#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown
Causes PKD-Phenotypes


Autosomal dominant polycystic kidney disease (ADPKD) is characterized by

formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2,

encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function

in primary cilia, but it is not well understood how these and other proteins are

targeted to cilia. Here, we provide the first genetic and biochemical link

between polycystins and the exocyst, a highly-conserved eight-protein membrane

trafficking complex. We show that knockdown of exocyst component Sec10 yields

cellular phenotypes associated with ADPKD, including loss of flow-generated

calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10

knockdown in zebrafish phenocopies many aspects of polycystin-2

knockdown—including curly tail up, left-right patterning defects,

glomerular expansion, and MAPK activation—suggesting that the exocyst is

required for pkd2 function in vivo. We observe

a synergistic genetic interaction between zebrafish sec10 and

pkd2 for many of these cilia-related phenotypes.

Importantly, we demonstrate a biochemical interaction between Sec10 and the

ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the

exocyst and polycystin-2 at the primary cilium. Our work supports a model in

which the exocyst is required for the ciliary localization of polycystin-2, thus

allowing for polycystin-2 function in cellular processes.


Vyšlo v časopise: The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes. PLoS Genet 7(4): e32767. doi:10.1371/journal.pgen.1001361
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1001361

Souhrn

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by

formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2,

encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function

in primary cilia, but it is not well understood how these and other proteins are

targeted to cilia. Here, we provide the first genetic and biochemical link

between polycystins and the exocyst, a highly-conserved eight-protein membrane

trafficking complex. We show that knockdown of exocyst component Sec10 yields

cellular phenotypes associated with ADPKD, including loss of flow-generated

calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10

knockdown in zebrafish phenocopies many aspects of polycystin-2

knockdown—including curly tail up, left-right patterning defects,

glomerular expansion, and MAPK activation—suggesting that the exocyst is

required for pkd2 function in vivo. We observe

a synergistic genetic interaction between zebrafish sec10 and

pkd2 for many of these cilia-related phenotypes.

Importantly, we demonstrate a biochemical interaction between Sec10 and the

ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the

exocyst and polycystin-2 at the primary cilium. Our work supports a model in

which the exocyst is required for the ciliary localization of polycystin-2, thus

allowing for polycystin-2 function in cellular processes.


Zdroje

1. Smyth

BJ

Snyder

R

Balkovetz

DF

Lipschutz

JH

2003

Recent advances in the cell biology of polycystic kidney

disease.

Jeon

KW

Int Rev Cytol

San Diego

Elsevier Inc

52

89

2. Consortium

TIPD

1995

Polycystic kidney disease: The complete structure of the PKD1

gene and its protein.

Cell

81

289

298

3. Mochizuki

T

Wu

G

Hayashi

T

Xenophontos

SL

Veldhuisen

B

1996

PKD2, a gene for polycystic kidney disease that encodes an

integral membrane protein.

Science

272

1339

1342

4. Gonzalez-Perrett

S

Kim

K

Ibarra

C

Damiano

AE

Zotta

E

2001

Polycystin-2, the protein mutated in autosomal dominant

polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective

cation channel.

Proc Natl Acad Sci U S A

98

1182

1187

5. Vassilev

PM

Guo

L

Chen

XZ

Segal

Y

Peng

JB

2001

Polycystin-2 is a novel cation channel implicated in defective

intracellular Ca(2+) homeostasis in polycystic kidney

disease.

Biochem Biophys Res Commun

282

341

350

6. Hanaoka

K

Qian

F

Boletta

A

Bhunia

AK

Piontek

K

2000

Co-assembly of polycystin-1 and -2 produces unique

cation-permeable currents.

Nature

408

990

994

7. Yamaguchi

T

Hempson

SJ

Reif

GA

Hedge

AM

Wallace

DP

2006

Calcium restores a normal proliferation phenotype in human

polycystic kidney disease epithelial cells.

J Am Soc Nephrol

17

178

187

8. Yamaguchi

T

Nagao

S

Wallace

DP

Belibi

FA

Cowley

BD

2003

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from

autosomal-dominant polycystic kidneys.

Kidney Int

63

1983

1994

9. Yamaguchi

T

Pelling

JC

Ramaswamy

NT

Eppler

JW

Wallace

DP

2000

cAMP stimulates the in vitro proliferation of renal cyst

epithelial cells by activating the extracellular signal-regulated kinase

pathway.

