Download Supplementary Information Directional Exosome Proteomes Reflect

Supplementary Information
Directional Exosome Proteomes Reflect Polarity-Specific Functions
in Retinal Pigmented Epithelium Monolayers
Mikael Klingeborn1,*, W. Michael Dismuke1, Nikolai P. Skiba1, Una Kelly1, W. Daniel Stamer1,2
and Catherine Bowes Rickman1,3,*
1
Department of Ophthalmology, Duke Eye Center, Duke University, Durham, NC 27710
Department of Biomedical Engineering, Duke University, Durham, NC 27710
3
Department of Cell Biology, Duke University, Durham, NC 27710
2
*
Corresponding authors: Mikael Klingeborn, Duke Eye Center, DUMC Box 3802, AERI Rm
5040, Durham, NC 27710; Phone: 919-668-0649, E-mail: [email protected];
Catherine Bowes Rickman, Duke Eye Center, DUMC Box 3802, AERI Rm 5010, Durham, NC
27710; Phone: 919-668-0648, E-mail: [email protected].
Supplemental Tables S1 and S2. Excel files containing lists of all the proteins identified in
two separate apical exosome preparations with pure-to crude ratio values and abundance
values. Proteins sorted according to enrichment into the exosome preparation are shown in the
tab “Exosome enrichment” and proteins sorted according to relative abundance in the exosome
preparation are shown in the tab “Exosome abundance”.
Supplemental Tables S3 and S4. Excel files containing lists of all the proteins identified in
two separate basolateral exosome preparations with pure-to crude ratio values and
abundance values. Proteins sorted according to enrichment into the exosome preparation are
shown in the tab “Exosome enrichment” and proteins sorted according to relative abundance in
the exosome preparation are shown in the tab “Exosome abundance”.
Supplemental Table S5. Excel file containing the 80 most enriched proteins identified in
apical and basolateral exosome preparations including measures of the coefficients of
variation of the data. Proteins sorted according to the aggregate enrichment in the two datasets
for apical and basolateral exosome preparations are shown with the standard variation and
coefficient of variation for each protein in the two datasets.
Supplemental Tables S6 and S7. Excel files containing lists of all the proteins identified in
apical (S6) and basolateral (S7) dense EV preparations with pure-to crude ratio values and
abundance values. Proteins sorted according to enrichment into the dEV preparation are shown
in the tab “Enrichment” and proteins sorted according to relative abundance in the dEV
preparation are shown in the tab “Abundance”.
Supplemental Table S8. Proteins enriched in apically released dense EVs. The most enriched
proteins in the purest apical RPE-derived dEV preparation (fraction 9) identified by protein
correlation profiling (PCP) in comparison to a crude apical EV preparation. Proteins were
normalized to the abundance of COL18A1, and sorted by fraction 9-to-crude ratio in descending
order. Proteins co-enriched within a three-fold of COL18A1 identified by at least two unique
peptides are shown.
Enrichment ranking
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Protein name
Prolyl 4-Hydroxylase Subunit Beta
Collagen Type XVIII Alpha 1 Chain
Pyruvate Dehydrogenase (Lipoamide) Beta
Collagen Type XIV Alpha 1 Chain
Galectin 3 Binding Protein
Angiopoietin-like protein 7
Ferritin
Heat Shock Protein 90 Beta Family Member 1 (GP96)
Glycogen synthase 1
Heat Shock Protein Family A (Hsp70) Member 9
Ribophorin I
Tenascin
Malate dehydrogenase
Isocitrate Dehydrogenase (NADP(+)) 2, Mitochondrial
ADAMTS Like 5
Collagen Type I Alpha 2 Chain
Atlastin GTPase 3
Slit Guidance Ligand 3
ATRX, Chromatin Remodeler
Collagen Type V Alpha 1 Chain
Nucleolin
Collagen Type I Alpha 1 Chain
Major Vault Protein
Slit Guidance Ligand 2
Heparan Sulfate Proteoglycan 2 (Perlecan)
Fibronectin
Nidogen 2
Collagen Type XI Alpha 1 Chain
Gene name
P4HB
COL18A1
PDHB
COL14A1
LGALS3BP
ANGPTL7
FTL
HSP90B1
GYS1
HSPA9
RPN1
TNC
MDH2
IDH2
ADAMTSL5
COL1A2
ATL3
SLIT3
ATRX
COL5A1
NCL
COL1A1
MVP
SLIT2
HSPG2
FN1
NID2
COL11A1
Pure/Crude*
1.14
1.00
0.95
0.94
0.82
0.75
0.69
0.67
0.66
0.60
0.59
0.57
0.56
0.54
0.53
0.52
0.48
0.47
0.46
0.44
0.42
0.41
0.40
0.37
0.35
0.34
0.33
0.33
*Ratio of the relative abundance in the dEV preparation to the relative abundance in the crude
EV preparation, normalized to COL18A1.
