1 Monitoring mitochondrial [Ca 2+ ] dynamics with rhod-2, ratiometric pericam and aequorin Rosalba I Fonteriz, Sergio de la Fuente, Alfredo Moreno, Carmen D. Lobatón, Mayte Montero and Javier Alvarez Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, SPAIN. Corresponding author: Javier Alvarez Instituto de Biología y Genética Molecular (IBGM) Departamento de Bioquímica y Biol. Mol. y Fisiología, Facultad de Medicina, Ramón y Cajal, 7 E-47005 Valladolid, SPAIN Tel: +34-983-423085 FAX: +34-983-423588 e-mail: [email protected]*Manuscript
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1
Monitoring mitochondrial [Ca2+
] dynamics with
rhod-2, ratiometric pericam and aequorin
Rosalba I Fonteriz, Sergio de la Fuente, Alfredo Moreno, Carmen D.
Lobatón, Mayte Montero and Javier Alvarez
Instituto de Biología y Genética Molecular (IBGM), Departamento de
Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina,
Universidad de Valladolid and Consejo Superior de Investigaciones
Científicas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, SPAIN.
Corresponding author:
Javier Alvarez Instituto de Biología y Genética Molecular (IBGM) Departamento de Bioquímica y Biol. Mol. y Fisiología, Facultad de Medicina, Ramón y Cajal, 7 E-47005 Valladolid, SPAIN Tel: +34-983-423085 FAX: +34-983-423588 e-mail: [email protected]
[19] R. Rizzuto, A.W. Simpson, M. Brini and T. Pozzan, Rapid changes of
mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin, Nature
358 (1992) 325–327.
[20] M.J. Barrero, M. Montero, J. Alvarez, Dynamics of [Ca2+] in the endoplasmic
reticulum and cytoplasm of intact HeLa cells, A comparative study. J. Biol. Chem.
272 (1997) 27694-27699.
[21] R. Rizzuto, P. Pinton, W. Carrington, F. S. Fay, K. E. Fogarty, L. M. Lifshitz, R. A.
Tuft, T. Pozzan. Close Contacts with the Endoplasmic Reticulum as Determinants
of Mitochondrial Ca2+ Responses. Science 280 (1998) 1763-1766.
[22] R. Rizzuto, C. Bastianutto, M. Brini, M. Murgia, T. Pozzan. Mitochondrial Ca2+
Homeostasis in Intact Cells. J. Cell Biol. 126 (1994) 1183-1194.
20
Figure Legends
Fig. 1. Monitorization of [Ca2+]M with mitRP. HeLa cells expressing mitRP were
stimulated with 100M histamine as indicated. The traces show a typical single
cell time course of the fluorescence obtained at 415nm and 485nm excitation, as
indicated. The higher trace shows the ratio F485nm/F415nm.
Fig. 2. Effects of two consecutive agonist stimulations on [Ca2+]M measured with rhod-
2, aequorin or pericam. A. Hela cells were loaded with 1M rhod-2 AM and then
stimulated with 100M histamine as indicated. Each trace is the mean of all the cells
present in the microscope field (13 and 16) in two different experiments. These traces
are representative of the results obtained in 22 similar experiments. B. Hela cells
expressing mitRP were stimulated with 100M histamine as indicated. The upper
traces correspond to measurements obtained in three different single cells. The lower
trace is the mean of all the cells present in the microscope field (16 cells). This
experiment is representative of 23 similar ones. C. Hela cells expressing mitmutAEQ
and reconstituted with coelenterazine n were stimulated with 100M histamine as
indicated. In the lower trace, cells were loaded with 2M rhod-2AM prior to the
experiment. The inset shows statistical data of the mean value of the first and second
[Ca2+]M peak obtained either in control cells (C1 and C2, n=11) or in cells loaded with
rhod-2 (R1 and R2, n=7).
