Supplementary materials

Supplementary information

Exocyst Dynamics During Vesicle Tethering and Fusion Syed Mukhtar Ahmed1*, Hisayo Nishida-Fukuda1,2,3,§, Yuchong Li4,5, W. Hayes McDonald6, Claudiu Gradinaru4,5 and Ian G. Macara1* Supplementary documents include: 7 supporting and additional data figures, 1 figure showing original immunoblots, and 1 supporting table showing peptide counts for proteins identified by mass spectrometry data shown in Fig 1E, and a supporting table of reagents described in methods.

______________________________________ 1Department

of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, 37240, USA, 2Department of Biochemistry and Molecular Genetics and 3Department of Hepato-Biliary-Pancreatic and 3Breast Surgery, Ehime University Graduate School of Medicine, Japan, 4Department of Physics, University of Toronto, Toronto, ON, M5S 1A7, Canada, 5Department of Chemical & Physical Sciences, University of Toronto Mississauga, Mississauga, ON, L5L 1C6, Canada, 6Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37240, USA. § Current address: Department of Genome Editing, Institute of Biomedical Sciences, Kansai, Medical University, Japan *Corresponding author

a

SEC3 (EXOC1)

Exon18

Exon19

SEC5 (EXOC2)

Exon20

Exon29

Exon28

SEC8 (EXOC4)

Exon18

non CDR

CDR CDR

non CDR

CDR

5’ -GCACAGTCCCACTAA-(42bp)- CCAGCAAACACTACGGAAAGAGTGAGCAAGT-3’ stop

non CDR

5’ -AACCACTGTGTGA-(33 bp)-CCAGTATGCACGGCCACAGACTCTAGCTGCTCA -3’

stop

5’ -TGAAAACATAGGCTCTGCAAGAAGAGCATCAGACATGGCGGTGGAGACTGT-3’ PAM stop

PAM

PAM

5’ -AAAATAACCACTGTGGGTACCGTGAGC******AAGTAAGAATTCTAGCTGCTCAGCATGG-3’ ATCGCACAGTCCCACGGTACCGTGAGC******AAGTAACTGCAGGAGCAAGTGCTCTGA

5’ homology arm (1 kbp)

sfGFP (717 bp)


AGCAGTCATGAAAACAGGTACCGTGAGC******AAGTAAGAATTCAGACTGTCCTCGTGCT



sfGFP (717 bp)

sfGFP (717 bp)



LITMUS 29 LITMUS 29

k SEC6 (EXOC3)

Exon11

EXO70 (EXOC7)

Exon12

Exon17

Exon16

Exon18

Mr(K) 150 CDR

non CDR

CDR

5’ -TCCTTAAGTAGCCCTGTCCTCCCTGCTGCCCTATGTGTCTTTACTGTGTGGC-3’ stop

IP: GFP-Trap WB: GFP

100

non CDR

5’-ACACCTCTGCTTGAGTCTGCCAGTGTTTCTGCCCAGTTCTGCCGGCATGCC-3’ stop PAM

PAM

SE C 5s EX fG O FP 70 sf G FP

LITMUS 29

Lysate WB: GFP

150 100

5’ -GCAAAGCTCCTTAAGTCTAGAGTGAGC******AAGTAAGGATCCGGCTTCCCTGTTGTCA-3’


sfGFP (717 bp)

5’-CCTCTGCTGGATCCGTGAGCAA***ACAAGTAACTCGAGCAGTTCTGCCGGCATGCC-3’

5’ homology arm (1.5 kbp) sfGFP (717 bp)


IP: GFP-Trap WB: SEC8

100

3’ homology arm (1.1 kbp)

Lysate WB: SEC8 IP: GFP-Trap WB: SNAP23 Lysate WB: SNAP23

100 25 LITMUS 29

b

SEC3sfGFP Clone# 1kbp Ladder

1 3 4 5

6 8

9 10 11 12 WT

LITMUS 29

c

d

SEC5sfGFP Clone# 1kbp Ladder

2

3

7

8

9

11

21

1kbp Ladder

23

22

25

26 WT

e

SEC6sfGFP Clone# WT

1kbp Ladder

1

2

5

3

6

EXO70sfGFP Clone#

2000 1500 1000

A4 (or B4)

WT

Ladder 1000

Primer M73/M74: WT: 262bp, KI: 955bp Primer M81/M82: WT: 397bp, KI: 1114bp 1kbp Ladder

