神経系における細胞骨格関連タンパク質エズリンの機能に関する研究

Novel functions of cytoskeletal protein ezrin in nervous system

2015

3

March, 2015

Doctoral Program In Advanced Life Sciences

Graduate School of Life Sciences

Ritsumeikan University

MATSUMOTO Yosuke

1 Dotti et al., 1988; Arimura and Kaibuchi, 2007 1-1 º 1 º 2 1 3 4/5

Rho GTPase RhoA Rac1 Cdc42

1-2 Govek et al., 2005

Dent and Getler, 2003; Gupton and Gertler, 2010 Rho GDP GTP

Rho

Rac1 Cdc42 p21-activated kinase PAK

Hayashi et al., 2007 RhoA

Rho kinase ROCK Redmond and Ghosh, 2001

II myosin light chain 2 MLC2

ROCK

Kollins et al., 2009 Rho

Schmidt and Hall, 2002; Tcherkezian and Lamarche-Vane,

2007 Rho guanine nucleotide exchange factor RhoGEF Rho

GDP GTP 1-3A Rho

GTPase-activating protein RhoGAP Rho GTPase GTP

GDP Rho 1-3B Rho guanine

nucleotide dissociation inhibitor RhoGDI Rho GDP

º Ezrin/Radixin/Moesin ERM

Tsukita and Yonemura, 1999 1-4 N

four-point-one, ezrin, radixin, moesin FERM C

II nf2 ERM FERM º 85% 84% 61% ERM ERM º ERM N C

phosphatidylinositol 4,5-bisphosphate PIP2

1-5

3T3

lysophosphatidic acid LPA ROCK C

564

FERM Matsui et al., 1998

RhoA/ROCK C

RhoA N FERM RhoGDI

Takahashi et al., 1997 RhoGDI

RhoA GDP RhoA

Vil2-/- Casaletto et al., 2011

LLC-PK1

Speck et al., 2003 RhoA

RhoA

RhoA/ROCK RhoA

ERM

13H9 ERM

ERM

Goslin et al., 1989; Everett and Nichol, 1990 º

1 Vil2kd/kd Tamura et al., 2005 Vil2+/+ Vil2kd/kd Vil2kd/kd Vil2kd/kd Vil2+/+ Vil2+/+ Vil2kd/kd RhoA 3 RhoA/ROCK MLC2 Rac1 Cdc42 ROCK II Vil2+/+ Vil2kd/kd 8 Vil2kd/kd V Vil2+/+ V RhoA/ROCK/MLC2

2 ERM

lrrk2 leucine-rich

repeat kinase 2 LRRK2 Jaleel et al., 2007: Parisiadou et al., 2009

6-hydroxydopamine 6-OHDA 6-OHDA

Vil2kd/kd Tamura et al., 2005 Vil2kd/kd C57BL/6J Jcl ICR 12 BKC Vil2kd/kd 2 3 1-6 Tamura et al., 2005 5% 10% Tamura et al., 2005

Hatano et al., 2013 Hatano et al., 2014

3 mm 50 mM 10

95ºC 1 M Tris-HCl pH 8.0

10 12,000 rpm PCR PCR

PCR 380 bp

290 bp PCR

Vil2+/+: Vil2+/kd: Vil2kd/kd = 1:2:1

15.5 HBSS

0.25% trypsin/EDTA Invitrogen 20 37ºC

Viesselmann et al., 2011 10% FBS Opti-MEM

Invitrogen 15

0.1 mg/ml poly-D-lysine 60 mm

24 B27 Invitrogen GlutaMAX Invitrogen 0.3% glucose

37.5 mM NaCl Neurobasal Invitrogen 37ºC

CO2 5% 40 µM Y-27632 Wako 50 µM blebbistatin Wako 10 mM pH 7.4 4% º 4% º 2 15% 100 mM 20 µm 0.3% TritonX-100 100 mM

4 C

2 4 4 C

IgG Vector IgG Vector

2 VECTASTIN ABC Vector

0.02% DAB 0.3% ( )

