ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5105–5114 doi:10.1242/jcs.160242
RESEARCH ARTICLE
The position of mitochondria and ER in relation to that of the
secretory sites in chromaffin cells
José Villanueva1, Salvador Viniegra1, Yolanda Gimenez-Molina1, Virginia Garcı́a-Martinez1,
Giovanna Expósito-Romero1, Maria del Mar Frances1, Javier Garcı́a-Sancho2 and Luis M. Gutiérrez1,*
Knowledge of the distribution of mitochondria and endoplasmic
reticulum (ER) in relation to the position of exocytotic sites is
relevant to understanding the influence of these organelles in tuning
Ca2+ signals and secretion. Confocal images of probes tagged to
mitochondria and the F-actin cytoskeleton revealed the existence of
two populations of mitochondria, one that was cortical and one that
was perinuclear. This mitochondrial distribution was also confirmed
by using electron microscopy. In contrast, ER was sparse in the
cortex and more abundant in deep cytoplasmic regions. The
mitochondrial distribution might be due to organellar transport,
which experiences increasing restrictions in the cell cortex. Further
study of organelle distribution in relation to the position of SNARE
microdomains and the granule fusion sites revealed that a third
of the cortical mitochondria colocalized with exocytotic sites
and another third located at a distance closer than two vesicle
diameters. ER structures were also present in the vicinity of
secretory sites but at a lower density. Therefore, mitochondria and
ER have a spatial distribution that suggests a specialized role in
modulation of exocytosis that fits with the role of cytosolic Ca2+
microdomains described previously.
KEY WORDS: Exocytosis, Chromaffin cell, Mitochondria,
Endoplasmic reticulum, Confocal microscopy
INTRODUCTION
Adrenomedullar chromaffin cells are widely used as a model to
study exocytosis of dense vesicles. After stimulation of the
splacnic nerve, these cells release catecholamines in response to
the elevation of the cytosolic Ca2+ concentration ([Ca2+]c) in a
process involving the transport of granules, translocation to the
plasma membrane, docking at the secretory sites and fusion of
membranes with extrusion of soluble contents (Burgoyne et al.,
1993). Major cellular structures, such as the cortical cytoskeleton,
play fundamental roles in different stages of the secretory cascade
(Gutiérrez, 2012), whereas organelles, such as the endoplasmic
reticulum (ER) and mitochondria, control and shape [Ca2+]c
elevations at the subplasmalemmal region (Garcı́a et al., 2006;
Garcı́a-Sancho et al., 2012). In addition, chromaffin granules
could be acting as a Ca2+ reservoir influencing vesicular transport
1
Instituto de Neurociencias, Centro Mixto Universidad Miguel Hernández-CSIC,
Cra de Valencia S/N, Sant Joan d’Alacant, 03550 Alicante, Spain. 2Instituto de
Biologı́a y Genética Molecular (IBGM), Universidad de Valladolid and CSIC, c/
Sanz y Forés, 3, 47003 Valladolid, Spain.
*Author for correspondence ([email protected])
Received 22 July 2014; Accepted 25 September 2014
and exocytosis (Camacho et al., 2008). According to the actual
vision, voltage-operated Ca2+ channels (VOCCs) of the plasma
membrane trigger fast Ca2+ elevations in the cytosol that are
amplified by the ER, thus generating high-Ca2+ microdomains in
the proximity of secretory sites (Garcı́a et al., 2006). Upon
exocytosis, nearby mitochondria take up this Ca2+, thus
restricting the levels of the messenger that reach the cell core
(Villalobos et al., 2002). Therefore, triads composed of plasma
membrane channels, ER and mitochondria optimize Ca2+ signals
in a subtle way (Montero et al., 2000; Garcı́a et al., 2006; Garcı́aSancho, 2012) and avoid large elevations of Ca2+ in the cell core,
which could otherwise trigger cell excitotoxicity (Duchen, 2012).
This view of the role of subcellular organelles in exocytosis is
mostly based on measurements of Ca2+ concentrations in the
interior of organelles and secretory behaviour during activation of
chromaffin cells (Núñez et al., 2007). However, there is a lack of
structural studies that have directly assessed association between
the location of secretory sites and organelle distribution.
In the present work, by using high-magnification confocal
microscopy, we have characterized the populations of
mitochondria and ER elements in cultured bovine chromaffin
cells in relation to their distances to the secretory apparatus or to
the exocytotic sites. Our results support the idea of functional
triads proposed previously and, in addition, we find distinct
subpopulations of organelles located in the immediate vicinity of
secretory sites, which are in position to generate both the
localized Ca2+ signals and the energy needed to sustain
exocytosis.