1

3

4

5

6

8

9

1kbp Ladder

2

3

Primer M12/M13: WT: 1114bp, KI: 1828bp

7 8 9 11 21 23 22 26 WT

1kbp Ladder

10 11 12 WT

1

2

3

5

500

6

2000 1500

300

1000

Primer M74/M75: WT: 1164bp, KI: 1881bp 1kbp Ladder

2

3


7 8 9 11 21 23 22 26 WT

Primer M81/M84: WT: 1250bp, KI: 1967bp 1kbp Ladder

1

3

4

5

6

8

9

10 11 12 WT

f

2000 1500 1000



g

SEC8sfGFP Clone# 8D

(kDa)

12F

2G

4D

4F

A3 A4 B1 B2 B3 B4 C1 C3 C4 WT A3 A4 B1 B2 B3 B4 C1 C3 C4 WT

2000

4H

5B

1500

6F

150

Sec8

100

SEC5Halo + EXO70sfGFP Clone# 1kbp Ladder

1000

Primer M73/M76 WT: 1193bp, KI: 2075bp


150

GFP

100

Gapdh

50

Primer M81

h

i

SEC5mScarleti + EXO70sfGFP Clone# (kDa)

A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4

j SEC10sfGFP

250

3

SEC

SEC15sfGFP

5

SEC

6

SEC

10

SEC

15

SEC

WT

(kDa)

150 100

150

75

100

Blot : anti-RFP antibody

75 1kbp Ladder

1kbp Ladder

A1 B2 C3 WT

1000 500

1kbp Ladder

A1 B2 C3 WT

1500 1000


A1

B2

C3

WT

Blot: anti-GFP antibody

1500 1000



Supplementary Figure 1. Design and Validation of Gene-Edited Exocyst Cell Lines. (a) Schematic of designs for targeting vectors to insert sfGFP at the C-terminus of exocyst subunits SEC3, SEC5, SEC6 and EXO70 genes. Targeting vector deletes the STOP codon and PAM sequence. (b) Genotypes of SEC3-GFP clones using primer sets listed in Supplemental Table 2. Bottom panel shows sequencing of PCR product of the C-terminal region of clone 10. (c-e) Genotypes of SEC5-GFP, SEC6-GFP and EXO70-GFP clones as indicated. (f) Genotype of SEC5-Halo/EXO70-GFP double knock-in clones as indicated. (g) Western blot of SEC8-GFP clones using either anti-SEC8 or anti-GFP antibodies. GAPDH antibodies used to assess loading. (h) Western blot analysis and PCR genotyping of SEC5-mScarlet/EXO70-GFP double knock-in clones. SEC5-mScarlet expression was assessed using anti-RFP antibodies. (i) Confocal images of SEC10-GFP and SEC15-GFP after gene editing and FACS. White arrowheads indicated cells with successful incorporation of sfGFP. (j) Western blot analysis of SEC3-GFP, SEC5-GFP, SEC6-GFP, SEC10-GFP and SEC15-GFP cell lines using anti-GFP antibodies. WT = wild type parental cells. (k) SEC5-GFP or EXO70-GFP pulldown using GFP-Trap beads followed by immunoblot analysis to determine binding of native SEC8 (anti-SEC8 antibody) and SNAP23 (anti-SNAP23 antibodies). Pulldowns of SEC5-GFP or EXO70-GFP were assessed using anti-GFP antibodies.

Exo70: VTDYIAEK

1.0 105

1500000 400000 300000 200000

300000 200000 100000

20000

0

0

Total AUC

anti-EXO70

SEC15 peptides

75000

10000

50000 0 R

K

LM FD VF

G EW AE

LT E

RalB

Q

D

SA ET YV

FD

SL

VF

N Q

D EL

RalA

VL VF

G FE

SP

LL AV D

EI EG AA H

G

R

K D

K YT

K LE PD D

AL

0

LM

H

K

25000

FQ

Lysate WB: EXO84

0

m

Gray values (a.u)

75

100

Ctrl SEC8sh EXO70sfGFP

10000

Counts

GFP Intersection

6000 4000 2000

h 6s C SE

on tro l

sh

Rab11/SEC8

C

10 SE C

C

on tro l

0

Rab11/EXO70

300

Ctrl

Ctrl SEC8sh SEC5Halo

Rab11

8000

400

n

EXO70sgRNA SEC8sfGFP

Exocyst colocalization with Rab11 1.0

p = 0.04

n.s.

