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Dynamic networks & clathrin-mediated endocytosis. Eva Schmid (LMB Cambridge). Marijn Ford (now at UC Davis). Gerrit Praefcke (now at Cologne). What is a Hub? Are they static? Why have them?.

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slide1

Dynamic networks

&

clathrin-mediated

endocytosis

Eva Schmid

(LMB Cambridge)

Marijn Ford

(now at UC Davis)

Gerrit Praefcke

(now at Cologne)

slide2

What is a Hub?

Are they static?

Why have them?

slide3

At the synapse speed and fidelity are important to ensure the quantal nature and reliability of synaptic vesicle exocytosis

speed

fidelity

slide4

What is

Fidelity?

Exo

Endo

slide5

Clathrin-mediated endocytosis

The overall process is a

series of linear steps

but at the same time it is a

series of simultaneous micro-reactions(e.g. cargo recruitment, membrane invagination and coat assembly occurring in parallel)

slide6

Clathrin-mediated endocytosis

The overall process is a

series of linear steps

but at the same time it is a

series of simultaneous micro-reactions(e.g. cargo recruitment, membrane invagination and coat assembly occurring in parallel)

slide7

Clathrin-mediated endocytosis

The overall process is a

series of linear steps

but at the same time it is a

series of simultaneous micro-reactions(e.g. cargo recruitment, membrane invagination and coat assembly occurring in parallel)

Involving clathrin, adaptors (AP2) and

at least 20 different accessory proteins

slide8

The endocytic interactome

Accessory Proteins

(over 20 different proteins bind to the AP2 a-appendage)

Hubs

slide10

The a-appendage:

two independent binding sites

W840

Top Site

Peptide containing an FxDxF motif

Binds with an affinity of 4.6mM

F740

Side Site

Peptide containing a WVxF motif

Binds with an affinity of 0.7mM

slide11

Endocytic accessory proteins have a similar overall structure

Structured

domains

  • Protein:protein interaction domains
  • with no obvious tertiary structure
  • Contain multiple motifs, short amino acid sequences,
  • Please don’t call them ‘unstructured domains’ as they may have some secondary structure!!

Motif

domains

Epsin

slide12

Structured

domains

Motif

domains

AP2 a-motifs

slide14

Eps15 affinity for the a-appendage

contains 15 repeats of the sequence DPF

slide15

Eps15 affinity for the a-appendage

+

2-3 sites of 16mM

1 site of 20nM

So not all 15 motifs are available for simultaneous interactions

slide16

Eps15 affinity for the a-appendage

2-3 sites of 16mM

1 site of 20nM

16mM

16mM

16mM

20nM

  • From mutagenesis we know that the 20nM affinity is due to occupation of both top and side sites of one appendage
    • Thus this is a novel way to gain high affinity yet a readily reversible interaction… ie. 2 linear peptides linked by a flexible linker
slide17

Eps15 affinity for the a-appendage

2-3 sites of 16mM

1 site of 20nM

  • Eps15 with its simultaneous interactions with 4 appendage domains could help to cluster AP2s at sites of endocytosis
slide18

Motif domains are not unstructured and linear

But neither are they stable globular domains.

They are designed to package motifs in an efficient manner, such that when one motif is occupied then further motifs are exposed

Motif

Motif domain

a-appen-

dage

‘structural cooperativity’ in motif binding

slide19

This low structural stability means that these motif domains can search a wide range of space for potential ligands

slide20

This low structural stability means that these motif domains can search a wide range of space for potential ligands

Like a fishing line with lots of hooks……

But for entropic and statistical reasons the domain will prefer a more compact fold

And thus the hooks will gather ligands back to the core folded domains

slide21

Motif:domain interactions

  • A novel way to gain relatively high affinity and yet reversibility
  • Give rise to dynamic instability (a necessary characteristic of many cellular processes)
  • Allow cross-linking/multimerisation of binding targets
  • Efficient packaging of many different interactions surfaces
  • Multiple interactions that filter noise
  • A way to search space and draw ligands to a point
slide22

The network behaviour makes sense…..