RESULTS
Mitochondria and ER distribute differently in chromaffin cells
In order to study mitochondria distribution, cultured bovine
chromaffin cells expressing RFP-tagged LifeAct, a 17-aminoacid peptide that binds to F-actin but does not interfere with
cytoskeletal dynamics (Riedl et al., 2008), were also labeled with
Mitotracker Green and visualized by confocal microscopy with a
high-numeric aperture objective (1006; NA 1.45). Sequential
slices were obtained every 0.3 mm along the z-axis from the cell
limits, marked by the presence of the cortical F-actin (left image
in Fig. 1A) to the equatorial plane (right image in Fig. 1A; 11
images taken along 3 mm). Mitochondria (in green) were
abundant in the cortical zone, less were present in the zone
defined by the middle images and their abundance increased
again in the region adjacent to the nucleus. The existence of these
two different populations was further supported when averaging
the normalized fluorescence intensity in experiments performed
in many cells, as depicted in Fig. 1B. From these measurements
the presence of a peripheral population of mitochondria present in
the cortical region (0–1 mm of the cell limit) is evident, and an
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ABSTRACT
RESEARCH ARTICLE
Journal of Cell Science (2014) 127, 5105–5114 doi:10.1242/jcs.160242
internal, perinuclear population, marked by increasing Mitotracker
fluorescence 2–3 mm deeper than the plasma membrane, is also
apparent. Using a different approach, the number of mitochondria
was counted after assigning particles that were above a threshold
level and placing them in groups according to their distance from
the plasma membrane (0–1, 1–2, and 2–3 mm; Fig. 1C). Again the
presence of a cortical population accounting for 40% of the total
mitochondria and a perinuclear population of a similar percentage
was apparent. The region between these two populations had a
smaller proportion of mitochondria (less than 17% of total;
Fig. 1C). The total fluorescence intensity values shown in Fig. 1B,
also gave very similar percentage figures to those for the number of
particles: 38% in the 0–1 mm z-range, 15% in the 1–2-mm region
and 47% in the 2–3-mm region. The distribution of mitochondria
contrasts with the distribution of RFP–LifeAct fluorescence, which
decreases monotonically from the cortical area to the equatorial
area (Fig. 1B).
As an alternative method, the distribution of mitochondria was
analyzed in equatorial images displaying the nuclear position, as
shown in Fig. 2. In this case, the usual position of the nucleus in
one pole of the cell provided evidence that a population of
mitochondria was positioned between the nucleus and the closest
plasma membrane, and therefore could be considered as both
cortical and perinuclear (CP, Fig. 2D). Estimation of this
population in several cells, based on fluorescence determination,
provided evidence that CP mitochondria made up 12%61
(6s.e.m.) of the total mitochondria (Fig. 2E), Therefore, the
mitochondria population associated with the cellular cortex was
determined as being between 28% and 38% depending on the
criteria used (whether CP mitochondria were assigned to the
perinuclear or to the cortical population, respectively).
A similar approach was used to investigate the distribution of
ER, which was labeled with ERtracker Green. In this case, we
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observed a much more uniform distribution in the z-plane, with a
slight increase in the ER fluorescence intensities when moving
from the external cortical region to the internal perinuclear area
(Fig. 3A–C). Averaging ER fluorescence intensities (Fig. 3D) or
counting discrete ER particles (Fig. 3E) showed that the amount
of ER increased gradually from the cortical zone to the cell
interior to reach its maximal value in the regions adjacent to the
nucleus. As above, LifeAct fluorescence decreased monotonically
with displacement towards the equatorial region (Fig. 3D).
In a morphometric analysis, the size and density of
mitochondria and ER elements present in the perinuclear and
the cortical areas were quantified and compared (Table 1). Both
organelles were significantly smaller in size in the cortical region
than in the perinuclear region (see Area column). Interestingly,
the density of mitochondria was higher in the cortical area,
whereas the ER density appeared to be similar in the two regions.
Therefore, the ER and mitochondrial distributions were also
shown to be markedly different using this approach: ER
distribution was relatively homogeneous, whereas mitochondria
showed two different subpopulations.
The existence of different mitochondrial populations in
chromaffin cells was further characterized using electron
microscopy (supplementary material Fig. S1A,B). In this case,
mitochondria were identified by observation of the characteristic
internal cristae (supplementary material Fig. S1C) and its size and
distance to the plasma membrane studied in ten cells.
Interestingly, this experiment provided evidence that the two
populations of mitochondria with cortical and perinuclear
localization each comprised 30–40% of the total number of
mitochondria (supplementary material Fig. S1D). Again, these
estimations depend on the size of the pool of mitochondria that
are assigned as being both perinuclear and cortical. This CP
population was found to constitute ,15% of the total.