0.8 0.6 0.4 0.2 0.0 on tro l SE C 10 sh C on tro l SE C 6s h

EXO70

200

C

IP: GFP-Trap WB: EXO84

k

p < 0.0001

Fraction colocalization

75

Intersection

100000

R

400000

Total AUC

Total AUC

600000

FP

SEC8

SEC8-GFP/Intersection

Sec8 shRNA

A3 R

100

mApple-Rab11/Intersection

Control

20000

N

A2 N

sh 0 C1

SE Lysate WB: GFP (SEC8-sfGFP)

SEC6

SEC8-GFP/mApple-Rab11

125000

300 Gray values (a.u)

150

IP: GFP-Trap WB: GFP (SEC8-sfGFP)

75

75

j

sg

A1 R sg

R

N

tro on C

Mr(K) 150

Co nt ro

l

sh l

tro

C

on

SE

SEC3sfGFP

800000

0

i

10

sh C

l

10 C

SE

tro on C

Mr (K) 150

l

GFP-Trap pulldown

30000

200000

l

TUBULIN

Input

D LP R

RALA and RALB peptides

EXO84 peptides 150000

12000 10000 8000 6000 4000 2000 0 sg

sg anti-EXO70

Normalized band intensity of EXO70

A2

A2

RN

g

RN

RN

sg

sg

Co

nt

ro

l

A1

TUBULIN

TUBULIN

Total AUC

TUBULIN

VQ

anti-SEC6

1000000

h

YI EL K

EL K

Q EI EH

LT D

FI

anti-GFP (SEC3-sfGFP)

f

400000 300000 200000 100000 0

TY Q SI

IF N

l

A

ro

RN

nt Co

sh

tro l RN A

Co n

e

sh

NA 2

d

sh R

l

NA 1

sh R

nt ro Co

c

2000000

LD G

31.0

2500000

LS D PS

30.5

Retention Time

Retention Time

3000000

N EF

30.0

6000 4000 2000 0

SEC10 peptides 3500000

YF

0.0

31.0

60000 40000 20000

Total AUC

5.0 103

K

SEC8 peptides 700000 650000 600000 550000 70000 60000 50000 40000 15000 10000 5000 0 TE R AL G LG PA K

30.5

1.0

104

SV PE PS IV LL R YL EV ST LV SK

5.0 103

30.0

SEC6 peptides

Total AUC

y6 - 751.3733+ y10 - 590.2768++

0.0

SEC8 shRNA 300000 150000

y7 - 880.4159+

104

1.0

1.5 104

Intensity

Intensity

y9 - 1108.5092+ y8 - 977.4687+

FW

H LN

SA

sfGFP: SAMPEGYVQER Control

LF EN

LA VV D

Retention Time

Retention Time

LV LS Q LP N

Q ER

K AE

28.8

G YV

28.7

M PE

28.6

VT D YI

28.5

D AK

0

1.5 104

40000

100000

0.0 28.4

0.0 28.3 28.4 28.5 28.6 28.7 28.8

Total AUC

400000

2000000

5.0 104

5.0 104

SEC5 peptides 140000 120000 100000

AR

Intensity

y4 - 460.2766+

SEC3 peptides

500000

2500000

1.5 105

y5 - 623.3399+

1.0 105

EXO70 and GFP (bait)

