen LJ, Kuhn M, Chaffron S, Doerks T, Kruger B, et al. STRING 7–recent developments in the integration and prediction of protein interactions. Nucleic Acids Res 2007;35: D358–62. [36] Salwinski L, Eisenberg D. The MiSink Plugin: Cytoscape as a graphical interface to the Database of Interacting Proteins. Bioinformatics 2007;23:2193–5. [37] Breitkreutz BJ, Stark C, Tyers M. Osprey: a network visualization system. Genome Biol 2003;4:R22. [38] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25: 3389–402. [39] Rengan R, Ochs HD. Molecular biology of the Wiskott–Aldrich syndrome. Rev Immunogenet 2000;2:243–55. [40] Rengan R, Ochs HD, Sweet LI, Keil ML, Gunning WT, Lachant NA, et al. Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells. Blood 2000;95:1283–92. [41] Yan M, Di Ciano-Oliveira C, Grinstein S, Trimble WS. Coronin function is required for chemotaxis and phagocytosis in human neutrophils. J Immunol 2007;178: 5769–78. [42] Yan M, Collins RF, Grinstein S, Trimble WS. Coronin-1 function is required for phagosome formation. Mol Biol Cell 2005;16:3077–87. [43] Jayachandran R, Gatfield J, Massner J, Albrecht I, Zanolari B, Pieters J. RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes. Mol Biol Cell 2008;19:1241–51. [44] Combaluzier B, Mueller P, Massner J, Finke D, Pieters J. Coronin 1 is essential for IgM-mediated Ca2+ mobilization in B cells but dispensable for the generation of immune responses in vivo. J Immunol 2009;182:1954–61. [45] Combaluzier B, Pieters J. Chemotaxis and phagocytosis in neutrophils is independent of coronin 1. J Immunol 2009;182: 2745–52. [46] Huang YW, Yan M, Collins RF, Diciccio JE, Grinstein S, Trimble WS. Mammalian septins are required for phagosome formation. Mol Biol Cell 2008;19:1717–26. [47] Carrizosa E, Gomez TS, Labno CM, Klos Dehring DA, Liu X, Freedman BD, et al. Hematopoietic lineage cell-specific protein 1 is recruited to the immunological synapse by IL-2-inducible T cell kinase and regulates phospholipase Cgamma1 Microcluster dynamics during T cell spreading. J Immunol 2009;183:7352–61. [48] Beekman JM, van der Poel CE, van der Linden JA, van den Berg DL, van den Berghe PV, van de Winkel JG, et al. Filamin A

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 01 1 ) 4 5 0 –4 68

[49]

[50]

[51]

[52]

[53] [54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65] [66]

[67]

stabilizes Fc gamma RI surface expression and prevents its lysosomal routing. J Immunol 2008;180:3938–45. Heikkinen OK, Ruskamo S, Konarev PV, Svergun DI, Iivanainen T, Heikkinen SM, et al. Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin. J Biol Chem 2009;284:25450–8. Lesourne R, Fridman WH, Daeron M. Dynamic interactions of Fc gamma receptor IIB with filamin-bound SHIP1 amplify filamentous actin-dependent negative regulation of Fc epsilon receptor I signaling. J Immunol 2005;174: 1365–73. Nomachi A, Nishita M, Inaba D, Enomoto M, Hamasaki M, Minami Y. Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A. J Biol Chem 2008;283:27973–81. Popowicz GM, Schleicher M, Noegel AA, Holak TA. Filamins: promiscuous organizers of the cytoskeleton. Trends Biochem Sci 2006;31:411–9. Farrants AK. Chromatin remodelling and actin organisation. FEBS Lett 2008;582:2041–50. Rosson GB, Bartlett C, Reed W, Weissman BE. BRG1 loss in MiaPaCa2 cells induces an altered cellular morphology and disruption in the organization of the actin cytoskeleton. J Cell Physiol 2005;205:286–94. Park H, Cox D. Cdc42 regulates Fc gamma receptor-mediated phagocytosis through the activation and phosphorylation of Wiskott–Aldrich syndrome protein (WASP) and neural-WASP. Mol Biol Cell 2009;20:4500–8. Stuart LM, Ezekowitz RA. Phagocytosis and comparative innate immunity: learning on the fly. Nat Rev Immunol 2008;8:131–41. Stuart LM, Boulais J, Charriere GM, Hennessy EJ, Brunet S, Jutras I, et al. A systems biology analysis of the Drosophila phagosome. Nature 2007;445:95–101. Tucholska M, Bowden P, Jacks K, Zhu P, Furesz S, Dumbrovsky M, et al. Human serum proteins fractionated by preparative partition chromatography prior to LC-ESI-MS/MS. J Proteome Res 2009;8:1143–55. Bowden P, Pendrak V, Zhu P, Marshall JG. Meta sequence analysis of human blood peptides and their parent proteins. J Proteomics 2010;73:1163–75. Diakonova M, Gerke V, Ernst J, Liautard JP, van der Vusse G, Griffiths G. Localization of five annexins in J774 macrophages and on isolated phagosomes. J Cell Sci 1997;110(Pt 10): 1199–213. Gerke V, Creutz CE, Moss SE. Annexins: linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol 2005;6:449–61. Papusheva E, Mello de Queiroz F, Dalous J, Han Y, Esposito A, Jares-Erijmanxa EA, et al. Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility. J Cell Sci 2009;122:656–66. Uruno T, Zhang P, Liu J, Hao JJ, Zhan X. Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization. Biochem J 2003;371:485–93. Hao JJ, Zhu J, Zhou K, Smith N, Zhan X. The coiled-coil domain is required for HS1 to bind to F-actin and activate Arp2/3 complex. J Biol Chem 2005;280:37988–94. Wolski KM, Haller E, Cameron DF. Cortactin and phagocytosis in isolated Sertoli cells. J Negat Results Biomed 2005;4:11. Wade R, Bohl J, Vande Pol S. Paxillin null embryonic stem cells are impaired in cell spreading and tyrosine phosphorylation of focal adhesion kinase. Oncogene 2002;21: 96–107. Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, et al. Integrin function: molecular hierarchies of

