orian shirihai mitochondrial workshop chile 2013
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Shape Connectivity Fusion Dynamics Autophagy
Orian Shirihai Boston University
Is my protein a mitochondrial protein?
Properties of mitochondrial targeting pre-sequence: • Positively charges amino acids • Hydrophobic domains • α helix
MitoTracker EGFP Merge
GFP
ABC-me
Protein trafficking to mitochondria
0.83+/-0.04
0.18+/-0.04
20µ
20µ
105105
0.81+/-0.07
0.33+/-0.18
0.5+/-0.1
0.81+/-0.07
0.5+/-0.1
0.77+/-0.07
R41A-L46Q-I47Q 0.15+/-0.08
Active drug
Inactive drug
Active Inactive
Effect of antibiotics on protein mitochondrial trafficking
Mitochondrial Morphology
Morphology Marker, suggestive a potential alteration in mitochondrial function
Bioenergetics Ca++ buffering apoptosis
Fusion/fission Dynamics
Why should one care to measure mitochondrial morphology and dynamics?
Nutrients Cause Changes in Mitochondrial Architecture
20 mM Glucose
11 mM Glucose 0.4 Palmitate
5 mM Glucose 0.4 Palmitate
20 mM Glucose 0.4 Palmitate
5 mM Glucose
The size of the individual unit is determined by the balance between fusion and fission
Fragmented
Connected
Super- connected
Fusion Fission Fusion Fission
Control + OPA1 +++ OPA1
INS1
Increased fusion rate may be accompanied by fragmentation
Approaches to the quantification of mitochondrial morphology: 1. Form factor and Aspect Ratio analysis (high throughput)
2. Tracing the matrix boundaries of individual mitochondria (accurate)
Conclusion: Mitochondrial network fragmentation can serve as a sensitive but non-specific marker for: • reduced fusion rate (common under metabolic or oxidative stress) • increased fusion/fission dynamics
Form Factor perimeter2/4πarea
Aspect Ratio major/minor
Branching & length long vs round (tubulation)
Original image
Binarized (0:1) image
.
Koopman W J H et al. Am J Physiol Cell Physiol 2005;289:C881-C890
©2005 by American Physiological Society
Fragmented connected
Beta Cell Mitochondrial Morphology is Altered by Nutrient Load
Effect of glucolipotoxicity on Mitochondrial architecture Quantified by calculating the relative occurrence of tubular vs round mitochondria using Aspect Ratio
Main drawback of morphometric analysis: A. Does not account for dynamics B. Crowded mitochondria may appear connected even if fragmented
Photo-activateable GFP can be used to label the boundaries of an individual mitochondrion
Photo-activatable GFP becomes fluorescent only after absorbing UV light
GFP
PA- GFP PA- GFP
GFP
Matrix-targeted photo-activatable -GFP
Visualizing Mitochondrial Networks
Size distribution of mitochondria as fraction of the total mitochondrial area
This approach is also useful in determining the relationship of the mitochondrial network with other organelles
Mitochondrial morphology of the brown adipocyte cells
MTG Nile red Merge BF
3D
10µm
10µm
5µm
Wikstrom, Sereda, et al unpublished
Z-stack Z-stack Single plane mtDsred
mtDsred
mtPAGFP Merge Single plane Merge
Photoactivation
10µm
5µm
Site of photo-activation
Quantification of fusion dynamics
0 200 400 600 800 10000
2
4
6
8
10
12
14
Time (Seconds)
fission
fusion
Cell-1
4
6
7
8
9
10
11
12
13 14
2
5
3
Fusion and fission are paired and occur as a cluster in which fission follows fusion Resting periods follow the clusters
Network Fusion
Fission
Solitary period
Fusion-fission Cluster
Life cycle of mitochondria : solitary period and the fusion-fission cluster
Mouli et al. Biophysical J. 2009
ER
The Mitochondria Life Cycle
/ 2 Mff/Drp1 Fis1
2cm
Plants Pigeons
The Mitochondria Life Cycle
5-20 MINUTES
1-3 HOURS
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80
MatrixIMM
GFP
F.I.
Time [min]
T=0 min t=55min
INS1
Nor
mal
ized
fluo
resc
ent i
nten
sity
Matrix protein
Inner membrane protein
LNK 999 Control
Quantification of mitochondrial dynamics
0.5
0.6
0.7
0.8
0.9
1
1.1
0 10 20 30 40 50
Time (min)
Average Intensity
(normalized)
FFA
WT /Control
Networked
Punctate and fragmented
Fis1 RNAi Rescues Mitochondrial Dynamics in Cell Exposed to HFG
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
0 10 20 30 40 50
FFA + Fis1 RNAi
Time (min)
Average Intensity
(normalized)
FFA
WT /Control
Networked
Punctate and fragmented
Molina et. al. Diabetes 2009
Morphology Marker, suggestive a potential alteration in mitochondrial function
Bioenergetics Ca++ buffering apoptosis
Fusion/fission Dynamics An essential process that may provide potential mechanism for altered function :bioenergetics etc.