SEC8 shRNA

y6 - 738.3668+

1.5 105

Intensity

b

2.0 105

EG VE

Control

FF

y7 - 839.4145+

Total AUC

2.0 105

Toral AUC

a

Rab11/SEC8 Rab11/EXO70

Supplementary Figure 2. Mammalian Exocyst Subunit Connectivity. (a) EXO70-GFP capture using GFP-Trap nanobodies. Shown are representative MRM spectra of intensity peaks features of the indicated EXO70 and sfGFP peptides in the control or SEC8 shRNA treated cells. (b) Quantifications of the area under the curve (AUC) for the indicated peptides for each protein species shown. Control pull-downs are denoted in blue whereas SEC8 depleted conditions are designated in red. (c) Knockdown efficiencies of two independent shRNAs targeting SEC3-GFP. α-Tubulin used as loading control. (d) Knockdown efficiency of SEC6 shRNA. (e) Knockdown efficiency of EXO70 shRNA. (f) Knockout efficiency with three independent guide RNAs targeting EXO70 gene loci in Exon1. (g) Quantification of panel F. (h) GFP-Trap pull-down of endogenous SEC3-GFP from untreated or SEC10 shRNA treated NMuMG cells. Western blots were immunoblotted with anti-GFP to detect SEC3-GFP, or with anti-SEC6, anti-SEC8 and anti-EXO70 antibodies to assess co-precipitations of unlabeled exocyst subunits. (i) GFP-Trap pull-down of endogenous SEC8-GFP from untreated or SEC10 shRNA treated NMuMG cells. Blots were probed with anti-GFP to detect SEC8-GFP, or with anti-EXO84 antibodies to assess amount of co-precipitation of endogenous unlabeled EXO84. (j) Quantification of fluorescence intensities from TIRFM images of SEC5-Halo and EXO70-GFP double knock-in cells treated with Control or SEC8 shRNAs. SEC5-Halo was labeled with JF585 Halo ligand. (k) Fluorescence intensity quantifications of SEC8-GFP from TIRFM images in untreated or EXO70 knockout cells. (l) Example of SEC8-GFP and mApple-Rab11 coincidence. Cells were transduced with mApple-Rab11 lentivirus. Spot diameters in the range of 0.30-0.35µm was used to identify GFP and Rab11 and the fraction of the particle that are both red and green were determined using NIS Elements spot detection algorithm. Scale bar = 5µm. (m) Total number of particles analyzed for Rab11, exocyst subunits and fraction that co-localize. (n) Quantification of the fraction of exocyst subunits SEC8-GFP or EXO70-GFP that coincide with mApple-Rab11. Ordinate axis = intersection/GFP. Cells were treated with scrambled, Sec10 or Sec6 targeting hairpins. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, data points are plotted as dots. Statistical significance was assessed using one-way ANOVA followed by Scheffe’s multiple comparison tests.

a

c

Cell proliferation data

SEC6sfGFP SEC3mScarleti Mr (K) Clone # WT A1

Day0 Day 1 Day 2 Day 3

2 106 1 106

250

SEC3mScarleti SEC3

150 Blot : SEC3 100 37

2000

SEC3sfGFP Cln 10

SEC3

Blot : GAPDH

GFP-TRAP

WT

3000

SEC3mScarleti + SEC6sfGFP Cln A1

SE

SEC3GFP/ SEC3mScarleti

Knock-in Clones (kDa) 150 Blot : SEC3 100 100

SEC6GFP

1000

SEC6

fG 3s C SE

fG 8s C SE

FP

0 FP

Intensity of Sec5Halo

SEC5Halo Intensity at the PM 4000

SEC3sfGFP Cln 10

C 5H al o

+S E

fG FP

C 5H al o

Input

C 3s

fG FP

d

WT

b

+S E

C 3s SE

C 8s

fG FP

SE

SE

C 8s

fG FP

0

SEC3mScarleti + SEC6sfGFP Cln A1

Cell Counts

3 106

100

Blot : SEC5 Blot : SEC6

75 100

Blot : SEC8

75

Blot : EXO70

Supplementary Figure 3. Functionality of Exocyst Knock-in Cell-Lines. (a) Cell proliferation of SEC8-GFP, SEC3-GFP, SEC8-GFP + SEC5-Halo and SEC3-GFP + SEC5-Halo knock-in cells over three days. Error bars indicate s.d. Statistical analysis was using one-way ANOVA to compare groups for each day. (b) Comparison of SEC5-Halo fluorescence intensity at the bottom PM (PM) in double knock-in cells. Intensities were measured using TIRFM. Halo tag was labeled with JF585-HTL. (c) Western blot analysis of SEC3-Scarlet+ SEC6-GFP double knock-in cells. Clone A1 is homozygous for SEC3. WT = wild-type parental NMuMG cells. (d) GFP-Trap pull-down and western blots analysis from double knock-in cell lines expressing SEC3-GFP, or SEC3-Scarlet + SEC6-GFP. WT = wildtype cells, Cln = clone.

a

EXO70sfGFP

Akt-CAHalo-JF646

c

Merge

GFP

Halo-JF646 Substate alone

b

SEC5-Halo-JF646

# of particles analyzed

8000 6000

sfGFP Halo Intersection

4000 2000 0

FP fG EX

O

70

fG

C

3s C

SE

SE

e

8s

FP

an

d

SE

C

a 5H s EX fG nd SE alo O FP C 70 a 5H s n al SE fGF d S P EC o C 8s a 5 fG nd Ha Ak lo FP an tHa d l Ak o tH al o

d

SEC5-Halo-585 photobleaching step detections

160 140 120 30

100

40

0

10

20

30

40

Time (s)

h

SEC8sfGFP photobleaching step detections 220 200 180 160 140 120 100

#2

Intensity

200 150 10

20

30

40

0

Time (s)

f

10

20

100 150

75 75

0

10

20

30

40

Time (s)

