Models of the plasma membrane - from the fluid mosaic to the picket fence model

Mario Schelhaas Institute of Cellular Virology The plasma membrane fulfils many dynamic functions Composition of the plasma membrane

• proteins, lipids, carbohydrates

• i.e. > 400 membrane proteins, 500 lipid species, 1000 sugar species The fluid mosaic model Singer and Nicolson 1972

• proteins are embedded in a lipid bilayer • lipids and proteins exhibit fluidity, i.e. they diffuse laterally in PM Lipids in the PM

• Lipids that spontaneously form a bilayer when mixed with water.. Phospholipids and some glycolipids

• Lipids that alone cannot form a bilayer, but can be dissolved into, and thus accommodated in, a bilayer Cholesterol, gangliosides, lysophosholipids, tri- and di- glycerides, isoprenoids, …

Lipid fluidity

• lipid composition determines fluidity at specific temperatures

• lipid composition is, in fact, adjusted to fine-tune the degree of fluidity in response for example to temperature changes • importance of fatty acid chain saturation for fluidity • importance of e.g. steroid content for rigidity How membrane proteins associate with the lipid bilayer

Membrane proteins are either ‘dissolved’ in the bilayer phase (in one leaflet only, or both), or attached to the surface of it. Concept of dissolving a transmembrane protein into lipid bilayers

Charged based interactions with phopho-head groups of lipids

Hydrophobic alpha-helices as membrane spanning part

Hydrophilic extra- / intracellular parts Concept of dissolving a transmembrane protein into lipid bilayers Types of membrane anchors Proteins are mobile within the plasma membrane Functionalization of the PM

• exchange of composition (exo- and endocytosis) The fluid mosaic model Singer and Nicolson 1972

• PM composed of lipids, proteins, carbohydrates • is a ‘fluid’ organelle • it can adapt to extra- and intracellular stimuli (exo-/ endocytosis) • exhibits polarity • compartmentalised • Criticism: How does compartmentalisation and polarity arise? Macroscopic compartmentalisation by diffusion restriction Lateral association of membrane proteins Lipid rafts form micro domains Local modifications of lipids

• Local synthesis allows association of specific PIP- binding proteins -> domain formation Lipid rafts contain a distinct set of integral proteins Certain raft domains do not exchange associated membrane proteins Certain raft domains do not exchange associated membrane proteins Domain formation leads to compartmentalisation A 20 year old enigma

• Why are macroscopic diffusion coefficient in the plasma membrane and artificial lipid bilayers so different?

• differences 5-50 fold ?

• needed single molecule tracking to understand Single molecule tracking

µ opioid receptor

phospholipid Hop diffusion matches size distribution of actin network ‘holes’ Hydro-dynamic friction effect The picket fence model Signaling receptor immobilisation

E-cadherin dynamics Compartmentalization in the mesoscale Virus Tracking Scattering and fluorescence detection

1 micrometer

interferometric scattering detection: iSCAT Combined detection

First observation: the trajectories do not overlap

Virus Tracking 4 types of lateral What we have not touched upon … movement exist Retrograde Transport of viruses along filopodia HPV-16 transport along actin protusions by retrograde flow

actin retrograde flow HPV-16

ECM myosin II

receptor clustering - signal transduction

actin retrograde flow HPV-16

ECM myosin II 4 types of lateral movement contribute to PM functionalization An updated version of the plasma membrane Virus entry HPV internalization is slow Individual HPV16 endocytosis events are quick

HPV16 clathrin HPV16 cell surface events: structural changes and receptor switching HPV secondary receptor candidates

Evander et al., Schelhaas et al., Scheffer et al., Woodham et al., J. Virol., 1997 PLoS pathog, 2012 J. Virol., 2013 PLoS one, 2012

A2t

ITGα6 EGFR CD151/CD63

microdomains Receptor Hopping? Functionalization of tetraspanin- enriched microdomains Are the structural changes rate-limiting for uptake? Structural changes contribute (in part) to asynchronous uptake Is the secondary receptor available for binding of fcHPV16? fcHPV16 binds inefficiently to HSPG-deficient cells HPV16 fcHPV16

the secondary receptor is not available for direct binding Becker et al., 2018 Receptor Hopping? x Virus engages receptors en route to a different structure HPV16 enters by a novel endocytic mechanism

Na+/H+ antiporter actin dynamics but no Rho GTPase activity

Schelhaas et al., 2012 Spoden et al., 2013 Endocytic vesicle formation

early stage intermediate stage late stage HPV16 endocytosis (WT) HPV16 micropinocytosis - working model

? EGFR

Abl2 Thank you

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