Master course Molecular & Cellular Life Sciences Part 1: Intracellular Membrane Processes...

Master course Molecular & Cellular Life Sciences

Part 1: Intracellular Membrane Processes

October 8, 2009

Paul van Bergen en Henegouwen

Mechanisms of clathrin-independent

internalization

Paul van Bergen en Henegouwen

Different endocytic portals

Characteristics

Endocytosis: what’s the problem?

bending fission

Five ways to bend a membrane

Clathrin-dependent internalization

Clathrin-coated and vesicles:Ultrathin section of plastic-embedded material

Clathrin-coated pits and vesicles by EMReplica of freeze-dried plasma membranes

Clathrin-coated vesicles:Replica of freeze-dried plasma membranes

Clathrin: Heavy- and light chains

Clathrin makes a network

How clathrin is bending?

AP-2Adaptor Protein-2 complex

a b2m2

s2

EarHinge

Trunk

100 kDa

50 kDa

17 kDa

a-adaptin b-adaptin

There are different AP-complexes on different sites in the cell

Golgi-complex Plasma membrane Golgi-complex

a b2m2

s2g b1m1

s1d b3

m3

s3

AP-1 AP-2 AP-3

Protein/protein interactions control CCV formation

a b2m2s2

Receptor

Yxxf

Eps15

DPF

Receptor

LL

LfxfD/E

Clathrin

AP-2

I

EHs

II

COIL

III

DPF/UIM

Epsin

NPF

Lipids

DPW

PIP2

Epsin induces bendingof liposomes

The netwerk forms a ballElectron tomography of a coated vesicle

Adaptor protein(AP2) complex

s chain m chain

b adaptin

a adaptin

Clathrin triskelion

Smith et al. (1998) EMBO J. 17, 4943

Overview of clathrin structures

Marsh and McMahon (1999) Science 285, 215-220

From bending to fission

Dynamin, a GTPase involved in fission

PIP2 Amphiphysin

Dynamin is forming a ring around the neck

Effect of pure dynamin on lipid vesicles

PIPK

Membrane curvature generation

AP2ClathrinEps15

IntersectinAmphiphysinEpsin Dynamin

Endophilin

Moleculen involved in the whole process

Possible role for actin filaments

PIPK

Membrane curvature generation

AP2ClathrinEps15

IntersectinAmphiphysinEpsin Dynamin

Endophilin

Actin

SynaptojaninAuxilinHsc70

Moleculen involved in the whole process

Overview of all interactions

EGFR internalization via a non-coated pathway!

Is the alternative route visible?

Caveolae

Caveolin

Caveolar internalization starting from cholesterol-rich domains

Trafficking of Caveolae

Phagocytosis:Internalization of large items

Overview of different internalization routes

Properties of different CI-routes

Classification of internalization routes

Questions

What does the bending?What does the fission?

Interleukin-2 receptor complex

IL-2R internalization is Clathrin-independent clathrin-inhibition by ΔEps15

IL-2R internalization is Dynamin-dependentDynamin-inhibition by Dyn-K44

TfR

wt

K44A

IL-2R is present in the Detergent Resistant Membrane (DRM) fraction