0.0045% 50 mM Tris-HCl pH 7.6

º º

FD Rapid GolgiStain Kit FD NeuroTechnologies

250 µm º º

Axioplan II, Carl Zeiss

º º 95% 15 70% 1

50% 1 1 2 0.5%

2 1 50% 1

4% 4% º 10 4ºC

0.1% Triton X-100 10 1%

BSA 30 2

4ºC fluorescein isothiocyanate FITC IgG

Jackson ImmunoResearch Alexa Fluor 633 IgG Vector

45 rhodamine phalloidin Invitrogen

FV-1000D FV-10i Olympus

Hirai et al., 2011

10 µm 1 40 µm 2 40 µm

3 2

Shelly et al., 2007 neuronal class III β-tubulin

ImageJ NeuronJ º

protease inhibitors Nacalai Tesque lysis buffer 5 mM Tris-HCl, pH 7.4, 250 mM sucrose

20 4ºC 17,500×g

protease inhibitors Cell BioLabs

phosphatase inhibitors Nacalai Tesque RIPA buffer 25 mM Tris-HCl pH 7.6, 150

mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS 10 4ºC

10 14,000×g SDS 50 mM Tris-HCl, pH 6.8, 2% SDS, 2% 2-mercaptoethanol, 20% glycerol, 0.01% BPB 5 95ºC SDS-PAGE PVDF 5% 60 2 4ºC HRP IgG Millipore HRP

IgG Millipore 1 Immobilon Western

Chemiluminescent HRP Substrate Millipore LAS-3000 Fujifilm

ImageJ

Rho

lysis buffer 25 mM HEPES, pH 7.5, 150

GST-PAK-PBD Cell BioLabs 1 4ºC

RhoA Rac1 Cdc42 lysis buffer

3 SDS 5 95ºC

SDS-PAGE PVDF RhoA Rac1 Cdc42

6 1 cm bregma 0.7 mm 0.8 mm 2.7 mm Uhobby 0.02% 6-OHDA 10 µg 2 µl 5 6-OHDA 6 18 2 6-OHDA 5 3 100 cm º 10 cm º 2 cm 24 C º 15

15 º º 10 1 3 I II III IV 4 I 1 cm º I º 10 cm 4 60 º

SMART º Bio Research Center

º 10 º 1 2 7 º 60 º º I % 2

HPLC

EDTA 50 mg/l 3,4-dihydroxybenzylamine hydrobromide

0.1 N 15 4ºC 15,000×g

Vil2+/+ Vil2+/+ 2-1 1 3 Vil2kd/kd Vil2kd/kd 1 Vil2kd/kd 2-2A Vil2+/+ 80 kDa 2-2B Vil2kd/kd Vil2+/+ 10 µg 1 µg 0.5 µg Vil2kd/kd Vil2kd/kd 5%

Vil2kd/kd

neuronal class III β-tubulin

rhodamine phalloidin 2-3A,B

48 Vil2+/+ Vil2kd/kd 2-3C Vil2kd/kd 1 Vil2+/+: 8.8 ± 1.4% Vil2kd/kd: 17.3 ± 2.3% 3 Vil2+/+: 55.8 ± 4.2% Vil2kd/kd: 43.1 ± 4.1% 3 Vil2kd/kd Vil2+/+: 2.7 ± 0.2 Vil2kd/kd: 1.5 ± 0.2 2-3D 2-3E,F 96 2-4A,B Vil2+/+ 48 Vil2kd/kd Vil2+/+: 4.2 ± 0.6 Vil2kd/kd: 1.4 ± 0.2 2-4C 2-4D,E Vil2kd/kd RhoA

