Campbell-Valois & Trost et al. 1 Formerly: MCP/2010/006734 MCP/2011/016378 Full Title: Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome François-Xavier Campbell-Valois *,#,††,‡‡ , Matthias Trost *, †,**,††,‡‡ , Magali Chemali * , Brian D. Dill ** , Annie Laplante * , Sophie Duclos * , Shayan Sadeghi * , Christiane Rondeau * , Isabel C Morrow ¶ , Christina Bell † , Kiyokata Hatsuzawa ǁ , Pierre Thibault †,§ , Michel Desjardins *,‡,‡‡ * Département de pathologie et biologie cellulaire, † Institute for Research in Immunology and Cancer (IRIC), ‡ Département de microbiologie et immunologie, § Département de Chimie, Université de Montréal, C.P. 6128, succursale Centre-ville Montréal, QC, H3C 3J7, Canada. ** MRC Protein Phosphorylation Unit, University of Dundee, Dundee, DD1 5EH, Scotland, UK ¶ Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Queensland 4072, Australia. ǁ Department of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan. # Present address: Pathogénie Microbienne Moléculaire Unit, Institut Pasteur, 75015 Paris, France †† These authors contributed equally to this work ‡‡ Corresponding authors: Prof. Dr. Michel Desjardins Phone: (514) 343-7250 Fax: (514) 343-5755 Email: [email protected]Dr. Francois-Xavier Campbell-Valois Phone: +33 1 45 68 83 00 Fax: +33 1 45 68 89 53 Email: [email protected]Dr. Matthias Trost Phone: +44 1382 386402 Fax: +44 01382 223778 Email: [email protected]Running Title: Quantification of organelles input to the phagosome proteome MCP Papers in Press. Published on March 15, 2012 as Manuscript M111.016378 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
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Campbell-Valois & Trost et al.
1
Formerly: MCP/2010/006734
MCP/2011/016378
Full Title: Quantitative proteomics reveals that only a subset of the endoplasmic reticulum
contributes to the phagosome
François-Xavier Campbell-Valois *,#,††,‡‡, Matthias Trost *,†,**,††,‡‡, Magali Chemali*, Brian D.
Dill**
, Annie Laplante*, Sophie Duclos*, Shayan Sadeghi*, Christiane Rondeau*, Isabel C
Morrow¶, Christina Bell†, Kiyokata Hatsuzawaǁ, Pierre Thibault†,§
, Michel Desjardins*,‡,‡‡
*Département de pathologie et biologie cellulaire, †Institute for Research in Immunology and
Cancer (IRIC), ‡Département de microbiologie et immunologie, §Département de Chimie,
MRC Protein Phosphorylation Unit, University of Dundee, Dundee, DD1 5EH, Scotland, UK ¶Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis, University of
Queensland, Brisbane, Queensland 4072, Australia. ǁDepartment of Cell Science, Institute of Biomedical Sciences, Fukushima Medical University
School of Medicine, Fukushima 960-1295, Japan. #Present address: Pathogénie Microbienne Moléculaire Unit, Institut Pasteur, 75015 Paris,
France ††These authors contributed equally to this work ‡‡Corresponding authors:
We wish to thank Olivier Caron-Lizotte for the clustering of proteomics data, Drs
Jacques Paiement,Isabelle Jutras, Marek Gierlinski and Nicholas Schurch for helpful discussions,
Dr Moïse Bendayan and Irene Londono for electron microscopy (EM), respectively; Michel
Lauzon for confocal microscopy and EM; Serge Sénéchal and Danièle Gagné for FACS and the
sequencing platform of the IRIC. This work was supported by the Canadian Institutes of
Health Research (CIHR). F.-X.C.-V. is a CIHR fellow. M.T. was funded by the Deutsche
Forschungsgemeinschaft (DFG), M.T. and B.D.D. are funded by the Medical Research Council
(MRC). P.T. and M.D. are the Canadian Research Chair in Proteomic and Bioanalytical Mass
Spectrometry and Cellular Microbiology, respectively.