Kidney Int

57

1460

1471

10. Praetorius

HA

Frokiaer

J

Nielsen

S

Spring

KR

2003

Bending the primary cilium opens Ca2+-sensitive

intermediate-conductance K+ channels in MDCK cells.

J Membr Biol

191

193

200

11. Praetorius

HA

Spring

KR

2001

Bending the MDCK cell primary cilium increases intracellular

calcium.

J Membr Biol

184

71

79

12. Pazour

GJ

San Agustin

JT

Follit

JA

Rosenbaum

JL

Witman

GB

2002

Polycystin-2 localizes to kidney cilia and the ciliary level is

elevated in orpk mice with polycystic kidney disease.

Curr Biol

12

R378

380

13. Yoder

BK

Hou

X

Guay-Woodford

LM

2002

The polycystic kidney disease proteins, polycystin-1,

polycystin-2, polaris, and cystin, are co-localized in renal

cilia.

J Am Soc Nephrol

13

2508

2516

14. Nauli

SM

Alenghat

FJ

Luo

Y

Williams

E

Vassilev

P

2003

Polycystins 1 and 2 mediate mechanosensation in the primary

cilium of kidney cells.

Nat Genet

33

129

137

15. Nauli

SM

Rossetti

S

Kolb

RJ

Alenghat

FJ

Consugar

MB

2006

Loss of polycystin-1 in human cyst-lining epithelia leads to

ciliary dysfunction.

J Am Soc Nephrol

17

1015

1025

16. Goetz

SC

Anderson

KV

2010

The primary cilium: a signalling centre during vertebrate

development.

Nat Rev Genet

11

331

344

17. Pedersen

LB

Rosenbaum

JL

2008

Intraflagellar transport (IFT) role in ciliary assembly,

resorption and signalling.

Curr Top Dev Biol

85

23

61

18. Kramer-Zucker

AG

Olale

F

Haycraft

CJ

Yoder

BK

Schier

AF

2005

Cilia-driven fluid flow in the zebrafish pronephros, brain and

Kupffer's vesicle is required for normal organogenesis.

Development

132

1907

1921

19. Krock

BL

Perkins

BD

2008

The intraflagellar transport protein IFT57 is required for cilia

maintenance and regulates IFT-particle-kinesin-II dissociation in vertebrate

photoreceptors.

J Cell Sci

121

1907

1915

20. Sun

Z

Amsterdam

A

Pazour

GJ

Cole

DG

Miller

MS

2004

A genetic screen in zebrafish identifies cilia genes as a

principal cause of cystic kidney.

Development

131

4085

4093

21. Tsujikawa

M

Malicki

J

2004

Intraflagellar transport genes are essential for differentiation

and survival of vertebrate sensory neurons.

Neuron

42

703

716

22. Duldulao

NA

Lee

S

Sun

Z

2009

Cilia localization is essential for in vivo functions of the

Joubert syndrome protein Arl13b/Scorpion.

Development

136

4033

4042

23. Kishimoto

N

Cao

Y

Park

A

Sun

Z

2008

Cystic kidney gene seahorse regulates cilia-mediated processes

and Wnt pathways.

Dev Cell

14

954

961

24. Serluca

FC

Xu

B

Okabe

N

Baker

K

Lin

SY

2009

Mutations in zebrafish leucine-rich repeat-containing six-like

affect cilia motility and result in pronephric cysts, but have variable

effects on left-right patterning.

Development

136

1621

1631

25. Sullivan-Brown

J

Schottenfeld

J

Okabe

N

Hostetter

CL

Serluca

FC

2008

Zebrafish mutations affecting cilia motility share similar cystic

phenotypes and suggest a mechanism of cyst formation that differs from pkd2

morphants.

Dev Biol

314

261

275

26. Zhao

C

Malicki

J

2007

Genetic defects of pronephric cilia in zebrafish.

Mech Dev

124

605

616

27. Shen

MM

2007

Nodal signaling: developmental roles and

regulation.

Development

134

1023

1034

28. Essner

JJ

Amack

JD

Nyholm

MK

Harris

EB

Yost

HJ

2005

Kupffer's vesicle is a ciliated organ of asymmetry in the

zebrafish embryo that initiates left-right development of the brain, heart

and gut.

Development

132

1247

1260

29. Bisgrove

BW

Snarr

BS

Emrazian

A

Yost

HJ

2005

Polaris and Polycystin-2 in dorsal forerunner cells and

Kupffer's vesicle are required for specification of the zebrafish

left-right axis.