GTPase RhoA Rac1 Cdc42

Govek et al., 2005; Negishi and Katoh, 2002

GTP RhoA Vil2kd/kd

RhoA Vil2+/+ 3

2-5A,D Rac1 2-5B,E Cdc42 2-5C,F

Vil2+/+ Vil2kd/kd RhoA RhoA Vil2kd/kd MLC2 2-6A,B MLC2 ROCK º

2-6C,D Vil2+/+ Vil2kd/kd ROCK

Y-27632 MLC2

2-7A,B ERM 2-7C-E

MLC2 ROCK º Vil2kd/kd ROCK II Vil2kd/kd RhoA RhoA ROCK Y-27632 Bito et al., 2000; Ishizaki et al., 2000; Da Silva et al., 2003; Peris et al., 2012 Y-27632

2-8A-D

DMSO Vil2kd/kd Vil2+/+

2-8E Vil2+/+ Vil2kd/kd Y-27632

DMSO Y-27632

DMSO 2-8E Y-27632

Vil2+/+ Vil2kd/kd

Y-27632-treated Vil2+/+: 4.0 ± 0.3, Y-27632-treated Vil2kd/kd: 3.4 ± 0.3 2-8E

Y-27632 2-8F,G

Vil2kd/kd ROCK

II blebbistatin 2-9A,B

Y-27632 Vil2+/+ Vil2kd/kd blebbistatin

DMSO blebbistatin DMSO

2-9C blebbistatin

Vil2+/+ Vil2kd/kd

blebbistatin-treated Vil2+/+: 7.7 ± 0.8, blebbistatin -treated Vil2kd/kd: 8.4 ± 1.0 2-9C ROCK

2-9D,E RhoA/ROCK/MLC2

Vil2kd/kd Vil2kd/kd Vil2kd/kd 2-11 Vil2kd/kd 2-12A Vil2kd/kd 2-12B Vil2+/+: 368.0 ± 16.1 µm, Vil2kd/kd: 299.3 ± 4.5 µm 2-12C Vil2kd/kd V

neuronal class III β-tubulin 2-13A microtubule associated proteins 2 MAP2

2-13B 2-14A,B 6 2/3 5 Larkum et al., 1999 1 1 2/3 5 Laramée et al., 2013 Vil2kd/kd V

2-14C Vil2+/+: 212.4 ± 8.5 µm, Vil2kd/kd: 159.9 ± 11.7 µm 2-14D Vil2+/+: 4.9 ± 0.3, Vil2kd/kd: 4.0 ± 0.2 6-OHDA 6 % º 6-OHDA 3-1A º 50% 6-OHDA 3-1B 6-OHDA 18 6 6-OHDA º % 3-2A 2 6 º 40% 6-OHDA 3-2B 6-OHDA

DOPA 6-OHDA TH 6-OHDA TH 3-3A DOPAC HPLC 6-OHDA DOPAC 3-3B,C 6-OHDA 6-OHDA º 3-4 6-OHDA 2 º

RhoA/ROCK/MLC2 RhoA ROCK

1C11

Da Silva et al., 2003 II RhoA

Dent et al., 2007; Kollins

et al., 2009 RhoA

II

RhoA MLC2 II

Amano et al., 1998; Krey et al., 2013 Vil2+/+

RhoA/ROCK MLC2 II

2-10A Vil2kd/kd

RhoA/ROCK/MLC2

2-10B Y-27632 blebbistatin ROCK

II Vil2kd/kd

2-8,9 blebbistatin Y-27632 Vil2+/+

Vil2kd/kd V

in

vitro in vivo

ERM

Goslin et al., 1989; Antoine-Bertrand et al., 2011; Marsick et al., 2012 º

Gonzalez-Agosti and Solomon, 1996 º

microscale chromophore-assisted laser inactivation micro-CALI

º º 30% Castelo et

al., 1999 º

Paglini et al., 1998 º

ERM

deleted in colorectal carcinoma DCC netrin-1

Netrin-1 DCC

ERM ERM

Antoine-Bertrand et al., 2011 C

55% netrin-1 netrin-1 º 5% Vil2kd/kd º 3

Everett and Nichol, 1990; Paglini et al., 1998

2-1

RhoA

GTPase RhoA Rac1 Cdc42

Govek et al.,

Speck

LLC-PK1 RhoA

Speck et al., 2003 Casaletto Vil2−/−

RhoA MLC2 Casaletto et al., 2011 RhoA Vil2kd/kd RhoA/ROCK/MLC2 Schmieder MDCK podocalyxin RhoGDI RhoA Schmieder et al., 2004 RhoA 1 Rac1 Cdc42 PAK