References
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37. Muller-Taubenberger, A., Lupas, A. N., Li, H., Ecke, M., Simmeth, E., and Gerisch, G. (2001) Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J. 20, 6772-6782. 38. Gotthardt, D., Blancheteau, V., Bosserhoff, A., Ruppert, T., Delorenzi, M., and Soldati, T. (2006) Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake. Mol.Cell Proteomics. 5, 2228-2243. 39. Stuart, L. M., Boulais, J., Charriere, G. M., Hennessy, E. J., Brunet, S., Jutras, I., Goyette, G., Rondeau, C., Letarte, S., Huang, H., Ye, P., Morales, F., Kocks, C., Bader, J. S., Desjardins, M., and Ezekowitz, R. A. (2007) A systems biology analysis of the Drosophila phagosome. Nature 445, 95-101. 40. Camacho, P., and Lechleiter, J. D. (1995) Calreticulin inhibits repetitive intracellular Ca2+ waves. Cell 82, 765-771. 41. Roderick, H. L., Lechleiter, J. D., and Camacho, P. (2000) Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b. J Cell Biol 149, 1235-1248. 42. Roos, J., DiGregorio, P. J., Yeromin, A. V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J. A., Wagner, S. L., Cahalan, M. D., Velicelebi, G., and Stauderman, K. A. (2005) STIM1, an essential and conserved component of store-operated Ca2+ channel function. J Cell Biol 169, 435-445. 43. Liou, J., Kim, M. L., Heo, W. D., Jones, J. T., Myers, J. W., Ferrell, J. E., Jr., and Meyer, T. (2005) STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol 15, 1235-1241. 44. Zhang, S. L., Yu, Y., Roos, J., Kozak, J. A., Deerinck, T. J., Ellisman, M. H., Stauderman, K. A., and Cahalan, M. D. (2005) STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 437, 902-905. 45. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., and Hogan, P. G. (2006) Orai1 is an essential pore subunit of the CRAC channel. Nature 443, 230-233. 46. Gronski, M. A., Kinchen, J. M., Juncadella, I. J., Franc, N. C., and Ravichandran, K. S. (2009) An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response. Cell Death Differ 16, 1323-1331. 47. Nunes, P., and Demaurex, N. The role of calcium signaling in phagocytosis. J Leukoc Biol 88, 57-68. 48. Ingmundson, A., Delprato, A., Lambright, D. G., and Roy, C. R. (2007) Legionella pneumophila proteins that regulate Rab1 membrane cycling. Nature 450, 365-369. 49. Celli, J., Salcedo, S. P., and Gorvel, J. P. (2005) Brucella coopts the small GTPase Sar1 for intracellular replication. Proc.Natl.Acad.Sci.U.S.A 102, 1673-1678. 50. Roy, C. R., Salcedo, S. P., and Gorvel, J. P. (2006) Pathogen-endoplasmic-reticulum interactions: in through the out door. Nat.Rev.Immunol. 6, 136-147. 51. Ndjamen, B., Kang, B. H., Hatsuzawa, K., and Kima, P. E. Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments. Cell Microbiol 12, 1480-1494.
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Figure Legends
Figure 1. A proteomics approach reveals the contribution of the various source organelles to the
phagosome proteome. Rationale of the MS strategy designed to assess the relative contribution
of diverse organelles to the phagosome membrane. Polystyrene Beads (PB) phagosomes
(Phago15/0 or 15/45) were purified from J774.1 cells on sucrose gradients in three independent
trials. In parallel, Total Membrane (TM) and Post-Nuclear Supernatant (PNS) were isolated. The
protein composition of all four samples was characterized by mass spectrometry (MS) and their
relative abundance within the TM, PNS, Phago15/0 and Phago15/45 fractions determined (A).
Proteins were assigned to five organelles (Plasma membrane (PM), Endosome/Lysosome,
Endoplasmic reticulum (ER), Golgi apparatus and Mitochondria) according to the UniProt
database. Protein fold-changes were then plotted according to their frequency within -10 and 10
at bins of 1. The distribution of fold-change for proteins of the TM vs. PNS (B), Phago15/0 vs.