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Fig. 1. Two populations of mitocondria are detected by confocal microscopy. (A) Representative confocal images from experiments performed in cultured
chromaffin cells expressing RFP–LifeAct (red channel) and labeled with 100 nM Mitotracker Green (green channel). Images of five confocal planes separated by
0.75 mm, starting in the basal cortical region (left) and ending in the equatorial plane (right) are shown. Scale bar: 3 mm. (B) Mean6s.e.m. values of normalized
fluorescence for the different confocal planes determined for RFP–LifeAct (n5104 images) and Mitotracker fluorescence (n5135 images), represented
as a function of the distance measured from basal cortical to the equatorial plane (3 mm in total). (C) Percentage (mean6s.e.m.) of measured mitochondrial
particles with respect to the total population, corresponding to the cortical population, extending from 0 to 1 mm from the cortical plane (41%64 of the total
particles), the intermediate population, located at between 1 and 2 mm (17%64), and the perinuclear population, present at 2 to 3 mm from the cell
limits (42%63).
RESEARCH ARTICLE
Journal of Cell Science (2014) 127, 5105–5114 doi:10.1242/jcs.160242
Fig. 3. Distribution of the ER in
cultured chromaffin cells.
(A–C) Confocal fluorescence images
depicting the distribution of ERtracker
Green (100 nM) in cells expressing
RFP–LifeAct (red fluorescence).
Shown are panels covering five
confocal planes taken every 0.75 mm
from the basal cell cortex (left) to the
equatorial perinuclear area (right) and
depicting, F-actin labeling (A), the
ERtracker distribution (B), and
combining both channels
(C). (D) Mean6s.e.m. values of
normalized fluorescence for the
different confocal planes determined
for RFP–LifeAct (n5202 images), and
ERtracker (n5184 images)
fluorescence, represented as a
function of the distance measured
from the cortical basal to the equatorial
plane (3 mm in total). (E) Percentage
(mean6s.e.m., n5184 images) of total
ER particles calculated for the cortical
population extending from 0 to 1 mm
(13%62 of the total ER particles), the
intermediate population located
between 1 and 2 mm (23%63), and
the perinuclear population present at 2
to 3 mm from the cell limits (64%65).
Scale bars: 3 mm.
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Fig. 2. Distribution of mitochondria in chromaffin cells with a polarized nucleus. (A) Representative confocal image from the equatorial plane of a cell with
the nucleus localized off-center (polarized nucleus). (B,C) Fluorescence profiles obtained from the same cell as A and showing that there are subpopulations of
cortical and perinuclear mitochondria separated by transition areas (labeled with 100 nM Mitotracker Green). (D) In order to determine the mitochondrial
density in different part of the cells, fluorescence intensity was determined in the cortical (C, extending 1 mm from the cell limits), the transition (T) and the
perinuclear zones (P). In one pole of the cell, the mitochondria can be considered as forming part of the cortical and perinuclear populations (CP population).
(E) Fluorescence determined for the different populations of mitochondria, expressed as a percentage of the total fluorescence (mean6s.e.m. from experiments
performed in 10 cells). Scale bars: 3 mm.
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Journal of Cell Science (2014) 127, 5105–5114 doi:10.1242/jcs.160242
Table 1. Morphometry of mitocondria and ER structures in cultured bovine chromaffin cells
Structures
Localization
Density (particles/mm2)
P-value
Area (mm2)
P-value
Number of structures
Mitochondria
Cortical
Perinuclear
Cortical
Perinuclear
0.4660.05
0.2160.03
0.9460.17
0.8760.26
P,0.005
0.2260.01
0.5160.06
0.2560.02
0.5760.04
P,0.0001
79
69
104
104
ER structures
P.0.05
P,0.0001
Results are mean6s.e.m. P-values were calculated with a non-parametric Mann–Whitney test.
The two mitochondrial subpopulations have different
mobility
Why do chromaffin cells display two different subpopulations of
mitochondria? Distinctive transport of organelles and retention in
the cortical region could play a role, as previously proposed for
vesicle distribution in chromaffin and PC12 cells (Neco et al.,
2003; Rudolf et al., 2003; Gutiérrez, 2012). To test this
hypothesis, we measured the motion of mitochondria and the
ER in different areas of the cytoplasm of chromaffin cells. For
this purpose, particles were created by thresholding of images,
and the centroid of such particles was tracked at 1-s intervals in
experiments performed at room temperature (21–22 ˚C). Motion
was computed from the slope of the plots of the mean square
displacement (MSD) against time for a large number of
mitochondria and ER particles, and the data were averaged to
generate Fig. 4D. In Fig. 4A–C, sequential images separated by
5-s intervals are shown. We illustrate representative examples of
the motion of mitochondria in the cell cortex (polar section;
Fig. 4A) or in an equatorial section deep in the cell interior
(Fig. 4B). ER motion in or an equatorial section is also illustrated
(Fig. 4C). Interestingly, the slopes of the averaged plots of MSD
against time (Fig. 4D) indicated that there was a higher
mitochondria mobility in the cell interior (perinuclear) as
compared with in the cortical region (cortical), suggesting that
motion is slower when mitochondria approach the plasma
Fig. 4. Motion of mitochondria and ER in different
regions of chromaffin cells. Chromaffin cells
expressing RFP–LifeAct were stained with either
Mitotracker Green or ERtracker Green as described in
the previous figures, and imaged at 1-s intervals using a
confocal microscope at room temperature (21–22˚C).
