Science - USA (2019-01-04)

Awasthiet al.,Science 363 , eaav1483 (2019) 4 January 2019 3of14

intensity (%)

time (s)

NH 4 Cl

NMDA

intensity (%)

AMPA

time (s)

NH 4 Cl

intensity (%)

0 Ca2+

NMDA NH 4 Cl

2mM Ca2+

intensity (%)

time (s)

0 Ca2+ 2 mM Ca2+

AMPA NH^4 Cl

A

0.0

0.5

1.0

2.0

internal/ surface Syt3

ctrl

AMPANMDA

**

*

1.5

internalized GFP-Syt3

control

AMPA

NMDA

C surface GFP-Syt3 merge

GFP-Syt3

CLC-DsRed

Homer-GFP GFP-Syt3

CLC-DsRed Homer-myc

D Homer-GFP / CLC-DsRed GFP-Syt3 / CLC-DsRed GFP-Syt3 / Homer-myc

GluA2 C-tail

EFCYFSRAEAKRMKVAKNPQNINPSSSQNSQNFATYKEGYNVYGIESVKI

AP-2 NSF BRAG2 GRIP and PICK1

inputSyt3 +

GRIP

AP-2

GluN2A

GluA2

Syt3 –

GluA1

GluA3

GluN1

GABAARα 1

Pick1

BRAG2

E F

G

0 s 100 s 300 s 0 s 100 s 300 s

B

GluA2

BRAG2

inputneg. ctr

l.

EG

TA

0 Ca

2+

10 μM Ca

2+

100 μM Ca

2+

1000 μM Ca

2+

AP2

140

120

100

80

60

40

20

0

140

120

100

80

60

40

20

0 100 200 300

0 50 100

140

120

100

80

60

40

20

(^00100200300)
140
120
100
80
60
40
20
time (s)
0 50 100
834 883
—
inputneg

. ctr

l.

100 μM Ca

2+

GluA2

Tat-GluA2-3Y: – +

H

Fig. 2. Syt3 endocytoses in response to stimulation and binds GluA2,
AP-2, and BRAG2.(A) pHluorin-Syt3 endocytoses in response to
stimulation with AMPA or NMDA (B) in 2 mM calcium (left), but not in the
absence of calcium (right) [n; ROI/neurons/cultures, AMPA 2 mM Ca2+37/
7/3 (left); 0 and 2 mM Ca2+42/6/3 (right); NMDA, 2 mM Ca2+35/3/3
(left); 0 and 2 mM Ca2+40/3/3 (right)]. NH 4 Cl dequenches internalized
pHluorin-Syt3 fluorescence. Scale bars, 5mm. (C) GFP-Syt3 expressed
in Syt3 knockout hippocampal neurons internalizes in response to
stimulation with AMPA and NMDA (n= 28, 25, and 26 neurons per 2
cultures for control, AMPA, and NMDA, respectively; one-way ANOVA,
Tu ke y’s test). (D) GFP-Syt3 colocalizes with CLC (clathrin light chain)–
Dsred at postsynaptic endocytic zones, and not with Homer-myc
at PSDs (n; ROI/neurons, 34/20 Homer-GFP/CLC-DsRed; 29/19

GFP-Syt3/CLC-Dsred; 34/18 GFP-Syt3/Homer-myc from three cultures).

Scale bars, 5mm. (E) Recombinant Syt3 C2AB pulls down GluA2, AP2, and

BRAG2 from brain homogenates. Syt3–is beads only. (F). Binding sites,

on the GluA2 C-terminal tail, of proteins implicated in receptor recycling

and tested in pull downs. (G) Calcium-dependence of Syt3 C2AB pull

down of GluA2, AP2, and BRAG2 from brain homogenate. In 0 Ca2+

conditions, brains were homogenized and solubilized in calcium-free

buffer, but endogenous buffered calcium in internal stores remains.

Addition of EGTA eliminates any potential effects of these additional

sources of calcium. (H) Western blot of GluA2 pulled down from brain

homogenate by Syt3 C2AB in 100mM calcium with or without

preincubation with 1mM Tat-GluA2-3Y peptide. Negative controls are

beads only.

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