MAMs - bfb

Technical Criteria to Evaluate
Mitochondria-ER Contact Sites (MAMs)
Giovanna Cenini (AG Voos)
Bonn 27.07.2016
ructure of ER Membrane-Contact Sites (MCSs)
Plasma
membrane
s and Voeltz 2016, Nature Reviews
Mitochondria-ER Contact Sites: MAMs
Mitochondrion
Giorgi et al 2015, Antiox & Redox Sign
MAMs: Not Just Structural Issue, But Also Function!!!
Lipid-biosynthesis
- Synthesis of PtdEt out of PtdSer in the mitochondrial membrane
- Lipid Transport occurs non-vesicular
Apoptosis
- Calcium reaching a cytotoxic concentration
caspases
Destruction of the Cell
Release of cytochrome C
Activation of
Regulation of mitochondrial fission
- ER forms tubules, that wrap around mitochondria and
determine the fission site
Calcium Transfer
- Activation of enzymes of the citric acid cycle
- Via IP3 signaling
Vance 2014, BBA Review
MAMs: Role in Alzheimer Disease (AD)
Gomez and Schon, 2016
MAMs: Not just Membranes, but also Protein Complexe
Phillips and Voeltz 2016, Nature Reviews
MAMs Complexes: VAPB-PTPIP51
• 
VAPB: Vesicle associated membrane protein-associated protein
• 
VAPB is ER membrane protein
• 
Mutation of VAPB causes ALS-type 8
• 
PTPIP51: Protein tyrosine phosphatase-interacting protein 5
• 
PTIPIP51 is mitochondrial OM protein
PTPIP51-VAPB interaction regulates Calcium homeostasis
MAMs: Technical Approaches
1. Microscopy Immunofluorescence
Density gradient + Western Blot
2. Biochemical and Analytical Methods
Density gradient
+
Quantitative Mass Spectrometry
MAMs: Microscopy Approach
• Morphology of ER, mitochondria and MAM
1. Transfection HeLa cells with VAPB or PTPIP51
or VAPB+PTPIP51DNA for 24h
2. Primary Antibodie: F1b (mitochondrial marker)
and VAPB (ER marker)
3. Secondary Antibody (Alexa488; Cy3)
4. Fluorescence Microscopy
•  Overexpression MAM markers to stabilize
ER-Mitochondria contacts
Morphology of Mitochondria and ER
β - Mitochondria
Sfl AMG Microscope (AG Voos)
α-VAPB - ER
Morphology of Mitochondria and ER
β - Mitochondria
can Zeiss Confocal Microscope
α-VAPB - ER
β
PIP51
PB
Sfl AMG Microscope (AG Voos)
MAMs Detection
α-VAPB
ovPTPIP51
+ VAPB
Another way to visualize MAMs: Scatter Plot
TPIP51+VAPB
can Zeiss Confocal Microscope
MAMs Morphology
α
α-F1β
ovPTPIP51+VAPB
ovPTPIP51+VAPB
an Zeiss Confocal Microscope
AG Voos Microscopy Criteria to Detected MAMs
1.  Stabilization of MAMs trough overexpression of MAMs components
2.  Co-localization of mitochondria-ER components
3.  Change of morphology of ER and mitochondria
4.  Linear distribution on a Scatter Plot
Principle Behind Density Gradients
Taken from Cells, A laboratory Manual.