Supplemental Table S9. Proteins enriched in basolaterally released dense EVs. The most enriched
proteins in the purest basolateral RPE-derived dEV preparation (fraction 9) identified by protein
correlation profiling (PCP) in comparison to a crude basolateral EV preparation. Proteins were
normalized to the abundance of COL18A1, and sorted by fraction 9-to-crude ratio in descending order.
Proteins co-enriched within a three-fold of COL18A1 identified by at least two unique peptides are
shown.
Enrichment ranking*
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Protein name
C-type lectin domain family 18 member A
Retinol Dehydrogenase 5
Annexin A4
ATP synthase subunit alpha, mitochondrial
Collagen Type XVIII Alpha 1 Chain
Complement C3
ADAMTS Like 5
ATP synthase subunit beta, mitochondrial
Growth Arrest Specific 6
Keratin 71
Amyloid Beta Precursor Protein
Actinin Alpha 4
Acyl-CoA Synthetase Short-Chain Family Member 3
Alpha-2-Macroglobulin
Ornithine carbamoyltransferase, mitochondrial
Keratin 25
Aldehyde dehydrogenase family 1 subfamily A3
Tubulin Alpha 1c
Myosin Heavy Chain 9
Sodium/potassium-transporting ATPase subunit beta
Cardiac muscle alpha actin 1
Solute carrier family 25 member 31
Phosphoglucomutase 1
Glutaryl-CoA dehydrogenase, mitochondrial
Propionyl-CoA carboxylase alpha chain, mitochondrial
Thrombospondin 3
Collagen Type V Alpha 1 Chain
MHC class I antigen
Arginase-1
Lactate Dehydrogenase A
Heparan Sulfate Proteoglycan 2 (Perlecan)
Glycogen phosphorylase, liver form
Phosphoglycerate kinase 1
Isocitrate Dehydrogenase (NADP(+)) 2, Mitochondrial
Aldehyde Dehydrogenase 1 Family Member A1
Isocitrate dehydrogenase [NADP]
Argininosuccinate Lyase
Aldehyde Dehydrogenase 7 Family Member A1
Aldehyde dehydrogenase family 8 member A1
Glutamate dehydrogenase 1, mitochondrial
Epoxide hydrolase 2
Actin Gamma 1
Hemoglobin subunit alpha
HtrA Serine Peptidase 1
Ketohexokinase
2,4-Dienoyl-CoA Reductase 1, Mitochondrial
Serpin Family E Member 2
Centrosomal protein 250kDa
Annexin A5
Tubulin Beta 4B Class IVb
Betaine--Homocysteine S-Methyltransferase
Acetyl-CoA Acyltransferase 1
Short-chain-specific acyl-CoA dehydrogenase, mitochondrial
Gene name
CLEC18A
RDH5
ANXA4
ATP5A1
COL18A1
C3
ADAMTSL5
ATP5B
GAS6
KRT71
APP
ACTN4
ACSS3
A2M
OTC
KRT25
ALDH1A3
TUBA1C
MYH9
ATP1B1
ACTC1
SLC25A31
PGM1
GCDH
PCCA
THBS3
COL5A1
SLA-3
ARG1
LDHA
HSPG2
PYGL
PGK1
IDH2
ALDH1A1
IDH1
ASL
ALDH7A1
ALDH8A1
GLUD1
EPHX2
ACTG1
HBA1
HTRA1
KHK
DECR
SERPINE2
CEP250
ANXA5
TUBB4B
BHMT
ACAA1
ACADS
Pure/Crude†
1.92
1.72
1.07
1.00
1.00
0.91
0.86
0.84
0.79
0.76
0.75
0.69
0.68
0.67
0.66
0.64
0.64
0.62
0.59
0.56
0.56
0.56
0.54
0.52
0.52
0.52
0.51
0.51
0.51
0.50
0.49
0.49
0.48
0.48
0.47
0.47
0.47
0.47
0.46
0.46
0.45
0.45
0.44
0.44
0.44
0.43
0.42
0.42
0.42
0.41
0.41
0.41
0.40
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
Annexin A6
Heat Shock Protein Family A (Hsp70) Member 5