20 10 0

g SEC8sfGFP pulldown

SEC8sfGFP

WT

SEC8sfGFP

WT

100

40

Time (s)

GFP-TRAP Input (2.4%) pulldown

Mr (K) 150

30

150

30

l

0

200

tro

100

2 steps detected

250

1-step detected

100

p = 0.01

80 SEC8 GFP (SEC8sfGFP) SEC6 EXO70

% bound

Intensity

#1

250

Intensity

0 steps detected

300

p > 0.9999

40

A

20

Time (s)

N

10

R

0

sh

40

r3

30

Pa

20

Time (s)

150

on

10

200

C

0

100

2 steps detected

250

#1 #2

# of Vesicle Fusions/min

120

180

Intensity

#1 #2

140

100

1-step detected

200

0 steps detected

160

Intensity

Intensity

180

60 40 20 0

SEC6

EXO70

Supplementary Figure 4. Fractional Interactions of Cellular Pools of Exocyst subunits. (a) AKT-Halo was expressed in EXO70-GFP cells as a negative control for interactions. Halotag was labeled with JF-646 HTL and and imaged to look at the distribution of expression prior to cell sorting. Scale bar = 20μm. (b) Gating used for cell sorting in panel c. (c) SEC3-GFP, SEC8-GFP and EXO70-GFP knock-in cells were transduced with lentivirus expressing Akt-Halo, and sorted for similar expressed levels than SEC5-Halo. Halo was labeled with JF-646 HTL. (d) Graph shows number of molecules analyzed in the data shown in Figure 5d. (e) Representative images of 0, 1 or 2 photobleaching steps detected by the step detection algorithm. (f) Representative experiment where SEC8-GFP was captured using GFP nanobodies and immunoblotted with anti-SEC8, anti-GFP, anti-SEC6 or anti-EXO70 antibodies. Blots on the left show 2.4% input of the total lysate and the blot of the right shows pulldown with nanobodies. (g) Quantification of the percentage of SEC6 and EXO70 bound to SEC8 from 3 independent experiment experiments. (h) Number of Vamp2-pHluorin fusions at the bottom of cells in control or Par3 depleted NMuMG cells assessed using TIRF microscopy. p value calculated using Student’s t-test.

b.

HaloTag

Cell by cell intensity scatter of NMuMG cells stably expressing Venus-Halo

Lentiviral expression FACS

105.5 105 GFP

NMuMG cells HaloTag labeling Cell lysis

104.5 104 103.5

Assess fraction labeled by single molecule counting

5

5

10 5.

10 5

10 4.

10 3

5

103

10 4

Venus

10 3.

a.

JF646-HTL

c.

d.

Effect of incubation time with Halo ligand on labeling efficiency 0.6

Fraction labeled

0.4 0.3 0.2 Bmax = 0.4 ± 0.014 100

200

300

0.0

400

Halo dye [nM]

e.

Incubation time

f.

Venus-Halo HTL-JF646

0.010

Cross correlation

0.005

0.8 0.6 0.4 0.2

ed

oJ

F6

Ve n

46

us

0

-1

10

10

-2

ou

pl

(s)

C

ou

pl

C

ed

-3

10

10

-4

0.0

10

-5

0.000

10

Venus-Halo labeling efficiency with HTL-JF646 using dcFCS

1.0

Venus

Fraction coupled

G( )

0.015

al

0

0.2

H

0.0

Kd = 16.93 ± 3.24 nM

0.4

18 h

0.1

p = 0.0079

1h

Fraction labeled

HaloTag labeling efficiency across different Halo-ligand-JF585 concentrations 0.5

Venus-Halo

0.04

0.02 0.01

0.6 0.4 0.2

H

al

o

co

co

up

up

le

le

d

d

0

us

(s)

10

-1

10

-2

10

-3

10

-4

0.0

10

10

-5

0.00

0.8

Ve n

G( )

1.0

HTL-JF585 Cross correlation

0.03

Venus-Halo labeling efficiency with HTL-JF585 using dcFCS

h.

Venus Fraction coupled

g.