  • Clathrin is an organising hub, not a protein recruitment hub. This ensures that empty clathrin cages do not form in the absence of membranes and cargo
  • AP2 does not self assemble, and only weakly binds to cargo. This ensures that cargo recruitment, membrane bending and polymerisation are tightly coupled.
slide23

Properties of endocytic and other biological networks

(feed forward and competitive loops)

Noise reduction:

Low affinity interactions ensure that processes are only activated on coincidence of several signals

Information processing:

The multimeric state of the AP2 hub allows it to bind multiple ligands according to their relative affinities and concentrations. Thus the hub integrates information. The competition between AP2 and clathrin also means that there is a sensing of the commitment along the endocytic pathway (the process gestalts).

slide27

Thus 4 potential ligand interaction sites on each AP2 complex.Does this make it a HUB? No

slide28

It is the concentration of AP2s on the membrane that gives it the ability to bind multiple partners according to affinities and concentrations

AP2 hub zone

slide29

AP2

in solution

Changing hubs gives directionality

Recruitment of AP2

to membrane

and concentration

Clathrin polymerisation

slide30

The clathrin hub

Miele et al 2004

Ter Haar et al 2002

b3 adaptor hinge

LLDLD

Amph WxxW

slide31

AP2

in solution

Changing hubs gives directionality

Recruitment of AP2

to membrane

and concentration

Clathrin polymerisation

  • Only on self-polymerisation does clathrin become a hub
slide32

Clathrin binding to the b -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)

Clathrin

b -appendage

slide33

Clathrin binding to the b -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)

Clathrin terminal domain

slide34

Clathrin binding to the b -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)

slide35

Clathrin binding to the b -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)

slide36

How clathrin-coated pits mature…

affinity

avidity

matricity

  • Sequential displacement of core and accessory proteins (affinity matures to avidity matures to matricity)
  • The process is pulled forward from the end
slide37

How clathrin-coated pits mature…

affinity

avidity

matricity

slide38

How clathrin-coated pits mature…

ATP

GTP

  • Sequential displacement of core and accessory proteins (affinity matures to avidity matures to matricity)
  • The process is pulled forward from the end
slide39

A Network view of clathrin-coated vesicle formation

A

AP2 adaptors sense

lipids, cargo,

accessory proteins

and other cargo adaptors

AP2

slide40

B

Building the cage: AP2

network hub is stabilized

through crosslinking by

accessory proteins

AP2

slide41

C

Clathrin is recruited and

polymerisation stabilises

the forming vesicle.

AP2 loses its position as a hub.

Clathrin is the new

organising hub

AP2

slide42

D

Dynamin and other late interacting partners (like uncoating factors) start to function

The point of no return.

AP2

slide43

E

Energy is used to re-prime the system for a new start.

AP2

slide44

AP2

in solution

Changing hubs gives directionality

Recruitment of AP2

to membrane

and concentration

Clathrin polymerisation

  • Only on self-polymerisation does clathrin become a hub
  • Note: in a clathrin-coated pit one has a snap-shot of the network at several different stages
slide46

In a coated-pit there may even be the beginning stages of uncoating, as the lipid phosphatase begins to work under the clathrin lattice

slide47

This means that fluorescent imaging will frequently not have the resolution to deduce the time dependence of recruitment

slide49

Early and late events can be predicted…

Time

1

2

Cage formation

3

Vesicle scission

3’

A short path-length gives an immediate response

To put a time delay in the response an additional interaction step is added

Uncoating and repriming

of molecules

slide50

This view maintains that:

Overexpression of a pathway hub will have little phenotype

Underexpression of a pathway hub will have a major phenotype

Overexpression of an accessory node will have a major phenotype

Underexpression of an accessory node will have little phenotype

Hubs