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

467

cytoskeletal and signaling molecules. J Cell Biol 1995;131: 791–805. Torres AJ, Vasudevan L, Holowka D, Baird BA. Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays. Proc Natl Acad Sci USA 2008;105:17238–44. Shafikhani SH, Mostov K, Engel J. Focal adhesion components are essential for mammalian cell cytokinesis. Cell Cycle 2008;7:2868–76. Khandani A, Eng E, Jongstra-Bilen J, Schreiber AD, Douda D, Samavarchi-Tehrani P, et al. Microtubules regulate PI-3K activity and recruitment to the phagocytic cup during Fcgamma receptor-mediated phagocytosis in nonelicited macrophages. J Leukoc Biol 2007;82:417–28. Steinckwich N, Schenten V, Melchior C, Brechard S, Tschirhart EJ. An essential role of STIM1, Orai1, and S100A8-A9 proteins for Ca2+ signaling and Fc(gamma)R-mediated phagosomal oxidative activity. J Immunol 2011;186:2182–91. Beliveau E, Guillemette G. Microfilament and microtubule assembly is required for the propagation of inositol trisphosphate receptor-induced Ca2+ waves in bovine aortic endothelial cells. J Cell Biochem 2009;106:344–52. Full SJ, Deinzer ML, Ho PS, Greenwood JA. Phosphoinositide binding regulates alpha-actinin CH2 domain structure: analysis by hydrogen/deuterium exchange mass spectrometry. Protein Sci 2007;16:2597–604. Rescher U, Ruhe D, Ludwig C, Zobiack N, Gerke V. Annexin 2 is a phosphatidylinositol (4,5)-bisphosphate binding protein recruited to actin assembly sites at cellular membranes. J Cell Sci 2004;117:3473–80. Gotthardt D, Warnatz HJ, Henschel O, Bruckert F, Schleicher M, Soldati T. High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases. Mol Biol Cell 2002;13:3508–20. Morrissette NS, Gold ES, Guo J, Hamerman JA, Ozinsky A, Bedian V, et al. Isolation and characterization of monoclonal antibodies directed against novel components of macrophage phagosomes. J Cell Sci 1999;112(Pt 24):4705–13. Lypowy J, Chen IY, Abdellatif M. An alliance between Ras GTPase-activating protein, filamin C, and Ras GTPase-activating protein SH3 domain-binding protein regulates myocyte growth. J Biol Chem 2005;280:25717–28. Leung R, Wang Y, Cuddy K, Sun C, Magalhaes J, Grynpas M, et al. Filamin A regulates monocyte migration through rho small GTPases during osteoclastogenesis. J Bone Miner Res 2010 May;25(5):1077–91. Lee WL, Cosio G, Ireton K, Grinstein S. Role of CrkII in Fcgamma receptor-mediated phagocytosis. J Biol Chem 2007;282:11135–43. Kim C, Dinauer MC. Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid. J Leukoc Biol 2006;79: 223–34. Scott CC, Dobson W, Botelho RJ, Coady-Osberg N, Chavrier P, Knecht DA, et al. Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis. J Cell Biol 2005;169:139–49. Hall AB, Gakidis MA, Glogauer M, Wilsbacher JL, Gao S, Swat W, et al. Requirements for Vav guanine nucleotide exchange factors and Rho GTPases in FcgammaR- and complement-mediated phagocytosis. Immunity 2006;24: 305–16. Zrihan-Licht S, Fu Y, Settleman J, Schinkmann K, Shaw L, Keydar I, et al. RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion. Oncogene 2000;19:1318–28. Seastone DJ, Zhang L, Buczynski G, Rebstein P, Weeks G, Spiegelman G, et al. The small Mr Ras-like GTPase Rap1 and the

468

J O U RN A L OF P ROTE O M IC S 7 5 ( 2 01 1 ) 4 5 0 –46 8

phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum. Mol Biol Cell 1999;10:393–406. [85] Touret N, Paroutis P, Grinstein S. The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma. J Leukoc Biol 2005;77:878–85. [86] Touret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, Pypaert M, et al. Quantitative and dynamic assessment of the

contribution of the ER to phagosome formation. Cell 2005;123: 157–70. [87] Bajno L, Peng XR, Schreiber AD, Moore HP, Trimble WS, Grinstein S. Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation. J Cell Biol 2000;149:697–706.