Hayashi et al., 2007; Redmond

and Ghosh, 2001 FERM

Rac1

Schulz et al., 2010 RhoA

RhoA/ROCK

RhoA

ROCK

Matsui et al., 1998;

Jeon et al., 2002 RhoA/ROCK

4-1A ROCK Y-27632 Vil2+/+

º 2-7A,C-E RhoA Vil2kd/kd º 2-6C,D ERM ROCK RhoA/ROCK 4-1B ERM phosphoinositide

3-kinase PI3 kinase protein kinase C PKC LRRK2 Gallo,

2008; Parisiadou et al., 2009; Kim et al., 2010

LRRK2 lrrk2

G2019S 2019

Jaleel et al., 2007 LRRK2 G2019S º

ERM 3-4 ERM º ERM Kashimoto et al., 2013 Vil2kd/kd

1. RhoA/ROCK/MLC2

2. ERM

Vil2kd/kd in vitro in vivo

Vil2kd/kd

HPLC

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Ezrin mediates neuritogenesis via down-regulation of RhoA activity in cultured cortical neurons.

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Excessive expression of hippocampal ezrin is induced by intrastriatal injection of 6-hydroxydopamine.

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17. Hatano R, Akiyama K, Tamura A, Hosogi S, Marunaka Y, Caplan MJ, Ueno Y, Tsukita S, Asano S. Knockdown of ezrin causes intrahepatic cholestasis by the dysregulation of bile fluidity in the bile duct epithelium. Hepatology. 2014 Oct 13.

18. Hatano R, Fujii E, Segawa H, Mukaisho K, Matsubara M, Miyamoto K, Hattori T, Sugihara H, Asano S. Ezrin, a membrane cytoskeletal cross-linker, is essential for the regulation of phosphate and calcium homeostasis. Kidney Int. 2013 Jan;83(1):41-9. 19. Hayashi K, Ohshima T, Hashimoto M, Mikoshiba K. Pak1 regulates dendritic branching

and spine formation. Dev Neurobiol. 2007 Apr;67(5):655-69.

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21. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000 May;57(5):976-83.

22. Jaleel M, Nichols RJ, Deak M, Campbell DG, Gillardon F, Knebel A, Alessi DR. LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity. Biochem J. 2007 Jul 15;405(2):307-17.

23. Jeon S, Kim S, Park JB, Suh PG, Kim YS, Bae CD, Park J. RhoA and Rho kinase-dependent phosphorylation of moesin at Thr-558 in hippocampal neuronal cells by glutamate. J Biol Chem. 2002 May 10;277(19):16576-84.

Phosphorylation of ezrin/radixin/moesin (ERM) protein in spinal microglia following peripheral nerve injury and lysophosphatidic acid administration. Glia. 2013 Mar;61(3):338-48.

25. Kim HS, Bae CD, Park J. Glutamate receptor-mediated phosphorylation of ezrin/radixin/moesin proteins is implicated in filopodial protrusion of primary cultured hippocampal neuronal cells. J Neurochem. 2010 Jun;113(6):1565-76.

26. Kollins KM, Hu J, Bridgman PC, Huang YQ, Gallo G. Myosin-II negatively regulates minor process extension and the temporal development of neuronal polarity. Dev Neurobiol. 2009 Apr;69(5):279-98.

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28. Laramée ME, Rockland KS, Prince S, Bronchti G, Boire D. Principal component and cluster analysis of layer V pyramidal cells in visual and non-visual cortical areas projecting to the primary visual cortex of the mouse. Cereb Cortex. 2013 Mar;23(3):714-28.