Spector, Goldaman, & Leinwand
MAMs: Biochemical and Analytical protocol
Percoll Gradient: Western Blot results -1Mouse Liver
Mouse Embryonic Fibroblast (MEF)
Nature/Vance Protocol 2009
= MAM marker
Nature/Vance Protocol performed in University of Fer
in 2013
Percoll Gradient: Western Blot Results -2Mouse Liver
Mitochondria
Markers
MAMs
Markers
PDI
Grp75
Pure
MAM
ER
Crude
Pure
HeLa
SY5Y
Mito
Mito
Crude
Mouse Brain
Mito
Mito
MAM
Pure
Crude
MAM
Crude
Pure
MAM
ER
57,1 KDA
75 kDa
IP3R
308 kDa
VDAC
32 kDa
Type1
Tom 40
40 kDa
Tim 23
23 kDa
Percoll Gradient: Quantitative Mass Spectrometry
iver and Brain Mouse
Ca.1700 proteins identified
Comparison with MAMs markers of literature
MS
Identified
Molecular chaperones
Calnexin
MS
MAM
Identified Enriched
bolic Enzymes
oA: Cholesterol acyltransferase (ACAT)
glycerol acyltransferase 2 (DGAT2)
o-6-phosphatase
chain fatty acid-CoA ligase type 4 (FACL4)
chain fatty acid-CoA ligase type 1 (FACL1)
hotidylserine synthase -1, -2 (PSS-1, PSS-2)
KB)
✔
✔
✗
✗
✔
✔ PSS-1
✔
✗
✔ liver
✗
✗
✗
✔ liver
✗
dine Receptors
endoplasmic reticulum Ca2+ -ATPase (SERCA)
/BiP
ERp57
Grp75/Mortalin-2
✔ liver
✗
✔
✗
✔ liver
✗
✔
✗
S-100 protein
S100B
Ubiquitin Ligase
MET30 E3 ubiquitin ligase
Autocrine motility factor receptor (AMFR)
Phosphoacidic cluster sorting protein (PACS-2)
✔
✔
✔
✗
✗
✔
✗
✗
✔ brain
✔ liver
✗
✗
✔
✗
✔
✔
✗
✗
✗
✗
✔
✔
✔
✗
✗
✗
Electron transport chain protein
Cytochrome c
Mitochondrial Fission proteins
✗
✗
Mitofusin 2
Dynamin-related protein 1 (DRP1)
Others
cular chaperones
-1 Receptor
Calreticulin
ERp44
✔
✗
Vesicular-sorting protein
channel or transporter
ceptors
MAM
Enriche
✗
✔
✗
✔ liver
Voltage-dependent anion channel 1 (VDAC1)
Voltage-dependent anion channel 2 (VDAC2)
Voltage-dependent anion channel 3 (VDAC3)
MAMs Isolation through Percoll gradient
  No reproducibility all the tested samples
  MAMs Literature Markers missing
  Is the Percoll highest ring really MAMs fraction??
Sucrose gradient
MAMs Isolation: Sucrose Gradient Protocol
1
Isolation of HeLa mitochondria through manual pottering
and differential centrifugation steps
Sucrose density gradient 20-35% UZ 1h 33000 RPM
4° C
2
3
4
5
6
7
8
Grp78
VAPB
PTPIP51
Tim23
3. Fractionation and TCA 72% precipitation
4. SDS-PAGE and Wester blot
5. Immunodecoration for ER marker (Grp78-Lumen);
Mitochondria marker (Tim23-IM); MAM Markers
(PTPIP51-VAPB)
Expected results:
• ER-Mitochondria (non-MAM) marke
in different fractions
• PTPIP51-VAPB MAM Markers in th
SAME fractions
Percoll vs Sucrose Gradient
Percoll
Sucrose
Colloidal silica
Advantages
l 
Disadvantages
l 
• 
• 
• 
• 
• 
• 
Lowerosmolarity
Fastprepara0on
Con0nuous
Disaccharide
• 
• 
• 
NoTCAprecipita0on • 
Requiresmore
star0ngmaterial
• 
Interferenceofgel • 
running
TCAprecipita0on
Requiresless
star0ngmaterial
Smallgradients:
fasterandeffec0ve
Prepara0ontheday
before
Pronetovaria0ons
Highviscosity
3.2 Results
Percoll vs Sucrose Gradient Results
ated crude Mitochondria from HeLa cells by pottering
GRP78
ER Lumen
ER
n
ER
PTPIP51
Mito OM
Mitochondria
MAM????
MAM???
VAPB
ER Membr.
ER
.
