CDC1348 lecture Krahn - Composition and structure of biological …sfb1348.uni-muenster.de/sites/default/files/2018-11... · 2018-11-22 · Cell membrane – functions Michael Krahn

Composition and structure of biological membranes, interaction of proteins with

membranes

Michael. P. KrahnMedical Cell Biology

Cell membranes - functions

Michael Krahn Composition of cell membranes

Limitation

BarrierIons (Na+, K+, Cl-, Ca2+)Water, metabolites,…..Pathogens……

Cell membrane – barrier function

Michael Krahn

Neurons: Excitation / action potential (Na+-influx depolarisation)

Cl- Na+

Cl- Na+

Cl-Na+

K+

K+

K+

K+Na+-channel

Cl- Na+

Cl- Na+

Cl-Na+

Cl- Na+

Cl- Na+

Cl-Na+

K+

K+

K+

K+

K+

K+

K+

K+

Na+

Na+

Na+

Na+Membrane potential

Animal cells ~ -70mV

Composition of cell membranes

Cell membrane – functions

Michael Krahn

Limitation

BarrierIons (Na+, K+, Cl-, Ca2+)

Polarity & directed transport (luminal <> basal)

water, metabolites,…..

Signal transduction

pathogens……


Cell membranes - Compartmentalization

Michael Krahn

Living room

Kitc

hen

office

corr

idor

WC


Cell membrane – composition

Michael Krahn

Cyto-Plasma

PlasmamembranPlasmalemma


Cell membrane – Lipid bilayer

Michael Krahn

“Amphipathic“

Hydrophilic head (polar or charded)

Lipophilic tail = fatty acid soluble(lipophilic, hydrophobic).

= water soluble (hydrophilic, lipophobic)

Micelle: hydrophobic centerMicelle: hydrophobic center

water

“Lipid monolayer“

Vesicel: hydrophilic centerVesicel: hydrophilic center

“Lipid bilayer“

energetically unfavorableenergetically unfavorable

Energetically favorableEnergetically favorable

LiposomeLiposome


Cell mebrane – dimension

Michael Krahn

Erythrocyte

Basal invagination of a tublar cell in the kidney

5 nm


Cell membrane – (Phospho)Lipids

Michael Krahn

Phospholipids

Hydrogentail

Glycerine

CholinePhosphate

Phosphoglycerides

Cell membranes: 500‐1000 different substances


Cell membranes – (Phospho)Lipids

Michael Krahn

Sphingosine Sphingo‐myelin

Phosphatidyl‐Serine

Phosphatidyl‐ethanolamine

Phosphatidyl‐Choline

Hydrogen tail

Glycerine

Choline

Phosphate

PhosphoglyceridesSphingo‐lipids

50% Phospholipids (mammalian cells)


Cell membrane – Asymmetry

Michael Krahn

cytoplasm

extracellular space

Phosphatidyl‐ethanolamins

Phosphatidyl‐Serins

Phosphatidyl‐Cholins

Sphingo‐myelin

Glykolipids‐


Cell membrane – Dynamics

Michael Krahn

Lateral diffusion (very frequent) = 2µm/s

Flexion Rotation

Flip-Flop (rare)


Cell membranes – stabilisation

Michael Krahn

Polar head group

Rigid region stabilized by Cholesterol

Region of increased fluidity

2. Cholesterol (~ 20%)(only in animal cells!!!)

1. Unsa urated FA↑ Fluidity↑

Hydrogentail

Glycerine

CholinePhosphate

rigid

3. Temperature(Bacteria/Yeast: Cold Unsa urated FA↑)


Cell membrane – stabilisation

Michael Krahn

Bacteria

Plants

No steroids

No cholesterol, but other steroids

Cell wall

Plasma membrane

Cell wall

Plasma membrane


Cell membranes – membrane proteins

Michael Krahn

Lipid bilayer

Integral membrane proteins 50%

50%

A wall without windows or doors is useless ……


Cell membrane –Lipid rafts

Michael Krahn

Liposomes

Phosphatidylcholine : Sphingomyelin

1:1

Phosphatidylcholine : Sphingomyelin :

Cholesterol1:1:1

Atomic-Force-Microscopy

GPI-anchored proteintransmembran protein Lipid-Raft – functions:

- Endocytosis

- Protein-sorting

- Signal transduction

- ……


Cell membranes - Phosphatidyl-inositols (PtdIns)

Michael Krahn

PO4

Phosphatidylinositol-4-phosphat (PtdIns-4P)

Phosphatidyl-inositol (PI)