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30. Marsick BM, San Miguel-Ruiz JE, Letourneau PC. Activation of ezrin/radixin/moesin mediates attractive growth cone guidance through regulation of growth cone actin and adhesion receptors. J Neurosci. 2012 Jan 4;32(1):282-96.

31. Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, Tsukita S, Tsukita S. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol. 1998 Feb 9;140(3):647-57.

32. Negishi M, Katoh H. Rho family GTPases as key regulators for neuronal network formation. J Biochem. 2002 Aug;132(2):157-66.

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34. Parisiadou L, Xie C, Cho HJ, Lin X, Gu XL, Long CX, Lobbestael E, Baekelandt V, Taymans JM, Sun L, Cai H. Phosphorylation of ezrin/radixin/moesin proteins by

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Primer Sequence

GenoA1 5’-CATGGTGCCACACAGGACTC-3’ EK29 5’-GTGTGGCACTCTGCCTTCAAG-3’ En2A 5’-AGCGGATCTCAAACTCTCCTC-3’

1 Primer sequences used in genotyping.

Antigen Source Species (Clone) Dilution (Application)

Ezrin Cell Signaling Technology Rabbit 1:1000 (WB) 1:100 (IF) Ezrin Acris Mouse (3C12) 1:1000 (WB)

1:100 (IHC) Radixin Gift from Dr. Tsukita Rat (R21) 1:1000 (WB) Moesin Gift from Dr. Tsukita Mouse (2287) 1:1000 (WB) pan-ERM Cell Signaling Technology Rabbit 1:1000 (WB) phospho-ERM Cell Signaling Technology Rabbit 1:1000 (WB) GAPDH Sigma Rabbit 1:5000 (WB) β-actin Cell Signaling Technology Rabbit 1:5000 (WB) α-tubulin Abcam Mouse 1:100 (IF) Class III β-tubulin Covance Rabbit 1:100 (IF) Class III β-tubulin Sigma Mouse (2G10) 1:5000 (IHC) MAP2 Chemicon Mouse (HM-2) 1:3000 (IHC) MLC2 Cell Signaling Technology Rabbit 1:100 (WB) Phospho-MLC2 Cell Signaling Technology Rabbit 1:100 (WB) RhoA Cytoekeleton Mouse 1:500 (WB) Rac1 Cell BioLabs Mouse 1:500 (WB) Cdc42 Cytoekeleton Mouse 1:500 (WB)

TH Sigma Mouse 1:10000 (IHC)

Stage 1 Stage 2 Stage 3 Stage 4/5

Filopodia

Neurites

Axon

Growth cone

Spines

Dendrites

Lamellipodia

1-1 Schematic representation of morphological changes in cultured neurons.

RhoA Rac1 Cdc42 PAK ROCK

Elongation

↓

Retraction

↓

↓

↓

↓

↓

↓

↓

↓

↓

↓

↓

↓

↓

↓

↓

Rho

→→→→→

Rho

↓

↓

↓

GEF

A RhoGEF

B RhoGAP

Rho

←←←←←

Rho

↓

↓

↓

GAP

C RhoGDI

Rho

→→→→→

Rho

↓

↓

↓

GDI

Rho activity:

Increased

Rho activity:

Decreased

Rho activity:

Decreased

586

85%

583

84%

577

Ezrin

Radixin

Moesin

Four-point-one, ezrin,

radixin, moesin (FERM)

domain

Actin

binding

domain

N

C

61%

591

Merlin

P-Thr

Inactive

“Closed form”

Active

“Opened form”

→→→→→→→→

Phosphorylation

F-actin

Membrane proteins

L1-CAM

DCC receptor

etc…

Ezrin

Radixin

Moesin

1-5 Activation of ERM proteins.

1-6 Targeting strategy of mouse ezrin gene (gene symbol Vil2).

Ezr in Ph al lo id in α -tu b u lin Me rg e

Stage 1 Stage 2 Stage 3

2-1 Distribution of ezrin was observed in wild-type cultured cortical neurons at the stages 1, 2 and 3 by immunofluorescence.

