Pathways Of Membrane Trafficking Some Basic Mechanisms And Regulations

Pathways of membrane trafficking Some basic mechanisms and regulations

Visit URL http://thierry.galli.free.fr/

Questions 1 Basic mechanisms of membrane 1. trafficking particularly membrane fusion 2. In vivo mechanisms and regulatory p processes

Answers 1. From genetics, and pharmacology and more particularly in vitro assay 2. From in vivo experiments

Basic mechanisms for intracellular membrane trafficking

Budding & fission

Fusion

Membrane deformations during budding/fission

Positive curvature- favored by cone-shaped lipids Negative curvature- favored by inverted cone-shaped lipids

1. The Golgi: a central platform in membrane t ffi ki trafficking

The Golgi is the central processing and sorting station of the secretory pathway

ER

ERGIC Golgi

ER

ERGIC

Golgi

Vesicle & Maturation Models

3. From NSF & SNAPs to SNAREs

Isolation of SNAREs

SNAREs: targets of clostridial NTs

SNAREs: targets g of clostridial neurotoxins

Inverted SNAREs  cell fusion

NSF revisited role

4 The SNARE complex 4.

(Sutton et al., Nature 1998)

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

SNAREs form 44-stranded coiledcoiled-coil “core” complex

SNARE: the generic model

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

5 Biophysics of Membrane Fusion 5.

Membrane deformations during fusion

Negative curvature- favored by inverted cone-shaped lipids

Positive curvature- favored by cone cone-shaped shaped lipids

Hydration barrier to spontaneous f i fusion

Annuall Reviews i

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

6. SSNAREs & membrane compartments 6 p Chaîne

Levure

Nématode

Drosophile

Mammifères

SNAREs

21 7

23 9

20 7

35 12

5

7

5

9

6

4

5

8

5

6

5

9

4 11

6 29

5 26

7 60

Qa Syntaxines

Qb Nter SNAP25

Qc Cter SNAP25

R V-SNARE

Sec1 Rab

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

SNAREs & membrane compartments

How do SNAREs get to where they are supposed to be?

Morphological p g Experiments p Suggest Role of Vesicle Coats

Each type yp acts at distinct locations

SNAREs and coats coats:: possible links  Se Several e a v-SNAREs S s interact e ac with molecular o ecu a coats:: VAMP4 coats VAMP4--AP1, VAMP7/TIVAMP7/TI-VAMPVAMP-AP3  The Longin domain of Sec22, Sec22 Ykt6, Ykt6 TI TI-VAMP/VAMP7 resembles a domain in AP2 subunits

Tetanus NeurotoxinNeurotoxin-Insensitive Vesicle Vesicle-Associated Membrane Protein (TI(TIVAMP)     

TI-VAMP/VAMP7 TIX-linked gene 25kD vv-SNARE ubiquitous Insensitive to NTs N-terminal extension off 100aa 100 called ll d Longin domain

Tetanus neurotoxin sensitive and insensitive exocytosis

V-SNARE

Sensitive to TeNT

Expressed in neurons

Synaptobrevin/VAMP2

Yes

Yes

Cellubrevin/VAMP3

Yes

No

TI--VAMP/VAMP7 TI

No

Yes

The e Longin o g family Ykt6

Sec22

SEDL

2

TI-VAMP

SEDL, component of the transport protein particle (TRAPP) involved in endoplasmic reticulum-to-Golgi vesicle transport Missense mutation X-linked spondyloepiphyseal dysplasia tarda

TI-VAMP TIinteracts with AP--3 AP

Plasma Membrane

t-SNARE AP-3 Brevin v-SNARE Longin v v-SNARE SNARE

(-)

(+)

TGN

Early/Recycling Endosomes TfR TfR+ « Rapidly Exocytic Compartment »

Late Endosomes /Lysosomes CD63 CD63+ « Slowly Exocytic Compartment »

AP3 is required to transport TI-VAMP to Late Endosomes

The mocha (mh) mouse is a null mutant for AP3 , th f therefore has h no AP3 complex l iin any cellll ttype -//

+/ +/-

Phenotypes: •coat and eye color dilution y •reduced levels of renal lysosomal enzymes in the urine •prolonged bleeding due to storage pool deficiency in the dense granules of platelets. •hyperactive and have an altered (hypersynchronized) theta wave ppattern in the electrocortigram. g •ZnT3 and ClC-3 reduced in mossy fiber terminals

TI--VAMP in the hippocampus TI

Lydia Danglot

Residual synaptic transmission after TeNT is asynchronous and AP-3 3 dependent

ctl+TeNT

mocha+TeNT

ctl+TeNT mocha+TeNT

Anita Scheuber, Jean-Christophe Poncer

TI-VAMP but not Syb2 is lost in mocha MF terminals

Lydia Danglot

stratum oriens (so) stratum pyramidale (sp) stratum radiatum (sr) stratum lucidum (sluc) stratum granulosum (sg)

TI-VAMP is in the Golgi area of mocha granule cells

Lydia Danglot

stratum oriens (so) stratum pyramidale (sp) stratum radiatum (sr) stratum lucidum (sluc) stratum granulosum (sg)

Conclusion  Presynaptic TITI-VAMP is lost in mocha Mossy fiber CA3 terminals  A form of TeNTTeNT-resistant asynchronous release l iis lost l t iin mocha h  Increased basal release in mocha

Regulation of TITI-VAMP endocytosis by Hrb

Regulation of TITI-VAMP endocytosis by Hrb