TM (C), Phago15/45 vs. TM (D) are shown. (E) Estimation of the contribution of each organelle
to phagosome and TM proteomes. Mean peptide intensities for all assigned proteins within an
organelle were averaged, yielding an estimate of the membrane contribution of the source
organelle. The intensities of all members of ER, PM, endosome/lysosome, mitochondria and
Golgi were set arbitrarily to 100 %, thus ignoring other endomembrane compartments.
Figure 2. SILAC experiment to identify potential contaminations to the phagosome. RAW264.7
macrophages were grown in light DMEM and phagocytosis induced for 30 min. These cells were
mixed with an equal number of cells grown in heavy labelled DMEM and lysed. Phagosomes
were isolated and analysed by quantitative MS. Subcellular localisation of proteins was obtained
from Uniprot and organelles were plotted according to their ratio of L:H in bins of 0.5. L:H
Campbell-Valois & Trost et al.
35
ration around one indicate potential source of contamination such as mitochondria and histones,
while most proteins associated to other organelles appear to be genuine phagosome components.
Figure 3. Validation of the label-free MS results by Western-Blotting. Similar amounts of
protein extracts from Phago15/0 and 15/45 and TM were probed with antibodies against
appropriate controls and ER annotated proteins that displayed various abundance levels
according to MS (A). The fold-changes obtained by MS were plotted against the relative
enrichment determined by densitometry of WB triplicates, yielding a linear correlation with R=
0.80 (B). ER and other organelles annotated proteins are represented by white and grey circles,
respectively. The numbers provide a link to the proteins blotted and the early and late status of
phagosomes probed in each instance (Table S7).
Figure 4. Validation of MS results by confocal microscopy indicates that only a sub-domain of
the ER is recruited to the phagosome. Early PB-IgG phagosomes were formed in J774A.1 cells
that were plated on fibronectin coated coverslips. After fixation and permeabilization, cells were
stained for various proteins detected on the phagosome (e.g. SRP54, Stx18 and SPTLC2) and
counterstained with Cnx antibody and phalloidin-bodipy to reveal nascent phagosomes
(Methods). The data indicate that the ER proteins SRP54 and Stx18 co-localize with Cnx on the
phagosome, while SPTLC2 does not (A). Quantification of the relative co-localization in whole
cells of putative phagosome markers over Cnx using the mean Pearson’s coefficients obtained by
the analysis of at least three representative fields for each staining reveal significant differences
in the distribution of several ER markers (B).
Campbell-Valois & Trost et al.
36
Figure 5. mVenus-Stx18, but not GFP-KDEL, is localized to the sub-region of ER implicated in
phagocytosis. Confocal microscopy of RAW264.7 stable cell line expressing GFP-KDEL in the
absence (top panel) or presence (bottom panels) of interferon-γ (IFN) which was used to flatten
the cell in order to improve the spatial resolution; GFP and Cnx were detected by
immunofluorescence. The bottom panel shows a flattened 3D image rendered from multiple
confocal sections obtained through the depth of the cell (the bar represents 10m). Note the
discrepancy in the co-localization of GFP and Cnx, particularly in the perinuclear region and at
the cell periphery (A). PB-IgG were internalized by RAW264.7 GFP-KDEL and J774A.1
mVenus-Stx18. Cells were stained for Cnx and F-actin was revealed by phalloidin-bodipy to
identify early phagosomes (B). WB using antibody against Cnx and GFP on phagosomes fraction
obtained from the same cell line as described in (B) were performed to compare the recruitment
of Cnx, GFP-KDEL and mVenus-Stx18 to the phagosme fraction.
Campbell-Valois & Trost et al.