ER
Mitochondria
Tim23
Mito IM
Mitochondria
30%
0%
ll
o
c
r
e
P
20%
se
o
r
c
u
S
MAMs Isolation: Sucrose Gradient + Digitonin
Isolation of HeLa mitochondria through digitonin (0.005%)
and differential centrifugation steps
Sucrose density gradient 20-35% UZ 1h 33000 RPM 4° C
Fractionation and TCA 72% precipitation
SDS-PAGE and Wester blot
Triton %
Digitonin%
0.5
0.1
0.05
0.5 0.1 0.05
0.01
0.5 0.1 0.005 0.001
0.5
0.1 0.005 0.001
mmunodecoration for ER marker (Grp78-Lumen);
ochondria marker (Tim23-IM); MAM Markers (PTPIP51PB)
M
G
T
Supernatant
Pellet
MAMs: Digitonin 0.005% vs Pottering
Pottering
ER Lumen
Digitoning 0.005%
PTPIP51 GRP78
ER
Mito OM
Mitochondria
MAM?
Tim23
VAPB
MAM?
ER
ER
Mitochondria
50
Mitochondria
35%
20%
e
s
o
r
c
Su
20%
se
o
r
c
u
S
•  Isolated Mitochondria from HeLa cells
MAMs: Destruction
Salt Treatment (2M NaCl) washing
• Destroy ionic interaction
• Wash away the ribosomes
• Wash away the ribosomes
Puromycin Treatment
Protein K digestion
• Digestion of outer membrane proteins
• Digestion protein-protein interaction
ated crude Mitochondria from HeLa cells by pottering
+
ER
-
MAM?
ER
ER
PTPIP51
Mito OM
ER
GRP78
ER Lumen
MAMs Destabilization through High Salt Concentration
+
VAPB
ER Membr.
-
MAM?
+
30%
0%
Tim23
Mito IM
Mitochondria
Mitochondria
35
ll
Perco
ted crude Mitochondria from HeLa cells by pottering
20%
e
s
o
r
c
Su
MAMs: Quantification of Sucrose Gradient Fractions
ER + Mitochondria
Intensity
Intensity fraction/Max
Intensity
Intensity fraction/Max
ER + Mitochondria
Fraction N
u
Fraction N
u
mb e r
mber
2M NaCl
Tim23 (Mitochondria IM)
Grp78/BiP (ER Lumen)
Tim23 (Mitochondria IM)
Grp78/BiP (ER Lumen)
MAM
Intensity
Intensity fraction/Max
Intensity
Intensity fraction/Max
MAM
Fraction N
u
PTPIP51 (Mitochondria OM)
(ER Membrane)
Fraction N
u
mber
PTPIP51 (Mitochondria OM)
(ER Membrane)
mber
2M NaCl
MAMs Destabilization by Puromycin and PK
Control
Puromycin
PK (50 µg/ml)
P78
umen
PIP51
o OM
VAPB
embr.
m23
to IM
ated crude Mitochondria from HeLa cells by pottering / Small Gradient (2 ml)
AG Voos Biochemical Criteria to Detected MAMs
1.  Co-localization of MAMs markers (i.e. PTPIP51-mito and VAPB-ER) in
the same fractions of density gradient
2.  The ER-mitochondria non-MAM markers should run in different
fractions depending on their density
3.  Destabilization or destruction of MAMs (i.e. 2M NaCl): the ER,
mitochondrial and MAM markers should be found in different fractions
4.  Stabilization of MAMs (i.e. overexpression of PTPIP51-mito and VAPBER): the co-localization of the ER and mitochondrial markers in the
same density gradient fractions should be even more pronounced
(work in progress)
Conclusions:
1.  PubMed and MAMs: 277 articles – new research field
2.  Detection of mitochondria-ER contacts is very challenging also
because it is very dynamic sub-organelle
3.  Stabilization and destruction
4.  Microscopy fluorescence seems a very good technique to detect
MAMs
5.  Sucrose density gradient, but not percoll, potentially could be a good
technique to isolate MAMs.
Thanks to:
AG Voos
§ 
Prof. Dr. Wolfgang Voos
§ 
§ 
§ 
§ 
§ 
§ 
§ 
§ 
Witold Jaworek
Michael Bruderek
Cornelia Rüb
Sabrina Yamoune
Anne Wilkening
Janina Friedel
Rita Furhmann
Karen Pollecker
§ 
§ 
Ursula Gerken
Mark Sylvester