Supplementary Figure 5. HaloTag Labeling Efficiency. (a) Schematic showing experimental design to determine Halo labeling efficiency in NMuMG cells. Halo fused to the C-terminus of of YFP was expressed in NMuMG cells using lentiviral expression system, and subsequently sorted for positive cells. (b) Correlation between fluorescence intensities of YFP and Halo labeled with JF-646-HTL. (c) Halo labeling efficiency across different concentrations (6.25, 25, 100, 150, and 400 nM) of HaloTag ligand conjugated to JF585 dye in NMuMG cells for 1.5h. (d) Halo labeling efficiency after NMuMG cells were labeled with 100nM of HaloTag ligand conjugated to JF585 for 1h or 18h. (e) Dual-color FCCS measurement of Venus-Halo labeled with 150nM HTL-JF646 for 2h in NMuMG cells. (f) Quantification of the fraction of Venus-Halo that are labeled with HTL-JF646. (g) Dual-color FCCS measurement of Venus-Halo labeled with 150nM HTL-JF585 for 2h in NMuMG cells. (h) Quantification of the fraction of Venus-Halo that are labeled with HTL-JF585.

0.04

Cross correlation

τ (s)

c

0

-1

τ (s)

SEC3sfGFP+SEC5Halo (bottom)

G (τ)

10

10

-5

0

10

-2

-3

-1

10

10

10

10

-4

0.00

-5

0.01

0.00

-2

0.02

0.05

10

SEC5Halo

10

0.10

SEC8sfGFP

-3

G (τ)

Cross correlation

0.05

10

SEC5Halo

0.15 G (τ)

SEC8sfGFP+SEC5Halo (bottom)

EXO70sfGFP

-4

0.20

b

10

EXO70sfGFP+SEC5Halo (bottom)

10

a

0.15

SEC3sfGFP

0.12

SEC5Halo Cross correlation

d Plasma membrane EXO70-SEC5 p = 0.0008

0.09

SEC8-SEC5

0.06

SEC3-SEC5

p = 0.0083

0

8

1.

6

0.

0.

4 0.

2

0. 0

10

-2

-3

-1

10

10

10

-4

10

10

-5

0.00

0.

0

0.03 Fraction bound

τ (s)

Supplementary Figure 6. Dual-Color FCCS Measurements of Exocyst Subunits near the base of the cells. (a) SEC5-Halo and EXO70-GFP, (b) SEC5-Halo and SEC8-GFP and (c) SEC5-Halo and SEC3-GFP FCS measurements near the base of the cells. (d) Statistics of fraction of GFP-tagged exocyst subunits near the bottom membrane of the cells. SEC8-GFP+SEC5-Halo: 64% ± 2.2%, SEC3-GFP+SEC5-Halo: 44% 2.6%, EXO70-GFP+SEC5-Halo: 39% ± 7.0% (mean ± s.e.m.). These numbers are probably underestimates as exocysts on the membrane are less diffusive. Also the measurements likely include both plasma membrane bound fraction as well as cytoplasmic fraction. HaloTag was labeled using JF646 Halo ligand (200 nM for 1.5h).

b

0

0.4

0.6

40

60

0

80

g

20

40

60

80

0

40

60

80

Time [s]

Pixel distance threshold used to take intensity measurements

i

h

5 pixels

0 3 6 9 12 15 18

Intensity (a.u) 0 5 10 15 20 25

pixel distance

pixel distance

j

Minimum resolvable peaks

20nm beads 50nm Z-steps

λ=488nm

0 3 6 9 12 15 18

y pixel distance

x

z y

lateral PSF, 244nm

x

z

Intensity

Intensity (a.u)

f

20

Time [s]

Intensity (a.u)

Mixed guassians from two close objects

25.68

0

Time [s]

Displacement (um)

e

20

0 20

0.8

30.23

23.52

40

SEC5-sfGFP

x 6

0.2

20

21.65

60

0.

1000 0 0.0

22.17

10

20.84

4

2000

40

0.

3000

CD86-3xsfGFP

80

60

30 20

100

2

4000

CD86-1xsfGFP

40

d CD86-2xsfGFP

80

0.

