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