Dev Biol

287

274

288

30. Obara

T

Mangos

S

Liu

Y

Zhao

J

Wiessner

S

2006

Polycystin-2 immunolocalization and function in

zebrafish.

J Am Soc Nephrol

17

2706

2718

31. Schottenfeld

J

Sullivan-Brown

J

Burdine

RD

2007

Zebrafish curly up encodes a Pkd2 ortholog that restricts

left-side-specific expression of southpaw.

Development

134

1605

1615

32. Pennekamp

P

Karcher

C

Fischer

A

Schweickert

A

Skryabin

B

2002

The ion channel polycystin-2 is required for left-right axis

determination in mice.

Curr Biol

12

938

943

33. McGrath

J

Somlo

S

Makova

S

Tian

X

Brueckner

M

2003

Two populations of node monocilia initiate left-right asymmetry

in the mouse.

Cell

114

61

73

34. Emmer

BT

Maric

D

Engman

DM

2010

Molecular mechanisms of protein and lipid targeting to ciliary

membranes.

J Cell Sci

123

529

536

35. Novick

P

Field

C

Schekman

R

1980

Identification of 23 complementation groups required for

post-translational events in the yeast secretory pathway.

Cell

21

205

221

36. Lipschutz

JH

Mostov

KE

2002

The many masters of the exocyst.

Curr Biol

12

R212

R214

37. Guo

W

Grant

A

Novick

P

1999

Exo84p is an exocyst protein essential for

secretion.

J Biol Chem

274

23558

23564

38. Rogers

KK

Wilson

PD

Zhang

X

Guo

W

Burrow

CR

2004

The exocyst localizes to the primary cilium in MDCK

cells.

Biochem Biophys Res Comm

319

39. Zuo

X

Guo

W

Lipschutz

JH

2009

The exocyst protein Sec10 is necessary for primary ciliogenesis

and cystogenesis in vitro.

Mol Biol Cell

20

2522

2529

40. Guo

W

Roth

D

Walch-Solimena

C

Novick

P

1999

The exocyst is an effector for Sec4p, targeting secretory

vesicles to sites of exocytosis.

EMBO J

18

1071

1080

41. Grindstaff

KK

Yeaman

C

Anandasabapathy

N

Hsu

S

Rodriguez-Boulan

R

1998

Sec6/8 complex is recruited to cell-cell contacts and specifies

transport vesicle delivery to the basal-lateral membrane in epithelial

cells.

Cell

93

731

740

42. Lipschutz

JH

Guo

W

O'Brien

LE

Nguyen

YH

Novick

P

2000

Exocyst is involved in cystogenesis and tubulogenesis and acts by

modulating synthesis and delivery of basolateral plasma membrane and

secretory proteins.

Mol Biol Cell

11

4259

4275

43. Lipschutz

JH

Lingappa

VR

Mostov

KE

2003

The exocyst affects protein synthesis by acting on the

translocation machinery of the endoplasmic reticulum.

J Biol Chem

278

20954

20960

44. Moskalenko

S

Henry

DO

Rosse

C

Mirey

G

Camonis

JH

2002

The exocyst is a Ral effector complex.

Nat Cell Biol

4

66

72

45. Shalom

O

Shalva

N

Altschuler

Y

Motro

B

2008

The mammalian Nek1 kinase is involved in primary cilium

formation.

FEBS Lett

582

1465

1470

46. Siroky

BJ

Ferguson

WB

Fuson

AL

Xie

Y

Fintha

A

2006

Loss of primary cilia results in deregulated and unabated apical

calcium entry in ARPKD collecting duct cells.

Am J Physiol Renal Physiol

290

F1320

1328

47. Grantham

JJ

1990

Polycystic kidney disease: neoplasia in disguise.

Am J Kidney Dis

15

110

116

48. Lipschutz

JH

1998

The molecular development of the kidney: a review of the results

of gene disruption studies.

Am J Kid Dis

31

383

397

49. O'Brien

LE

Tang

K

Kats

ES

Schutz-Geschwender

A

Lipschutz

JH

2004

ERK and MMPs sequentially regulate distinct stages of epithelial

tubule development.

Dev Cell

7

21

32

50. Omori

S

Hida

M

Fujita

H

Takahashi

H

Tanimura

S

2006

Extracellular signal-regulated kinase inhibition slows disease

progression in mice with polycystic kidney disease.