Neurons at the stages 1, 2 and 3 were stained with an anti-ezrin antibody, rhodamine phalloidin, and an anti-α-tubulin antibody, respectively. In the bottom lane, neurons were triple stained with an anti-ezrin antibody (green), rhodamine phalloidin (red) and an anti-α-tubulin antibody (blue). Scale bars, 50 µm.

A

B

+/+

kd/kd

2-2 Detection of ezrin in the Vil2+/+ and Vil2kd/kd neurons.

A, Immunofluorescence of the Vil2+/+ and Vil2kd/kd neurons at stage 1 using an anti-ezrin antibody. Scale bar, 50 µm. B, Western blotting of cell extracts (10 µg, 1 µg, 0.5 µg or 0.2 µg) from the Vil2+/+ and Vil2kd/kd neurons (2 DIV) with an anti-ezrin antibody.

A

B

+/+

kd/kd

βIII-tubulin/Phalloidin

2-3 Neuritogenesis is impaired by ezrin knockdown.

A, B, The Vil2+/+ (A) and Vil2kd/kd (B) neurons were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm.

N eu ro n s in s ta g e (% )

C

E

D

F

***

**

*

C, Stacked bar graph showing stage progression in the Vil2+/+ (n = 153) and Vil2kd/kd (n = 162) neurons. Stages of cells were defined by the length of the longest neurite as reported

previously. D-F, Quantitation of number (D) and length (E) of neurites, and length of axon (F) in the Vil2+/+ (gray columns, n = 50) and Vil2kd/kd (green columns, n = 50) neurons. Three independent experiments were performed. *p<0.05, **p<0.01, ***p<0.001, Student's t test. Data represent mean ± SE.

A

C

D

E

**

+/+

kd/kd

B

2-4 Morphological abnormalities in Vil2kd/kd neurons in 4 DIV.

A, B, The Vil2+/+ (A) and Vil2kd/kd (B) neurons were fixed at 4 DIV and stained with an anti-neuronal class III β-tubulin antibody. Scale bars, 50 µm. C-E, Quantitation of number (C) and length (D) of neurites, and length of axon (E) in the Vil2+/+ (white columns, n = 5) and

Vil2kd/kd (black columns, n = 5) neurons. Three independent experiments were performed.

A

C

B

D

*

E

F

A-C, The amounts of active and total RhoA (A), Rac1 (B) and Cdc42 (C) from cell lysates of the Vil2+/+ and Vil2kd/kd neurons (2 DIV). Representative patterns were presented. D-F, The ratios of active RhoA (D), Rac1 (E) and Cdc42 (F) to total amount of proteins were compared between the Vil2+/+ (white columns) and Vil2kd/kd (black columns) neurons. Each experiment was performed in triplicate. *p<0.05 , Student's t test. Data represent mean ± SE.

A

B

C

D

Ezrin Radixin Moesin

**

A, Western blotting of the Vil2+/+ and Vil2kd/kd neurons (2 DIV) using an antibody recognizing phospho-MLC2 (Ser19, top), MLC2 (middle) and GAPDH (bottom), respectively.

Representative blotting patterns were shown. 8 µg of cell lysate was applied onto each lane. B, The ratio of phosphorylated MLC2 to total MLC2 in the lysate of the Vil2+/+ (white column) and Vil2kd/kd (black column) neurons was shown. C, Western blotting of the Vil2+/+ and

Vil2kd/kd neurons (2 DIV) using an antibody recognizing phospho-ERM (top), pan-ERM (middle) and GAPDH (bottom), respectively. Representative blotting patterns were shown. 8 µg of cell lysate was applied onto each lane. D, The ratios of phosphorylated ezrin, radixin and moesin to each total protein in the lysate of the Vil2+/+ (white columns) and Vil2kd/kd (black columns) neurons were shown. Each experiment was performed in triplicate.

0 0.2 0.4 0.6 0.8 1 1.2 1.4

A

-

+

-

+

B

*

C

D

E

*

2-7 Phosphorylation was affected by Y-27632 in the MLC2, not in the ERM proteins.