(A,B) Sequence of representative images of
mitochondria separated by 5 s taken either at the polar
cortical plane (A) or at the equatorial plane (B). Images of
ER taken at the equatorial plane are shown in
(C). Motion was analyzed, after creation of particles by
using an appropriate image threshold, by tracking of
particle center and measuring displacement. (D) Plots
(mean6s.e.m.) of MSD against time for a large number
or mitochondria (MITO, n5202 particles) and ER
particles (n5148) in both the cortical and the perinuclear
regions. The plots indicate that ER particles were almost
immobile, whereas mitochondria presented faster motility
in the perinuclear than in the cortical region. Scale bars:
3 mm. (E,F) Cells were incubated with 2 mM
jasplakinolide (JASP) or 1 mM taxol for 30 min to study
the role of cytoskeletal systems in mitochondrial mobility.
The apparent mobility coefficient (mean6s.e.m.) was
estimated as the slope of the plot of MSD against time
(Qian et al., 1991, slope54D). In the perinuclear region,
mitochondria dynamics is sensitive to F-actin inhibitors
and microtubule stabilizers, whereas in the cortical
region mitochondria motion seems to only be controlled
by F-actin. **P,0.01, ***P,0.001 (one-way ANOVA
Kruskal–Wallis test when compared to control values).
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Interestingly, the size estimated for the averaged perinuclear
mitochondria particle is 0.11660.004 mm2 (mean6s.e.m.;
n5184), whereas this value decreased markedly for cortical
mitochondria (0.04960.002 mm2, n5201).
membrane. When compared to mitochondria, ER motion through
the cell cytoplasm was extremely slow, both in the cortical and in
the perinuclear region (Fig. 4D).
The mitochondrial transport seems to involve cytoskeletal
proteins, as the mobility coefficient in the perinuclear region
(calculated from the slope of the MSD against time plot) was
reduced by incubation of the cells with the F-actin inhibitor
jasplakinolide or with the microtubule stabilizer taxol (Fig. 4E).
In contrast, in the cortical region, the motion of mitochondria
seems to be controlled only by F-actin given that taxol did not
significantly influence the mobility coefficient (Fig. 4F).
To summarize, perinuclear mitochondria are transported
through F-actin and microtubular structures and experience
increasing mobility restrictions when approaching the cortical
region, which is formed by a dense meshwork of F-actin. This
retention could be the mechanism responsible for stabilizing the
location of a mitochondrial subpopulation in the vicinity of the
secretory sites.
A further demonstration of the retentive role of cortical F-actin
in stabilizing a mitochondrial population was obtained when we
found that Latrunculin A, an stabilizer of monomeric actin
(Spector et al., 1983), affects the distribution of chromaffin cell
mitochondria, decreasing the cortical population and favoring the
mitochondria in transition (supplementary material Fig. S2).
Journal of Cell Science (2014) 127, 5105–5114 doi:10.1242/jcs.160242
The distribution of cortical mitochondria has a relationship
with the location of the secretory machinery
The cortical mitochondria are best positioned to support the needs
of energy supply and Ca2+ clearance in the proximity of the
secretory apparatus (Montero et al., 2000; Villalobos, 2002;
Garcı́a et al., 2006). In order to address this idea directly, we
expressed RFP–SNAP-25 in cultured chromaffin cells; this
protein dimerizes with syntaxin-1 at the plasma membrane, and
the position of the dimers define the place of granule fusion
(Rickman et al., 2004; López et al., 2009; López-Font et al.,
2010). After cell treatment with Mitotracker Green, the cortical
region was observed in polar images (see example in Fig. 4), and
the distances between the fusion machinery (RFP–SNAP-25
microdomain labeling) and the nearest visualized mitochondria
(the nearest pixel corresponding to mitochondria labeling) were
measured. Interestingly, 25–30% of the SNAP-25 microdomains
colocalized with mitochondria and therefore no distance could be
measured under our optical resolution (Fig. 5A,B). The
distribution of distances shown in Fig. 5B, suggested, in
addition, that there was a second subpopulation of mitochondria
that were located within 0.3 mm, the average diameter of a
chromaffin granule. Therefore, it seems that separate
subpopulations of mitochondria could be distinguished in the
cortical region.