J Am Soc Nephrol

17

1604

1614

51. Calvet

JP

2008

Strategies to inhibit cyst formation in ADPKD.

Clin J Am Soc Nephrol

3

1205

1211

52. Sweeney

WE

Jr

Avner

ED

2006

Molecular and cellular pathophysiology of autosomal recessive

polycystic kidney disease (ARPKD).

Cell Tissue Res

326

671

685

53. Torres

VE

Harris

PC

Pirson

Y

2007

Autosomal dominant polycystic kidney disease.

Lancet

369

1287

1301

54. Friedrich

GA

Hildebrand

JD

Soriano

P

1997

The secretory protein Sec8 is required for paraxial mesoderm

formation in the mouse.

Dev Biol

192

364

374

55. Boletta

A

Qian

F

Onuchic

LF

Bhunia

AK

Phakdeekitcharoen

B

2000

Polycystin-1, the gene product of PKD1, induces resistance to

apoptosis and spontaneous tubulogenesis in MDCK cells.

Mol Cell

6

1267

1273

56. Follit

JA

Tuft

RA

Fogarty

KE

Pazour

GJ

2006

The intraflagellar transport protein IFT20 is associated with the

Golgi complex and is required for cilia assembly.

Mol Biol Cell

17

3781

3792

57. Jurczyk

A

Gromley

A

Redick

S

San Agustin

J

Witman

G

2004

Pericentrin forms a complex with intraflagellar transport

proteins and polycystin-2 and is required for primary cilia

assembly.

J Cell Biol

166

637

643

58. Pazour

GJ

Dickert

BL

Vucica

Y

Seeley

ES

Rosenbaum

JL

2000

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney

disease gene tg737, are required for assembly of cilia and

flagella.

J Cell Biol

151

709

718

59. Moyer

JH

Lee-Tischler

MJ

Kwon

HY

Schrick

JJ

Avner

ED

1994

Candidate gene associated with a mutation causing recessive

polycystic kidney disease in mice.

Science

264

1329

1333

60. Jonassen

JA

San Agustin

J

Follit

JA

Pazour

GJ

2008

Deletion of IFT20 in the mouse kidney causes misorientation of

the mitotic spindle and cystic kidney disease.

J Cell Biol

183

377

384

61. Yoder

BK

Tousson

A

Millican

L

Wu

JH

Bugg

CE

Jr

2002

Polaris, a protein disrupted in orpk mutant mice, is required for

assembly of renal cilium.

Am J Physiol Renal Physiol

282

F541

552

62. Lunt

SC

Haynes

T

Perkins

BD

2009

Zebrafish ift57, ift88, and ift172 intraflagellar transport

mutants disrupt cilia but do not affect hedgehog signaling.

Dev Dyn

238

1744

1759

63. Huang

P

Schier

AF

2009

Dampened Hedgehog signaling but normal Wnt signaling in zebrafish

without cilia.

Development

136

3089

3098

64. Fu

X

Wang

Y

Schetle

N

Gao

H

Putz

M

2008

The subcellular localization of TRPP2 modulates its

function.

J Am Soc Nephrol

19

1342

1351

65. Finetti

F

Paccani

SR

Riparbelli

MG

Giacomello

E

Perinetti

G

2009

Intraflagellar transport is required for polarized recycling of

the TCR/CD3 complex to the immune synapse.

Nat Cell Biol

11

1332

1339

66. Shibazaki

S

Yu

Z

Nishio

S

Tian

X

Thomson

RB

2008

Cyst formation and activation of the extracellular regulated

kinase pathway after kidney specific inactivation of Pkd1.

Hum Mol Genet

17

1505

1516

67. Mintzer

KA

Lee

MA

Runke

G

Trout

J

Whitman

M

2001

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally

and zygotically in dorsoventral pattern formation.

Development

128

859

869

68. Taulman

PD

Haycraft

CJ

Balkovetz

DF

Yoder

BK

2001

Polaris, a protein involved in left-right axis patterning,

localizes to basal bodies and cilia.

Mol Biol Cell

12

589

599

69. Thisse

C

Thisse

B

2008

High-resolution in situ hybridization to whole-mount zebrafish

embryos.

Nat Protoc

3

59

69

70. Barile

M

Pisitkun

T

Chou

C

Verbalis

M

Knepper

M

2005

Large-scale protein identification in intracellular aquaporin-2

vesicles from renal inner medullary collecting duct.

Mol Cell Prot

4

1095

1106

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2011 Číslo 4
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#