Mitochondrial aldehyde dehydrogenase 2
Beta-Ureidopropionase 1
Elongation factor 1-alpha
Aldehyde Oxidase 1
Carbamoyl-Phosphate Synthase 1
Heat Shock Protein 90 Alpha Family Class B Member 1
Dihydropyrimidinase
Fumarylacetoacetate Hydrolase
UDP-glucose 6-dehydrogenase
Collagen Type VIII Alpha 2 Chain
Integrin beta 1
Transferrin
Collagen Type VIII Alpha 1 Chain
ATPase Na+/K+ Transporting Subunit Alpha 1
Ubiquitin B
ATPase H+/K+ Transporting Non-Gastric Alpha2 Subunit
Fibulin 2
ANXA6
HSPA5
ALDH2
UPB1
EEF1A
AOX1
CPS1
HSP90AB1
DPYS
FAH
UGDH
COL8A2
ITGB1
TF
COL8A1
ATP1A1
UBB
ATP12A
FBLN2
0.40
0.40
0.40
0.40
0.39
0.39
0.39
0.39
0.39
0.38
0.38
0.37
0.37
0.37
0.35
0.35
0.34
0.34
0.34
*Fibronectin was the most enriched protein in the basolateral fraction 9 preparation. However,
the enrichment ratio appeared to be abnormally high (see Table S10), which prompted us to not
exclude it from this table.
†
Ratio of the relative abundance in the dEV preparation to the relative abundance in the crude
EV preparation, normalized to COL18A1.
Supplemental Table S10. Bidirectionally released proteins found among the 50 most
enriched proteins in apical and basolateral dEV preparations (fraction 9) from polarized
RPE monolayers. Proteins are shown in order of the average of enrichment in apical and
basolateral dEV preparations.
Protein name
Collagen Type XVIII Alpha 1 Chain
ADAMTS Like 5
Fibronectin
Collagen Type V Alpha 1 Chain
Isocitrate Dehydrogenase (NADP(+)) 2, Mitochondrial
Heparan Sulfate Proteoglycan 2 (Perlecan)
Gene name
COL18A1
ADAMTSL5
FN1
COL5A1
IDH2
HSPG2
Enrichment ranking*
Apical Basolateral
2
6
15
8
26
1
20
28
14
35
25
32
*Enrichment ranking in the dEV preparation by descending fraction 9-to-crude ratio of the
relative abundance of each protein.
Supplemental Table S11. Excel file containing all proteins identified by mass spectrometry
in lysate of transwell cultures of differentiated RPE cells. Proteins sorted according to number
of unique peptides identified for each protein. Proteins uniquely expressed by differentiated RPE
cells are highlighted in yellow.
Supplemental Figure S1. Morphology of primary porcine RPE (pRPE) monolayers grown
on inserts of varying pore size. pRPE cultures grown on cell culture inserts with 0.4 (a-b), 1.0
(c-d) and 3.0 µm (e-f). Phalloidin (red) stains F-actin along cell borders, displaying the
hexagonal cell shape. The widespread cytosolic staining for the RPE-specific marker RPE65
(green) in cultures grown on 0.4 µm pore inserts indicates highly differentiated RPE cells, see
panel a. Antibody to RPE65 was omitted in panels b, d and f. In RPE monolayers grown on 1.0
and 3.0 m filters, phalloidin staining shows actin stress fibers (panels d & f), and lower or
lacking RPE65 staining indicates non-differentiated cells (c and e). Nuclear Hoechst staining is
in blue. Scale bars are 50 µm.