SEC3-sfGFP SEC8-sfGFP EXO70-sfGFP SEC5-Halo

p < 0.0001

Intensity

# values

5000

c

Intensity

Histogram of displacement length

Intensity

a

Distance (μm)

Supplementary Figure 7. Determination of Criteria Used to Count Exocyst Subunit Molecules from Fluorescence Intensities. (a) Distribution of vector displacements between the first and last frames of the particle trace. Data are shown from >20,000 object for each exocyst subunit as indicated. Statistical significance was measured by Kruskal-Wallis non-parametric test followed by Dunn’s post-hoc test. Photobleaching steps of (b) CD86-1xsfGFP (c) CD86-2xGFP and (d) CD86-3xsfGFP in cells fixed with 3.7% paraformaldehyde followed by methanol and immunolabeled with anti-GFP-biotin antibodies and Streptavidin-ATTO488 dye. (e) Fluorescence landscape showing point-spread function of a single tetraspec bead spread on glass. (f) Fluorescence landscape of multiple TetraSpeck beads close together. Arrows point to two close but resolvable peaks from which individual intensities could be measured. (g) 2D intensity plots of the peaks described in F. (h) Empirically determined point spread function of Apo TIRF 60X objective, 1.49NA measured using 20nm TetraSpeck beads. Data shows lateral PSF = 244nm measured at 488nm wavelength, which was used for counting molecules. Pixel size, 120nm per pixel. (i) Intensity trace shown in panel is shifted to show minimal separation in pixel distance (5 pixels) that was used to measure intensities of the objects in cells. (j) Distributions of average intensity traces over a 20nm bead (n=3) or a SEC5-GFP particle at vesicle fusion sites (n=5).

Figure 1f

Figure 1c

Figure 5f

top : GFP bottom : GAPDH Sec 5

Sec6

Sec3 and tubulin

Sec 3

Exo70 Figure 1c

Supplementary Figure 1k

Supplementary Figure 1g

Sec8

Tubulin

Sec8 Supplementary Figure 1h

Sec6 and tubulin

GFP (green), GAPDH (red)

Supplementary Figure 2h

Supplementary Figure 2c and e

SNAP23 and Sec8 Supplementary Figure 2d

RFP Supplementary Figure 1j

Top : Sec3 Bottom : Sec6

Top : Sec8 Bottom : Exo70

Supplementary Figure 2f

GFP

Supplementary Figure 2i

Supplementary Figure 3d

Sec8

Sec6

Sec3

Sec5 Exo70

Supplementary Figure 4e Supplementary Figure 3c

Sec3 GAPDH

Top : Sec8 Bottom : Exo70

Top : GFP Bottom : Sec6

Supplementary Figure 8. Original uncropped images of western blots used in the paper. Each set of immunoblots are annotated with the figure and panel numbers. Where appropriate the region that was cropped out is shown with dashed boxes.