Fig. 5. Distances of cortical
mitochondria and ER to the
secretory machinery. Cells
expressing RFP–SNAP-25 were
incubated with Mitotracker Green or
ERtracker Green as described for the
previous figures. Then cortical
distributions where imaged using a
confocal microscope to study the
distances between the RFP–SNAP25 microdomains and the
mitochondria and ER structures.
Depicted are cortical polar confocal
planes of chromaffin cells showing
mitochondria and SNAP-25
microdomains (A) or ER and SNAP25 microdomains (C). Scale bars:
3 mm. These images where used to
calculate the distributions of
distances (expressed as
mean6s.e.m.) between SNAP-25
clusters and the nearest mitochondria
(B, 107 measurements from 10 cells)
or ER structure (D, 118
measurements from 17 cells) using a
0.1-mm bin size.
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The same approach was used to study the distribution of ER in
relation to the position of the secretory machinery (Fig. 5C).
Again, distances to the nearest RFP–SNAP-25 microdomain were
calculated. The distribution for a large number of ER particles
is shown in Fig. 5D. Approximately 20% of ER structures
colocalized with the secretory machinery patches, whereas a
second pool of cortical ER particles was found within the distance
of a chromaffin granule diameter (0.3 mm; Fig. 5D). The
existence of these two pools is reminiscent of the mitochondrial
organization, suggesting that the same organizing influences
apply to mitochondria and ER.
Exocytotic sites are located in the immediate vicinity of
cortical mitochondria
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Fig. 6. Distances of cortical mitocondria to the secretory sites. Cultured
chromaffin cells incubated with Mitotracker Red were stimulated by
depolarization with a 59 mM KCl solution for 1 min at room temperature (21–
22˚C), and then the secretion was stopped by lowering the temperature by
mixing with ice-cold buffer. Then secretory sites were labeled using an antiDBH antibody followed by a secondary antibody coupled to Cy2 (green
fluorescence) in ice-cold buffer to prevent endocytosis. The distribution of
cortical mitochondria and secretory sites was studied by acquiring confocal
images of the apical cortical area in polar sections (A) to calculate distances
in the membrane plane (XY, 122 measurements from 8 cells), or equatorial
sections (B) to calculate the radial distances (Z, 225 measurements from 12
cells). These distances were used to build a distribution (mean6s.e.m.)
using 0.1-mm bin size (C). Scale bars: 3 mm.
DISCUSSION
In chromaffin cells, mitochondria distribute within two
distinct pools
Two major conclusions can be derived from our study, first
that mitochondria distribute within two major populations, a
perinuclear and a cortical group, whereas the ER seems to
distribute in a relatively homogeneous manner throughout the
cytosol of chromaffin cells. Second, that in relation to the position
of exocytotic sites, both, cortical mitochondria and ER particles
form two distinctly distributed subpopulations, a first one that is
closely associated with the secretory sites and a second one that is
located ,0.3 mm away from the exocytotic sites. These two local
cortical subpopulations could be directly involved it the
regulation of Ca2+ signals and ATP supply in the immediate
vicinity of exocytotic sites.
It is accepted that different subpopulations of mitochondria can
exist in a variety of cellular types, such as synaptic and nonsynaptic mitochondria in neurons (Hollenbeck, 1996), and
sarcolemmal and miofibrillar mitochondria in myocytes
(Chikando et al., 2011). In secretory tissues, the bestdocumented case is the existence of three different
Journal of Cell Science
A more direct procedure to evaluate the proximity of cortical
mitochondria and ER to secretory sites is to estimate the
distances between these organelles and the fusion sites, which
can be labeled with anti-dopamine-b-hydroxylase (DBH)
antibody because DBH incorporates into the plasma
membrane after exocytosis (Patzak et al., 1984; Lopez et al.,
2007). In these experiments, chromaffin cells labeled with
either Mitotracker Red or ERtracker Red were stimulated by
cell depolarization for 1 min by perfusion with 59 mM KCl
solution, Then DBH was immobilized in the membrane by
sudden lowering of the temperature to 2 ˚C prior to DBH
staining with anti-DBH antibody for 2 h in ice-cold buffer.
After cell permeabilization and fixation, incubation with a
secondary antibody coupled to Cy2 (which emits green
fluorescence) allowed simultaneous observation of the fusion
sites (DBH) and organelle distribution (in red). The results are
illustrated in Figs 6 and 7, which compare polar planes
(Fig. 6A; Fig. 7A) and equatorial planes (Fig. 6B; Fig. 7B).