Supplemental Figure S2. Primary pRPE cultures grown on cell culture inserts with 0.4 µm
pore size. (a) Staining of F-actin with phalloidin (red) along cell borders display hexagonal cell
shape and staining for the tight junction-associated protein ZO-1 (green) overlaps with the
phalloidin staining and appears yellow. The antibody against ZO-1 was omitted in (b). (c)
Widespread cytosolic staining for the epithelial cell-specific marker cytokeratin (green) indicates
that these cells are epithelial cells. Antibody to cytokeratin was omitted in (d). Nuclear Hoechst
staining is in blue. Scale bars are 50 µm.
Supplemental Figure S3. Confocal immunofluoresecence staining in porcine RPE-choroid
punches (dia=6mm) (a and c), and porcine RPE transwell cultures (b and d), stained for Na+/K+ATPase alpha (green) and ZO-1 (orange) in a and b; Bestrophin-1 (green) and CD36 (red) in c
and d. In each panel the leftmost image is a merge of the separate channels shown to the right.
En face (x-y) views of optical section (z slice) close to the apical (a and b) and basal (c and d)
RPE plasma membrane are shown, respectively. (a-d) Location of the optical section is indicated
with a yellow line in the x-z view (lower panels). Location of the x-z view (z stack) is in turn
indicated with a yellow line on the en face view (upper panels). (a and b) Na+/K+-ATPase alpha
staining is apical in both porcine RPE-choroid and porcine RPE transwell cultures. (c and d) The
majority of Bestrophin-1 staining is localized to the basal membrane. In addition, some lateral
(side) Bestrophin-1 staining localized along cell borders (en face view in c and d) accounted for
the majority of the apparent staining in the apical portion of the x-z view (lower panels in c and
d). CD36 is found localized both apically and basolaterally. Z-stack is composed of 33 and 31
(a-b) or 33 (c-d) optical sections spaced 0.25 µm apart and imaged using a 100X oil immersion
objective. Ap = Apical, Bl = Basal.
Supplemental Figure S4. Effects of cell culture insert pore size on barrier function and
ultrastructure of pRPE cultures. (a) TEER was measured in 3.5-wk-old primary pRPE
monolayers grown on cell culture inserts with different pore sizes. SEM is indicated, three to
fourteen replicates per pore size. Dashed line indicates representative transepithelial electrical
resistance (TEER) (43±5 Ωcm2) in ARPE-19 cultures on 0.4 µm cell culture inserts1. *** p <
0.001. (b-c) Electron micrographs of porcine RPE cells grown on 1.0 µm pore cell culture insert
(b) Area close to the basolateral membrane is shown. Asterisks indicate multivesicular
endosomes (MVEs) containing vesicles with sizes corresponding to exosomes. Inset shows one
of the MVEs at higher magnification where the individual vesicles can be seen more clearly. (c)
Apical microvilli and basal infoldings (inset) can clearly be seen, indicating terminal
differentiation of RPE cells in these cultures. Inset shows several basal infoldings at higher
magnification of the area in the lower right corner of the image. Scale bars are 500 nm (b) and 1
µm (c), respectively.
Supplemental Figure S5. Size distribution of EVs released from polarized RPE monolayers.
(a) EVs released apically from polarized RPE grown on cell culture inserts with 0.4 µm (red and
cyan traces) and 1.0 µm pore sizes (orange and light cyan traces). EVs released under B-27- (red
and orange) and FBS-supplemented culture conditions (cyan and light cyan) are indicated. Note
that the modal particle size is very similar between all of the apically released EVs. (b) EVs
released basolaterally from polarized RPE grown on cell culture inserts with 0.4 µm (black and
green traces) and 1.0 µm pore sizes (gray and light green traces). EVs released under B-27(black and gray) and FBS-supplemented culture conditions (green and light green) are indicated.
Note that the modal particle size is very similar between all of the basolaterally released EVs.
Size distributions displayed are averages of three or more separate experiments.
References
1
Ablonczy, Z. et al. Human retinal pigment epithelium cells as functional models for the RPE in
vivo. Invest Ophthalmol Vis Sci 52, 8614-8620, doi:10.1167/iovs.11-8021 (2011).