Fig. 3. Effects of two consecutive Ca2+ additions in permeabilized cells on [Ca2+]M
measured with rhod-2, rhodFF, aequorin or pericam. A. Hela cells were loaded with
1M rhod-2AM and then permeabilized and stimulated twice with a 10M [Ca2+] buffer
as indicated. As in the rest of the panels of this figure, EGTA-containing intracellular
medium was perfused before and in the intervals between Ca2+ applications. The trace
21
shown is the mean response of 20 cells present in the microscope field and is
representative of 11 similar experiments. B. Hela cells were loaded with 2M rhodFF-
AM and then permeabilized and stimulated twice with a 10M [Ca2+] buffer as
indicated. The trace shown is the mean response of 12 cells present in the microscope
field and is representative of 7 similar experiments. C. Hela cells expressing mitRP
were permeabilized and stimulated twice with a 10M [Ca2+] buffer as indicated. The
trace shown is the mean response of 22 cells present in the microscope field and is
representative of 10 similar experiments. D. Hela cells expressing mitmutAEQ and
reconstituted with coelenterazine n were permeabilized and stimulated twice with a
10M [Ca2+] buffer as indicated. In the right panel, cells were loaded with 2M rhod-
2AM prior to the experiment. These experiments are representative of 3 similar ones of
each type. Temperature was 22ºC.
Fig. 4. Effects of Ca2+ addition to permeabilized cells after mitochondrial
depolarization. Hela cells expressing mitmutAEQ and reconstituted with
coelenterazine were permeabilized and stimulated with 10, 20 or 100M [Ca2+] buffers
in the presence or in the absence of 2M FCCP, as indicated. Temperature was 22ºC
in panels A, B and C, and 37ºC in panels D and E. The experiments shown are
representative of 3-4 similar ones of each type. In panel F, Hela cells expressing mitRP
were permeabilized and stimulated with a 10M [Ca2+] buffer in the presence of 2M
FCCP. The trace shown is the mean response of 15 cells and is representative of 4
similar experiments.
Fig. 5. Changes in mitochondrial morphology induced by rhod-2 loading. Hela cells
expressing mitEGFP were imaged with a confocal microscope before and at different
times during rhod-2 loading. In panel A, the effects of loading with 1 or 5M rhod-2AM
are shown. In panel B, cells were loaded with 2M rhod-2AM for 30 min and then the
22
dye was washed and further images were taken 6, 25 and 40 min after that. The
experiments shown are representative of 7-17 similar experiments of each type.
Fig. 6. Effect of loading with rhod-2 or rhod-FF on agonist-induced cytosolic and
mitochondrial [Ca2+] peaks. In the upper panel, Hela cells expressing cytAEQ were
reconstituted with coelenterazine, loaded with different concentrations of rhod-2AM or
rhodFF-AM and then stimulated with 100M histamine to measure the cytosolic [Ca2+]
peak. In the lower panel, Hela cells expressing mitmutAEQ were reconstituted with
coelenterazine, loaded with different concentrations of rhod-2-AM or rhodFF-AM and
then stimulated with 100M histamine to measure the mitochondrial [Ca2+] peak. The
insets on the right shows statistical data (mean s.e.m.) of several similar experiments
of each type (number of experiments indicated over each bar). Significance was
obtained by the ANOVA test (*, p<0,05; **, p<0,005; ***, p<0,0005).
Fig. 7. Effect of rhod-2 loading on mitochondrial membrane potential measured with
TMRE. Hela cells were loaded with 100nM TMRE and imaged in a confocal
microscope. Then, 5M rhod-2AM was added and images were taken 5, 15 and 30 min
after that. Mean fluorescence intensity was 78%, 58% and 47% of the initial one after
5, 15 and 30 min of rhod-2 loading, respectively. This experiment is representative of
10 similar ones.
Fig. 8. Effect of illumination and rhod-2 loading on mitochondrial morphology. HeLa
cells expressing mitEGFP and loaded or not with 1M rhod-2AM were imaged
with a confocal microscope. After taking an initial image (panel A, control; panel
D, rhod-2 loaded), cells were epi-illuminated for 1 min through a bandpass 515-
560nm filter. Then, two additional images were taken 1 min (panel B, control;
panel E, rhod-2 loaded) and 5 min (panel C, control; panel F, rhod-2 loaded) after
23
the end of the illumination period. The images shown are representative of the
results obtained in 6 experiments with control cells and 10 experiments with
rhod-2 loaded cells.
Fig 1
40
60
80
100
120
140
160
180
Flu
ore
scen
ce
(a
.u.)