Polar sections of the cortical region were used to measure the
distances separating exocytotic sites and the nearest organelle
in the plane of the membrane (xy-distance), whereas equatorial
sections allowed measuring the distance from the cell
membrane to the nearest organelle (z-distance, perpendicular
to the membrane plane). The distributions found are compared
in Fig. 6C and Fig. 7C. Interestingly, both distance distributions
were similar revealing that: (1) 30–35% of secretory sites
colocalized with mitochondria and, again, that (2) there is an
important population of mitochondria centered around the
300 nm distance. Taking into consideration that the cortical
mitochondria amounted to 40% of total in the cell (Fig. 1), this
means that ,12–14% of total mitochondria localize within a
extremely short distance of active sites, whereas a second
population, corresponding to 25% of the total mitochondria, are
located in close vicinity (a distance of one to two granule
diameters away) to the secretory sites.
A similar analysis of the ER distributions (Fig. 7) indicated
that ,10% of the cortical population is in close association
with exocytotic sites (and again the same results are obtained
when calculating distances in both polar or equatorial
distributions). As for mitochondria (see Fig. 6C), there is a
substantial ER subpopulation that locates within the range of
one to two vesicle diameters away from the secretory sites
(Fig. 7C). Taken together, our results indicate that two distinct
subpopulations of cortical mitochondria and ER exist that can
be stated as being closely associated with the secretory sites.
This physical proximity could be very important for regulation
of the exocytotic process in chromaffin cells, as discussed
below.
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mitochondria and ER structures as being in the immediate
vicinity of the secretory apparatus, thus meaning it is feasible that
they strictly control of fusion events in the local microdomains,
shape [Ca2+]c signals and also regulate the supply of energy for
the needs associated with exocytosis. Could these conclusions be
extended to chromaffin cells in other species? At least in guinea
pig chromaffin cells, a cortical population of mitochondria
constituting around 20% of the total has been described using
electron microscopy (Inoue et al., 1996). This percentage is of the
same order as that described here (between 28% and 38%), and
indicates that chromaffin cells from different species could have
cortical mitochondria.
Fig. 7. Distance of the cortical ER structures to the secretory sites.
Cultured chromaffin cells incubated with ERtracker Red (100 nM) were
stimulated by depolarization with a 59 Mm KCl solution for 1 min at room
temperature (21–22˚C). Secretory sites were labeled in green using antiDBH as indicated in Fig. 6. The distribution of cortical ER and secretory sites
was studied acquiring confocal images of the cortical area in polar sections
(A) to calculate distances in the membrane plane (XY, 122 measurements
from 15 cells), or equatorial sections (B) to calculate the radial distances
(Z, 222 measurements from 23 cells). These distances were used to build a
distribution (mean6s.e.m.) using a 0.1 mm-bin size (C). Scale bars: 3 mm.
mitochondrial subpopulations, namely, perinuclear, perigranular
and sub-plasmalemmal mitochondria, in pancreatic acinar cells
(Park et al., 2001). In these cells, the distinct mitochondria
subpopulations ensure the independent control of cytosolic Ca2+
levels in the different regions of the cell. For example, the
entry of Ca2+ though voltage-dependent Ca2+ channels at
the plasmalemma is rapidly buffered by subplasmalemmal
mitochondria, whereas the nuclear Ca2+signal is spatially
limited by the perinuclear population. By contrast, the [Ca2+]c
signal that controls exocytosis of secretory granules is confined to
the secretory pole by the perigranular mitochondrial pool
(Petersen and Tepikin, 2008; Petersen, 2012). Therefore, even
if we envision mitochondrial structures as forming part of a wider
reticular network, it also seems evident that mitochondria could
be modulating distinct physiological processes in different
regions of the cytosol.
In neuroendocrine chromaffin cells, a number of contributions
have provided evidence for the functional coupling of
mitochondria and ER to VOCCs, resulting in subtle regulation
of [Ca2+]c signals in the subplasmalemma (Montero et al., 2000;
Garcı́a-Sancho et al., 2012). This coupling is so tight and efficient
that it even affects the characteristics of individual fusion events
as measured by amperometry (Miranda-Ferreira et al., 2009). Our
present study precisely locates a substantial subpopulation of
The data reported here could be used to complete and redefine the
role of mitochondria and the ER in shaping Ca2+ signals during
stimulation in the neuroendocrine model of chromaffin cells and
its impact on secretion. Our findings are summarized in the model
shown in Fig. 8. Mitochondria are distributed in three different
groups: (1) cortical (subplasmalemmal), (2) transitional, and (3)
perinuclear (Fig. 8). In addition, the cortical subpopulation can be
subdivided into two further subpopulations: one that is closely
associated with the secretory machinery, and a second that is
located at a distance of one to two granule diameters away from
the plasma membrane, which would be embedded into the
cortical cytoskeleton. The cortical mitochondria can be
distinguished from the perinuclear not only by the distribution
within the cell, but also by their average size and density
(Table 1); cortical mitochondria are smaller and more densely
packed, perhaps because of the reduced space left by cytoskeletal
structures to accommodate organelles in this region (Giner et al.,
2007).