PO4

Phosphatidylinositol-3,4-bi-phosphat (PtdIns(3,4)P2

Phosphatidylinositol-3,4,5-tri-phosphat (PtdIns(3,4,5)P3

Phosphatidylinositol-4,5-bi-phosphat (PtdIns(4,5)P2PO4PO4

PO4


Cell membranes - Phosphatidyl-inositols (PtdIns)

Michael Krahn

Phosphatidyl-inositol (PI)

PtdIns-4P

PI4-Kinase PI4-Phophatase

PtdIns(4,5)P2

PI5-Kinase

PI5-Phosphatase

PtdIns(3,4,5)P3

SHIP1/2

PtdIns(3,4)P2

PI3-PhosphatasePI3-Kinase

PTENPI5-Kinase

PI3-Kinase


PtdIns-3PPI3-K

PtdIns(3,5)P2

PI3P-5K

PtdIns-5P

PI5-K

PI5P-4K

Cell membranes - Phosphatidic-acid (PA)


Hydrogentail

Glycerine

CholinePhosphate

Phosphatidyl choline

Hydrogentail

Glycerine

acidPhosphate

Phosphatidic acid

OH

Phosphatidic acid - synthesis


Hydrogentail

Glycerine

acidPhosphate

Phosphatidic acid

OH

Phosphatidic acid – Three ways of synthesis


Glycerine

acidPhosphate

Phosphatidic acid

OH

R R

Phosphatidyl cholinePhospho‐lipase D (PLD1/2)

Glycerine

acidPhosphate

1‐acyl‐sn‐glycerol‐phosphate = lysophosphatidic acid

OH

R OH

Glycerine

acidPhosphate

sn‐Gycerol‐3‐Phosphate

OH

OH OH

Acyl‐transferase I(GPAT1‐4)

Acyl‐Transferase II(LPAT1‐5)

Glycerol

(1,2)Diacyl glycerol (DAG)

OH

R R

DAG‐Kinase

timely & spatially regulated = signalling

Phospholipids-distribution in (mammalian) cells


Lemmons 2008

G. van Meer et al. 2008



Lemmons 2008Cinosi et al. 2011

Phosphatidylethanolamine ca. 15%Phosphatidylcholine ca. 30%

Sphingomyelin ca. 15%Cholesterol ca. 25%



Idevall‐Hagren and Camilli 2016

Membrane targeting of proteins


1. Transmembrane proteins

2. GPI-anchored proteins

3. Protein modifications: Lipid anchors- Farnesylation: CAAX- Palmitoylation: Cys- Myristoylation: M-Gly

4. Lipid-binding domains

Phospholipid-binding domains


Kahn and Lambright 2015



1. PH (Pleckstrin homology) domains (ca. 100aa, 285 in Hs):

- Pleckstrin- Phospholipase Cδ

- Akt (PKB) - PDK1- BTK- GRP1 (GEF for Arf6)

…..GEFs, GAPs, kinases

PtdIns(4,5)P2

PtdIns(3,4,5)P3

Promiscuity of PH domains


Bruntz 2014



3. FYVE domain (+ Zn2+):- endosomal proteins(e.g. EEA1, Vps27p/Hrs)

2. ENTH (Epsin N-terminal homology)- Epsin

PtdIns(4,5)P2

5. C1 domain (+ Zn2+):- PKCδ

DAG

4. PX domain:- Sorting nexins (Snx)

PtdIns-3P

6. C2 domain (+ Ca2+) :- PKCs- KIBRA

PtdSer



7. Annexin domain

8. BAR-domain- Sorting nexins- GAPs

9. F-BAR-domain- Syndapin

Acidic PhospholipidsSensingmembranecurvature

Stahelin 2009



Stahelin 2009

Phospholipid-binding domains – Polybasic motifs


Cox 2015



Dogliotti et al. 2017

GFP-LKB1 DE-Cad DlgMerge +

DAPI

DrosophilaGFP-LKB1

Lysophosphatic acid

Lysophosphocholine

PtdInsPtdIns(3)P

PtdIns(4)P

PtdIns(5)PPhosphatidylethanolamine(PE)

Phosphatidylcholine (PC)