A, Western blotting of the DMSO- or Y-27632-treated Vil2+/+ and Vil2kd/kd neurons (2 DIV) using an antibody recognizing phospho-MLC2 (Ser19, top), phospho-ERM (middle) and GAPDH (bottom), respectively. Representative blotting patterns were shown. 8 µg of cell lysate was applied onto each lane. B-E, The ratios of phosphorylated MLC2, ezrin, radixin and moesin to GAPDH in the lysate of the DMSO-treated (white columns) and

Y-27632-treated (black columns) Vil2+/+ and Vil2kd/kd neurons were shown. Each experiment was performed in triplicate. *p<0.05, Student's t test. Data represent mean ± SE.

β III -tu b u lin / Ph al lo id in

A

B

C

D

2-8 Y-27632 rescues neuritogenesis.

A-D, The Vil2+/+ and Vil2kd/kd neurons treated with DMSO (A,C) or 40 µM Y-27632 (24 h, B,D) were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm.

0 50 100 150 200 250

E

F

G

**

***

###

***

*

*

**

E-G, The number (E) and length (F) of neurites, and length of axon (G) were quantified in the

Vil2+/+ and Vil2kd/kd neurons treated with DMSO (white columns, n = 30) or 40 µM Y-27632 (black columns, n = 30). Three independent experiments were performed. *p<0.05, **p<0.01,

***p<0.001 (DMSO-treated vs. Y-27632-treated), ###p<0.001 (DMSO-treated Vil2+/+ vs. DMSO-treated Vil2kd/kd), Student's t test. Data represent mean ± SE.

A

B

+/+ + Blebbistatin

kd/kd + Blebbistatin

2-9 Blebbistatin rescues neuritogenesis.

A, B, The Vil2+/+ and Vil2kd/kd neurons treated with 50 µM blebbistatin (24 h) were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody. Scale bar, 50 µm.

C

E

***

#

***

D

C-E, The number (C) and length (D) of neurites, and length of axon (E) were quantified in the

Vil2+/+ and Vil2kd/kd neurons treated with DMSO (white columns, n = 10) or 50 µM blebbistatin (black columns, n = 10). Three independent experiments were performed.

***p<0.001 (DMSO-treated vs. blebbistatin-treated), #p<0.05 (DMSO-treated Vil2+/+ vs. DMSO-treated Vil2kd/kd), Student's t test. Data represent mean ± SE.

A Vil2

_neurons

Ezrin

RhoA

ROCK

MLC2

Neuritogenesis

B Vil2

_neurons

Ezrin

RhoA

ROCK

MLC2

Impairment of

Neuritogenesis

2-10 Schematic representation of the relationship between ezrin and RhoA/Rho kinase/MLC2 pathway in neuritogenesis.

A

Vil2

_Vil2

B

2-11 Ezrin is not detected in Vil2kd/kd mice brain.

Immunohistochemical analyses of ezrin expression in adult (8 week-old) wild-type (A) and

Vil2kd/kd (B) mouse brains using an anti-ezrin antibody (clone, 3C12). Scale bars, 1 mm. Cx, cerebral cortex; Hip, hippocampus; Am, amygdala; Hy, hypothalamus.

0 50 100 150 200 250 300 350 400 450

B

C

***

A

Vil2

_Vil2

2-12 Structure of cerebral cortex in the adult Vil2+/+ and Vil2kd/kd mice.

A, Nissl staining in the cerebral cortex of Vil2+/+ and Vil2kd/kd mice. The length of cortical layers (B), and the number of Nissl-positive neurons (C) in Vil2+/+ and Vil2kd/kd mouse brains. Three independent experiments were performed. ***p < 0.001, Student’s t test. Data

A

Vil2

_Vil2

βIII-tubulin

a

a

b

b

2-13 Impairment of neuronal morphology in Vil2kd/kd cerebral cortex.