SEC5-sfGFP

SEC3-sfGFP

EXO70-sfGFP

SEC5-sfGFP

SEC3-sfGFP

EXO70-sfGFP

SEC5-sfGFP

SEC3-sfGFP

EXO70-sfGFP

SEC5-sfGFP

SEC3-sfGFP

EXO70-sfGFP

Supplementary Table 1. LC-MS/MS analysis of exocyst subunits interactions

Unique Unique Accession # Protein Name Mol wt peptides Spectra Total Spectra % Coverage A6H5Z3 Exocyst complex component 6B (EXC6B), SEC15L 94 kDa 52 47 48 83 68 69 178 163 116 76 67 68 GFP Green fluorescent protein 27 kDa 6 7 7 9 12 13 46 35 23 32 34 34 O35250 Exocyst complex component 7 (EXOC7), EXO70 80 kDa 47 43 45 91 68 71 239 167 128 68 65 67 O35382 Exocyst complex component 4 (EXOC4), SEC8 111 kDa 53 67 63 93 126 128 237 452 391 72 81 77 Q9D4H1 Exocyst complex component 2 (EXOC2), SEC5 104 kDa 45 57 54 65 97 99 162 396 377 61 68 69 Q6KAR6 Exocyst complex component 3 (EXOC3), SEC6 86 kDa 38 52 50 64 88 95 145 472 274 61 68 69 Q8R3S6 Exocyst complex component 1 (EXOC1), SEC3 102 kDa 40 51 49 72 96 105 145 372 268 59 72 68 Q3TPX4 Exocyst complex component 5 (EXOC5), SEC10 82 kDa 39 36 35 71 56 55 192 144 94 62 65 63 Q8R313 Exocyst complex component 6 (EXOC6), SEC15 93 kDa 36 30 32 62 42 41 146 95 73 62 53 56 Q6PGF7 Exocyst complex component 8 (EXOC8), EXO84 81 kDa 31 24 27 44 27 27 77 55 49 55 41 45 P63321 RALA 24 kDa 4 4 4 5 4 6 6 5 7 50 40 50 Q9JIW9 RALB 23 kDa 9 6 9 12 6 13 14 7 19 47 33 48 Q8R361 Rab11 family-interacting protein 5 (RFIP5) 70 kDa 5 0 3 5 0 3 5 0 3 10 0 6.2 Q3UPH7 Rho guanine nucleotide exchange factor 40 (ARH40) 165 kDa 7 14 7 7 14 7 7 16 7 5.5 12 5.6 Q91YM2 Rho GTPase-activating protein 35 170 kDa 0 6 0 0 6 0 0 6 0 0 6.3 0 Q60875 Rho guanine nucleotide exchange factor 2(ARHG2) 112 kDa 1 2 0 1 2 0 1 2 0 2 2.2 0 Q9Z0U1 Tight junction protein ZO-2 131 kDa 2 18 13 2 18 13 2 25 14 2.1 17 13 Q811D0 Disks large homolog 1 (DLG1) 100 kDa 12 26 25 12 30 27 14 48 36 20 44 42 O09044 Synaptosomal-associated protein 23 (SNAP23) 23 kDa 6 6 6 - 39 Q8BVD5 MAGUK p55 subfamily member 7 (MPP7) 66 kDa 1 6 8 1 6 8 1 7 9 2.1 15 18 Mass spectrometry analysis of tryptic peptides fragments upon GFP-TRAP pull-down of EXO70-sfGFP, SEC3-sfGFP and SEC5-sfGFP from CRISPR knockin NMuMg cell lines. Only peptides determined with > 99%, and protein determined with >95% probability are shown.

Supplementary Table 2. Peptides and primers sequences 1. Peptides used in MRM-MS Uniprot Protein Name ID Q8R3S SEC3_MOUS 6 E

M15

SEC6

Reverse

DLAVVDAK

M73

SEC5

Forward

M74

SEC5

Reverse

ATGGGCATCTTTCTGCACCA CC

M75

SEC5

Forward

AGAATTCCATAGGCACTGGC TT

NIFSVPEIVR

M76

SEC5

Reverse

ACAGGGTACATATGCTGTGC TC

LTDPSLLYLEVSTLV SK TYQSITER

M81

SEC3

Forward

M82

SEC3

Reverse

CAGCCGCTCGAAAACACAA G

YFNEFLDGELK

M83

SEC3

Forward

CTGAGGGCCGAGACATGGA G

LSDPSDLPR

M84

SEC3

Reverse

CAATGAGCTAATGGGCAGC C

E21

EXO70

Forward

E22

EXO70

Reverse

LNHFFEGVEAR Q9D4H 1

SEC5_MOUS E

LVLSQLPNFWK LFENYIELK

Q6KAR 6 O35382

SEC6_MOUS E SEC8_MOUS E

FIQEIEHALGLGPAK Q3TPX 4 Q8R31 3

SEC10_MOUS E SEC15_MOUS E

FPFQDPDLEK HAAEGEIYTK

O35250

EXO70_MOUS E

HDFSTVLTVFPILR VTDYIAEK

Q6PGF 7

EXO84_MOUS E

GTTCATGCTGCCTTCTCTCC

Peptide Sequences

DFEGAVDLLDK

3.

AGCCCGTCACTGTCAGGATG

GGTGGTGTGGCACTCCATGC

GGTATGGCAGCGTGCCCTTC GGGGCTCCTGGTTTGGGCA C

sgRNA sequences to generate knock-ins

Subunits

sgRNA sequences (5’ -> 3’) PAM

SEC3

ACTCTTTCCGTAGTGTTTGCTGG

SEC5

AAGAGCATCAGACATGGCGGTGG

SEC6

CCTATGTGTCTTTACTGTGTGG

SEC8

GAGTCTGTGGCCGTGCATACTGG

SEC10

TCTCAGAAGGCCTCGGTAATGGG

SEC15

TATTGTCTCACGACCAGTGCTGG

EXO70

GTGTTTCTGCCCAGTTCTGCCGG

QLTEVLVFELSPDR 2.

Primers for genotyping

Prime r#

M12

Exocys t Subuni t SEC6

Primer directio n

Sequence (5’ -> 3’)

Forward

CTGAACGTGGCAAAGCTCCT TA GCAGACACAATCCACTCACT GG

M13

SEC6

Reverse

M14

SEC6

Forward

GCCTCTGGACATGGCTGTAT