Sphingosine-1phosphate

PtdIns(3,4)P2

PtdIns(3,5)P2PtdIns(4,5)P2

PtdIns(3,4,5)P3

Phosphatidic acid (PA)Phosphatidylserine (PS)blank

LKB1 LKB1LB

GFP-LKB1LB DE-Cad DlgMerge + DAPI

DrosophilaGFP-LKB1LB

NH2 - - COOH

VKKKGSALKRRAKKLTSCISVRKLSHCRTS538 567

5671kinase domainDrosophila LKB1

LKB1

PA PIP2 PIP3

P L P L P L

LKB1∆LB

Liposomesflotation assay

GFP-LKB1LB Baz MirMerge + DAPI

GFP-LKB1 Baz MirMerge + DAPI

DrosophilaGFP-LKB1



Krahn et al. 2010Kullmann and Krahn 2018

PDZ 1 PDZ 2 PDZ 3

1 1464

Par3

aPKC‐BD

P‐ Par1

P‐ Par1

SSRAKKPSILRGIGHMFRFGKNRKD

Michael Krahn

Tight Junctions (TJ)

Adherens Junctions (AJ)

apical

basal

PtdIns(4,5)P2

PtdI

ns(3

,4,5

)P3

PtdIns(3,4,5)P3

PtdIns(4,5)P2

GFPGFP Green fluorescent proteinGFPGFP

Martin Belmonte….. and Keith Mostov 2007

Plekstrin-homology domain(Phospholipase Cδ)

Plekstrin-homology domain(Akt1)

GFPGFP

Phospholipids of the cell membrane as determinants of polarity


Michael Krahn



Michael Krahn

TJ

AJ

apical

basal

PtdIns(4,5)P2

PtdI

ns(3

,4,5

)P3

PtdIns(3,4,5)P3

PtdIns(4,5)P2


Ax2Ax2

Ax2Ax2 Annexin-2Cdc42Cdc42

aPKCaPKC

Cdc42Cdc42 Cdc42 (kleine GTPase)

aPKCaPKC Atypische Kinase C



Michael Krahn

TJ

AJ

apical

basal

PtdIns(4,5)P2

PtdI

ns(3

,4,5

)P3

PtdIns(3,4,5)P3

PtdIns(4,5)P2

Ax2Ax2

Ax2Ax2 Annexin-2Cdc42Cdc42

aPKCaPKC

Cdc42Cdc42 Cdc42 (kleine GTPase)

aPKCaPKC Atypische Kinase C

Ax2Ax2

aPKCaPKC

Cdc42Cdc42







Michael Krahn

TJ

AJ

apical

basal

PtdIns(4,5)P2

PtdI

ns(3

,4,5

)P3

PtdIns(3,4,5)P3

PtdIns(4,5)P2

Ax2Ax2

Cdc42Cdc42aPKCaPKC

aPKCaPKC atypical proteinkinase C

Microdomains of the plasma membrane as signaling hubs

aPKCaPKC

Kinase activity↑

aPKC(closed

conformation)

= inactive

aPKC(open

conformation)

= active


Michael Krahn

TJ

AJ

apical

basal

PtdIns(4,5)P2

PtdI

ns(3

,4,5

)P3

PtdIns(3,4,5)P3

PtdIns(4,5)P2

Ax2Ax2

Cdc42Cdc42aPKCaPKC

PDK-1PDK-1

PDK-1PDK-1Phospho-inositol-dependentKinase

PTENPTEN

PI3-KPI3-K

PDK-1PDK-1AktAktP

Proteinsynthese ↑Zellwachstum ↑

Zellproliferation↑

PI3-KPI3-KPhospho-inositol-3-Kinase

PTENPTEN Phosphatase andtensin homolog

Tumor cells: PI3K↑PTEN↓

PI3-KPI3-K

Protein synthesis ↑Cell growth ↑

Cell proliferation↑





Kölsch et al. 2008

AMPKAMPKP

AMP

LKB1LKB1

mTORP

P

PIP3PIP3

AktAktP

PLDPCPC

Balanced cell growth and proliferation

PAPA

Healthy tissue

PIP3PIP3

AktAktP P P

PCPCPLD

PAPA

AMPKAMPK

LKB1LKB1

Cell proliferationCell growth

Tumor progression

mTORP

P P

Tumor cells

Dogliotti et al., 2017Kullmann and Krahn 2018

Michael Krahn

PAPA Phosphatidic acid

PCPC Phosphatidylcholine

PLD Phospholipase D



Michael Krahn

Detection of Phospholipids


Hagren and Camilli 2016

Michael Krahn

Manipulation of Phospholipids - Chemicals



Michael Krahn

Manipulation of Phospholipids - Optogenetics



Documents

CDC1348 lecture Krahn - Composition and structure of biological …sfb1348.uni-muenster.de/sites/default/files/2018-11... · 2018-11-22 · Cell membrane – functions Michael Krahn