Immunohistochemistry of Vil2+/+ and Vil2kd/kd cerebral cortices using antibodies against class III β-tubulin (A) and MAP2 (B). High magnification images in Vil2+/+ (a) and Vil2kd/kd (b) were shown. Scale bars, 50 µm.

B

Vil2

_Vil2

MAP2

a

a

b

b

Immunohistochemistry of Vil2+/+ and Vil2kd/kd cerebral cortices using antibodies against class III β-tubulin (A) and MAP2 (B). High magnification images in Vil2+/+ (a) and Vil2kd/kd (b) were shown. Scale bars, 50 µm.

C

_**

D

*

A

Vil2

_Vil2

_Vil2

Vil2

B

2-14 Ezrin knockdown causes abnormal dendritic outgrowth.

Golgi staining of Vil2+/+ and Vil2kd/kd cerebral cortices. Representative images of apical dendrites (A) and basal dendrites (shown by triangles, B) of layer V pyramidal neurons were shown. C, Quantification of length of apical dendrites (C) and number of basal dendrites (D) in the Vil2+/+ and Vil2kd/kd layer V pyramidal neurons. *p < 0.05, **p < 0.01, Student’s t test. Data represent mean ± SE. Scale bar, 5 µm.

1 day

7 day

Vehicle (6 w)

1 day

7 day

6-OHDA (6 w)

A

Vehicle (6 w)

6-OHDA (6 w)

***

B

3-1 Intrastriatal Injection of 6-OHDA Impairs Long-Term Spatial Memory at 6 weeks.

A, Representative traces of the swimming paths of vehicle- and 6-OHDA-microinjected mice at the 1 and 7 d of acquisition trials. 6-OHDA-microinjected mice had significantly impaired water maze performance that was associated with (B) a preference of the target quadrant (quadrant I). ***p < 0.001, Student’s t test. Data represent mean ± SE.

1 day

7 day

Vehicle (18 w)

1 day

7 day

6-OHDA (18 w)

A

Vehicle (18 w)

6-OHDA ( 18 w)

*

B

3-2 Intrastriatal Injection of 6-OHDA Impairs Long-Term Spatial Memory at 18 weeks.

A, Representative traces of the swimming paths of vehicle- and a 6-OHDA-microinjected mouse at the 1 and 7 d of acquisition trials. 6-OHDA-microinjected mice had significantly impaired water maze performance that was associated with (B) a preference of the target quadrant (quadrant I). *p < 0.05, Student’s t test. Data represent mean ± SE.

Striatum Striatum

Substantia nigra Substantia nigra

Vehicle

6-OHDA

A

3-3 Immunohistochemical and neurochemical analyses in the nigrostriatal DA system.

A, Representative photomicrographs of striatum and substantia nigra of vehicle- or 6-OHDA-microinjected mice at 6 weeks after the microinjection. These slices were immunostained by an antibody against TH. Scale bars, 1 mm (in striatum), 200 µm (in substantia nigra).

0 5 10 15 20 25 30 DA levels ( pmol /m g ti ss u e) DOP AC levels ( pmol /m g ti ss u e) 0 1 2 3 4 5 6

B

C

Vehicle 6-OHDA Vehicle 6-OHDA

*

_*

3-3 Continued.

B, C, Striatal dopamine (DA) and DOPAC contents after intrastriatal injection of 6-OHDA. Amounts of DA (B) and DOPAC (C) in the striatum from treated mice were measured using an HPLC-ECD system. *p<0.05, Student's t test. Data represent mean ± SE.

0 50 100 150 200 250 300 % o f ve h ic le

B

Ezrin Radixin Moesin

*

A

3-4 Level of ERM proteins in the hippocampus.

A, Western blotting for ezrin, radixin, moesin and GAPDH (control protein) presented protein bands of 82, 80, 75 and 37 kDa, respectively. B, Quantitative results were obtained by measuring the optical density of each band using computerized image analysis. *p<0.05, Student's t test. Data represent mean ± SE.

A

B

Phosphorylation

Inhibition