The ER distribution in the cytoplasm is different and increases
in density from the cell periphery to the perinuclear region
(Fig. 3D; Table 1). This distribution is consistent with that
expected for a reticular network and seems to be relatively static,
with very slow motion that seems does not seem to be sensitive to
perturbation of the transport systems based in cytoskeletal
structures. In any case, ER appears as a dense network that
becomes somewhat more tightly packed on approaching the
perinuclear area in our confocal images. Particle analysis
indicates that this increase is mainly due to the increase in size
of the average ER structure rather than a change in density, which
seems relatively homogeneous through the cytosolic space.
The non-homogeneous distribution of mitochondria could be
due to the transport mediated by F-actin and microtubular
structures, and their retention in the F-actin-dense cortex; a
similar mechanism has been proposed for other organelles such as
dense core granules (Neco et al., 2003; Rudolf et al., 2003;
Gutiérrez, 2012). How do the discussed ER and mitochondria
distributions affect the management of exocytotic Ca2+ signals in
chromaffin cells? Existence of two mitochondrial populations at
subplasmalemmal and cell core locations had been proposed
previously on the basis of the extremely bimodal mitochondrial
Ca2+ uptake generated by Ca2+ entry though VOCCs during cell
depolarization. This was attributed to the building up of two
different cytosolic microdomains with [Ca2+]c values near to
50 mM (subplasmalemmal) and 1 mM (cell core), respectively
(Montero et al., 2000; Garcı́a et al., 2006). The subplasmalemmal
mitochondrial pool would include ,50% of mitochondria, which
would be packed within the first 1 mm beneath the plasma
5111
Journal of Cell Science
A model to understand the impact of mitochondria and ER
populations in shaping Ca2+ signals during stimulation
RESEARCH ARTICLE
Journal of Cell Science (2014) 127, 5105–5114 doi:10.1242/jcs.160242
Fig. 8. A model of the distribution of
mitochondria and ER structures in chromaffin
cells. Subpopulations of mitochondria located at
the cortical (i), intermediate (ii) and perinuclear
(iii) regions are represented, taking into account
their calculated mean size and density (Table 1).
Subplasmalemmal mitochondria and ER
structures show a high degree of colocalization
with the secretory machinery at the cortical area.
A second pool of both organelles was embedded
in the F-actin cortex, represented as a grey
meshwork. The ER density is relatively uniform
throughout the entire cytosol. The increase in
fluorescence in the perinuclear area (Figs 1 and
2) is due to the increased size of the ER
structures. The distances of the different
mitochondria and ER pools to the secretory sites
are shown schematically in the right panel.
5112
takes longer for the subplasmalemmal mitochondria than for the
cell core mitochondria (Villalobos et al., 2002; Garcı́a et al., 2006).
In summary, our study supports the hypothesis that there are
specialized mitochondrial populations serving different roles in
the cortical and perinuclear areas of neuroendocrine cells, and
gives structural support for idea of fine tuning of Ca2+ signals by
the plasma-membrane–mitochondria–ER triads (Montero et al.,
2000) in the proximity of secretory sites. Future studies modeling
the influence of the organelle distributions proposed here and the
insertion of organelles in the cytoskeletal cages surrounding
secretory sites (Torregrosa-Hetland et al., 2011) will be needed to
understand the impact of such distributions in the fine tuning of
Ca2+ signals and secretion.
MATERIALS AND METHODS
Chromaffin cell preparation and culture
Chromaffin cells were isolated from bovine adrenal glands by
collagenase digestion, and they were further separated from the debris
and erythrocytes by centrifugation on Percoll gradients as described
previously (Gutierrez et al., 1988; Gil et al., 2002). The cells were
maintained as monolayer cultures in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum, 10 mM
cytosine arabinoside, 10 mM 5-fluoro-29-deoxyuridine, 50 U/ml
penicillin and 50 mg/ml streptomycin. The cells were harvested at a
density of 150,000 cells/cm2 in 22-mm diameter coverslips coated with
poly-lysine. Cells were used between the third and sixth day after plating.