Hyperpolarization
F Intensity
Imaging Mitochondrial Membrane Potential (MMP) R123- Excellent loading; Fast response, Not ratiometric, Substrate of MDR
A. Comparative loading- dependent on MMP, permeability & accessibility
B. Quenching of the preloaded dye- reports fast changes in MMP
depolarization
Hyperpolarization depolarization
F Intensity
Protocol for comparative loading: Incubate cells with R123 ~1µM or less for 30 minutes Wash dye by replacing media 3 times Acquire images of whole cells Analysis: intensity of dye inside mitochondria, use threshold to locate mitochondria No time-lapse, no sequential treatments Control: load parallel a samples in the presence of FCCP , Oligomycin The FCCP treated samples will load less, the OM treated sample will load more Load in the presence of MDR inhibitor, examples: Verapamil, CsA
Protocol for quenching mode: Incubate cells with R123 ~5uM or more for 30 minutes Wash dye by replacing media 3 times Acquire images of whole cells using epi-fluorescence microscopy (wide filed) Analysis: intensity of whole cell Use for time-lapse and sequential treatments Controls: After loading, treat with FCCP, expect an increase in Fluorescence intensity
Normal Islet Diabetic Islet
30µm
Normal Islet Diabetic Islet
30µm
0
5
10
15
20
25
30
0 0.2 0.4 0.6 0.8 1
Tota
l per
cent
cel
ls
0.40
0.50
0.60
0.70
0.80
0.90
2 4 6 8
Mid-low regularity cells
High regularity cells
Regularity bins
Period (min) R
egul
arity
Phase analysis shows Shifted Centroid
High Regularity population
Molecular Probes Inc.
High potential
Low potential
JC-1 emission wavelength is membrane potential dependent
Going subcellular: JC1- Ratiometric dye; Low permeability, Slow response
Mitochondria within a single cells are functionally heterogeneous
JC1 artifact
JC1 wonderful artifacts Beautiful artifacts of JC1
COS7 INS1
Islet Islet
Mito
chon
dria
+
+ +
+
+ +
+
+
+
+ +
+
+
+
Verapamil
+
+ +
+
+ +
+
+
Vm=58Log Dye(out) Dye(in)
+
MDR
TMRE / TMRM
Nuc (A)
Mt (A) 61Log
Nuc (B)
Mt (B) 61Log
Condition A Condition B
MitoTracker Green
Ratio image for ∆Ψ
TMRM
Merge Green/Red- β-cell
MTG TMRE Ratio
Focus dependent
∆Ψ Dependent
+7mV
-7mV
Red (TMRM) Green (Mitotracker)
=R
Ratio image
V
AVG =0mV
∆Ψ =58log R (AVG)
R (each mito)
Heterogeneity standard deviation
Average # pixels
mV
1SD 2SD
0
10
20
30
-22 -18 -14 -10 -6 -2 2 6 10 14
3mM
8mM
1 5 4
2
3
6 oligomycin
3 mM
+
4
5 6 7
3
2
1
8 mM
OM
mV
Frac
tion
pixe
l cou
nt
[%]
Glucose decrease heterogeneity
When membrane potential drops ATP synthase reverses its function: ATP is consumed (ATPase) and protons are pumped out, keeping Ψ intact.
ATP
ADP
Ψ Ψ
Two pathways can generate proton gradient across the inner membrane of the mitochondria
OLIGOMYCIN H+
H+ ATP
ADP
Ψ H+ H+
H+ Ψ H+ H+
H+
H+
H+
H+ H+
Ratio map
Ratio map 8mM Glu + Oligomycin
8mM Glu
Effect of Olygomicin, a blocker of ATP synthase on mitochondrial membrane potential
INS1, 5mM glu
β Cell
H+ ATP
ADP
Ψ H+ H+
H+
Ψ H+ H+
H+
H+
H+
Oligomycin
Summary: Measure MMP when the result can be interpreted and when confounding factors such as uncoupling are addressed Image MMP when subcellular heterogeneity of mitochondria is of interest Use TMRE or TMRM, Don’t use JC1 Always control with Oligomycin and with CCCP or FCCP Consider artifact generated by MDR and dye quenching Use image analysis software to quantify the changes
Uncoupling Resp chain Mt Fuel Avail ATP syn
Res
piro
met
ry
Basal
Oligomycin
FCCP
Mt biogenesis
Mechanism
Demand
Mt morphology (size)
ATP/ADP
Glycolysis (lactate)
∆Ψ
ROS
NADH
M.Me.Suc Reversed by
Mito content (protein/DNA)
Ass
ay
Sam Sereda Guy Las Marc Liesa Linsey Stiles Gilad Twig Jakob Wikstrom Anthony Molina Alvaro Elorza Boston University Barbara Corkey Neil Ruderman Susan Fried
Buck Inst David Nicholls Link Medicine Peter Lansbury Stockholm University Barbara Cannon Jan Nedergaard Seahorse Bioscience David Ferrick
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