1 min
415nm
485nm
1,5
2,0
2,5
3,0
3,5
4,0
4,5
Excitation r
atio (
485/ 415 n
m)
His
Figures
F/F
0 5
45n
m[C
a2
+] M
(M
)
C10
50
100
150
C2 R1 R2
0
50
100
150
200his
his
+rhod-2
2 min0
50
100
150
200 his his
control
C
Fig 2
A his
his0,9
1,0
1,1
0,8
2 min
his
his
0,7
0,8
0,9
1,0
1,1
rhod-2
aequorin
F485/F
415
flu
ore
scen
ce r
ati
o
cel 2
2,0
2,5
3,0
3,5
4,0
2 min
Mean 16 cells
1,5
2,0
2,5
3,0
3,5
4,0
2,0
2,5
3,0
3,5
4,0
4,5
cel 1
his hisB pericam
2,5
3,0
3,5
4,0
4,5
5,0
5,5
cel 3
Ca2+ 10μM
Ca2+
10μM
0,75
1,00
1,25
1,50F
/F0 5
45nm
Ca2+
10μM
mean 20 cells
2 min
A rhod-2
Ca2+
10μM
mean 11 cells
F4
80/F
415
ratio
mean 22 cells
2 min
0,5
1,0
1,5
2,0
2,5
3,0
3,5Ca2+
10μMCa2+
10μM
C pericam
0
200
400
600
800
1000
5 min
[Ca
2+] M
(M
)
Ca2+
10MCa2+
10M
Ca2+
10MCa2+
10M
rhod-2
loaded
D aequorin
0,9
1,0
1,1
1,2
2 min
F/F
0 5
45nm
Ca2+ 10μM
Ca2+
10μM
Ca2+
1mM
rhod-FFB
mean 12 cells
Fig 3
0
100
200
300
400
Ca2+
10M
[Ca
2+] M
(M
)
Fig 4
A
2 min0
2
4
6
8
10
Ca2+
FCCP
10M
20M
B
0
2
4
6
8
10
Ca2+ 10M
FCCP
37ºC
E
0
50
100
150
200
250Ca2+
10MD
22ºC0
10
20
30
40
50Ca2+ 100M
FCCPC
2,0
2,5
3,0
3,5
4,0
FCCP
Ca2+ 10μMR
atio F
480/F
415
F
t=0 15 min 30 min
rhod2-AM 5μM
rhod2-AM 1μM
t=0 15 min 30 min
A
Fig 5A
rhod2-AM 2μM loading
t=0 15 min 30 min
wash6 min 25 min 40 min
B
Fig 5B
1 min
+rhod2
2μM
+rhodFF
2μM
His
+rhod2-10uM
+rhodFF10
His
+rhod2
10μM
His+rhodFF
10μM
His
0
10
20
30
40
Control
His
Control
0.0
0.2
0.4
0.6
0.8
1.0 His
[Ca
2+] c
(M
)[C
a2
+] M
(M
)
Ct
rh2 5M
rh2 1
0M
rhF
F 1
0M
0,0
0,2
0,4
0,6
0,8
1,0
[Ca
2+] c
(M
)Ct
0
10
20
30
40
[Ca
2+] M
(M
)
*
*****
rh2 1
0M
rhF
F 1
0M
rhF
F 2M
rh2 5M
rh2 2M
Fig 6
6 54
4
12 4
6
3
3
3
t = 0 min t = 5 min
t = 15 min t = 30 min
Fig 7
Fig 8
control rhod-2 loaded
A D
EB
C F
t=0
t=+1 min
t=+5 min
I can certify that all authors of the manuscript entitled “Monitoring mitochondrial [Ca2+] dynamics with rhod-2, ratiometric pericam and aequorin” have seen and approved the manuscript as submitted. Dr. Javier Alvarez Professor of Biochemistry Corresponding author
*Co-author Approval Statement
I can certify that there are no conflicts of interest of any kind regarding the manuscript entitled “Monitoring mitochondrial [Ca2+] dynamics with rhod-2, ratiometric pericam and aequorin”. Dr. Javier Alvarez Professor of Biochemistry Corresponding author