Master class Biomolecular Sciences Part 1: Molecular Cell Biology. 4 September 2007 Paul van Bergen en Henegouwen

Master class Biomolecular Sciences Part 1: Molecular Cell Biology. Sept-Nov : Paul van Bergen en Henegouwen. Mechanisms of clathrin-independent endocytosis : Willem Stoorvogel. Endocytosis and MHC classII : X-track symposium : Ger Strous. Ubiquitination and endocytosis : Catherine Rabouille (coordinator). Classical and unconventional secretion :Madelon Maurice. Wnt pathway and membrane traffic : ABC-seminar I&I +IB Aio dag : Fulvio Reggiori. Autophagy : Bernd Helms.Lipid droplet biogenesis : Exam

Classical secretion unconventional Non clathrin mediated endocytosis

Before I start, few statements: - The course is compulsary. If you have no choice of missing ONE class, contact the teacher and the coordinator by (on website) - Each lecture will be posted on the website few days after each class - Article presentation. Each of you will study one article per block in a group of persons. Prepare the article as well as possible while contacting the teacherat least one week in advance. When you meet, the preparation should be done and you come for questions and for sorting out details. - Read the article : Download the article from the web for the colour pictures before coming to the course using the guidelines, your brain and your knowledge. The more you have thought of it, the better the discussion. - Exam. There will be 7 questions related to the 7 articles presented, hence the above. The more you participate, the easiest. - Switch off your mobile. No ringing, no SMS.

Mechanisms of clathrin- independent internalization Paul van Bergen en Henegouwen

Endocytosis: what’s the problem? bendingfission

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 EM Replica 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

Adaptor Protein-2 complex, AP-2     Ear Hinge Trunk 100 kDa 50 kDa 17 kDa  -adaptin  -adaptin

There are different AP-complexes on different sites in the cell Golgi-complex Plasma membrane Golgi-complex            AP-1 AP-2 AP-3

Protein/protein interactions control CCV formation     Receptor Yxx  Eps15 DPF Receptor LL L  x  D/E Clathrin AP-2 I EHs II COIL III DPF/UIM Epsin NPF Lipids DPW PIP 2

The netwerk forms a bal: Electron tomography of a coated vesicle ©John Wiley &Sons,Inc Adaptor protein (AP2) complex  chain  chain  adaptin  adaptin Clathrin triskelion Smith et al. (1998) EMBO J. 17, 4943

Overview of clathrin structures Marsh and McMahon (1999) Science 285,

From bending to fission

Dynamin, a GTPase involved in fission PIP 2 Amphiphysin

Dynamin is forming a ring around the neck

Effect of pure dynamin on lipid vesicles

PIPK Membrane curvature generation AP2 Clathrin Eps15 Intersectin Amphiphysin Epsin Dynamin Endophilin Moleculen involved in the whole process

Mogelijke rol van actine filamenten

PIPK Membrane curvature generation AP2 Clathrin Eps15 Intersectin Amphiphysin Epsin Dynamin Endophilin Actin Synaptojanin Auxilin Hsc70 Moleculen betrokken bij CCV vorming

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

Internalization of large items: Phagocytosis

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-dependent Dynamin-inhibition by Dyn-K44 TfR wt K44A

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

Overview of different internalization routes