37
Table 1. Mass Spectrometry statistics using Uniprot annotations
Proteins identified
Peptides Total PM Endo-
/Lysosome
ER Mitochondria Golgi
TM/PNS 13,647 1,764 65 76 117 168 29
Phago
15_0/TM
16,021 1,969 78 94 125 170 40
Phago
15_45/TM
16,003 1,955 70 104 125 174 36
Phago 15_0/TM
0
10
20
30
70
-10 -8 -6 -4 -2 2 4 6 8 10Phago/TM (fold-change)
TM/PNS
TM/PNS (fold-change)
0
10
20
30
40
-10 -8 -6 -4 -2 2 4 6 8 10
C
D Phago 15_45/TM
E
A
Figure 1
0
10
20
30
60
-10 -8 -6 -4 -2 2 4 6 8 10
Phago/TM (fold-change)
Phagosomes(phago)
0
10
20
30
40
50
PM Endo/lyso
ER Mito Golgi
Phago 15_0Phago 15_45TM
B
Time m
/z
MS
MS/MS
Ion profiling
Protein identificationLTQ-Orbitrap XL
b3
y3b4 y4
b5y5
y6
y7
VV N Y
Phago TM PNSFr
eque
ncy
PM
Mitochondria
EREndosome
Fold change (Phago/TM)0
FCGR1
CISY
CISY FCGR1
MitoERPM
TotalMembrane
(TM)
Post-nuclearsupernatent
(PNS)C
ell f
ract
ionn
atio
n LC
-MS/
MS
Abun
danc
eC
hang
es
Prot
ein
num
ber (
%)
Prot
ein
num
ber (
%)
Prot
ein
num
ber (
%)
Org
anel
le c
ontr
ibut
ion
(%)
n=3 n=3 n=3Time Time Time
m/z
Phago
m/z
TM PNSHie
rarc
hica
l cl
uste
ring
MS/MS
LC
PMEndo/Lyso ERMitoGolgi
38
Campbell-Valois & Trost et al.
Figure 239
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10
PM (n=116)
ER (n=38)
Golgi (n=16)
Mito (n=38)
Histones (n=9)
Endo/Lyso (n= 143)
L (phagosomal)/H (contaminant)
% o
f ide
ntifi
ed p
rote
ins
of o
rgan
elle
s
Campbell-Valois & Trost et al.
Figure 3
A
15/0 15/45 TM
EEA1
Rab1a
Rdh11
Cnx
SPTLC2
LAMP1
α/βSNAP
CD51
Na/K ATPase
SRPRb
Sec22b
SRP54
Stx18 WB
Abu
ndan
ce (l
og2)
B
y= -0.41+0.61x R=0.8
-6
-4
-2
0
2
4
6
-6 -4 -2 0 2 4
1
2
34 5
6
7
8
9
1011
12 13
16
14
15
17
1819
20
21
22
23
24
2526
27
28
29
30
31
MS Abundance (log2)
40
Campbell-Valois & Trost et al.
Figure 4
SRP54 Stx18 SPTLC2
0
0.2
0.4
0.6
0.8
A
F-actin
Cnx
Merge
F-actin F-actin
Cnx Cnx
Merge Merge
B
Pea
rson
’s c
oeffi
cien
t
SR
P54
SN
APα/β
Stx
18S
PTL
C2
Rab
1aS
tx4
EE
A1
LAM
P1
41
Campbell-Valois & Trost et al.
Figure 5
IB Cnx
IB GFP
A B
C GFP-KDEL Venus-Stx18
PhTCL
F-actin
GFP-KDEL
Cnx
F-actin
Cnx
mVenusStx18
PhTCL
Cnx GFP-KDELR
elat
ive
Abu
ndan
ce (P
hago
/TC
L)
Cnx GFP0-
2-
Cnx GFP0-
1-
42
Campbell-Valois & Trost et al.
Campbell-Valois & Trost et al.
43
Location in the text of each figure and table
Figure1: After the introduction, beginning of results section; in page 2 of the article.
Table1: After Figure 1 in page 2 as well.
Figure 2: In the Results section in page 3.
Figure 3: In the Results section; side-by-side with figure 4.
Figure 4: In the Results section; side-by-side with figure 3.