For experiments, the cell culture medium was replaced with Krebs/
HEPES (K/H) basal solution containing (in mM): NaCl, 134; KCl, 4.7;
KH2PO4, 1.2; MgCl2, 1.5; CaCl2, 2.5; glucose, 11; ascorbic acid, 0.56
and Na-HEPES, 15, pH 7.4. Cells were stimulated for 1 min using a
depolarizing solution with high levels of K+ (59 mM; obtained by
isosmotically replacing NaCl by KCl) in the K/H basal solution.
Dynamic confocal images of the F-actin cytoskeleton,
mitochondria and the ER
F-actin structures were labeled by expression of enhanced GFP- or RFPtagged LifeAct, a 17-amino-acid peptide that binds to F-actin without
altering its dynamics in vivo (Riedl et al., 2008) or in vitro studies. In these
experiments, chromaffin cells were transfected using the Amaxa basic
nucleofector kit for primary mammalian neuronal cells according to the
manufacturer’s instructions (Program 0-005, Amaxa GmbH. Koehl,
Journal of Cell Science
membrane (Villalobos et al., 2002). The maximal capacity of the
mitochondrial Ca2+ uptake is so high that 10% of the total
mitochondria, occurring at only 0.25 mm beneath the plasma
membrane, would be enough to sink essentially all the Ca2+
entering though maximally activated VOCCs (Villalobos et al.,
2002). These data fit with the pools and sizes proposed here. The
existence of the two subpopulations of cortical mitochondria and
a dense but relatively homogeneous ER ensures the presence of
both (1) a general system for amplification of Ca2+ signals using
Ca2+-induced Ca2+ release (CICR), and (2) an specialized
mitochondrial-buffering system controlling the progression of
the [Ca2+]c wave at two levels: the immediate space surrounding
Ca2+ channels and the fast-releasable vesicle pool of the secretory
machinery (Heinemann et al., 1994; Montero et al., 2000;
Villalobos et al., 2002; Garcı́a et al., 2006; Garcı́a-Sancho et al.,
2012), and a second level controlling the lower Ca2+ levels
needed for recruiting new vesicles embedded in the cortical
network (Montero et al., 2000; Villalobos et al., 2002; Garcı́a
et al., 2006; Garcı́a-Sancho et al., 2012). In addition, these
cortical subpopulations of mitochondria will be supplying the
ATP needed for active transport, as well as for vesicular priming
and maturation (Gunter et al., 1994; Kumakura et al., 1994;
Rizzuto et al., 2000; Villalobos et al., 2002).
The perinuclear mitochondria could fulfill a different role,
protecting the nucleus from the [Ca2+]c signals generated in the
periphery by limiting progression to the nucleoplasm (Chamero
et al., 2002; Gerasimenko and Gerasimenko, 2004; Alonso et al.,
2006). The relatively small increase in the Ca2+ concentration
inside the matrix of these mitochondria might nevertheless be
enough to stimulate respiration, thus also increasing the ATP
supply at the cell core (Villalobos et al., 2002). This energy supply
might be required for mobilizing new secretory vesicles from the
reserve pool as well as supporting the activity of other perinuclear
organelles, such as the Golgi, nucleoplasm or even the nuclear
structures (Montero et al., 2000; Villalobos et al., 2002; Garcı́a
et al., 2006; Garcı́a-Sancho et al., 2012). The size of the
mitochondrial Ca2+ load provides a ‘memory’ that forces an
increase of energy production that lasts until clearance of the Ca2+
debt by the mitochondrial Na+/Ca2+ exchanger is completed. This
Germany) as described previously (Villanueva et al., 2012). At 2 days after
cell transfection, cells were incubated either with a 1 mM Mitotracker
Green or Red or with the same concentration of ERtracker Green
(BODIPY–FL-glybenclamide) or ERtracker Red (BODIPY–TR-gglibenclamide) for a 15-min period at room temperature.
Fluorescence confocal images were obtained using an Olympus
Fluoview FV300 confocal laser system mounted on an IX-71 inverted
microscope incorporating a 1006 PLAN-Apo oil-immersion objective
with 1.45 NA. Excitation was achieved using argon and helium-neon
visible light lasers. Images were processed using the ImageJ program
with different plugins [particle centroid tracking, region of interest (ROI)
measurements, image average, multiple channel image comparison, and
colocalization analysis]. For the analysis of the motion of the
mitochondria and the ER, particles were generated by image
thresholding, obtaining ellipsoid forms, and the centroid position was
tracked at 1-s intervals. Further analysis such as the mean square
displacement (MSD) determinations according to Qian et al., 1991, was
performed using in-house macros for Igor Pro (WaveMetrics Inc., Lake
Oswego, OR). The plot of MSD against time was used to analyze the area
explored by a particle in motion from an initial position, and it is essential
to calculate the diffusion coefficient and to define the motion as confined
or not confined (Qian et al., 1991).
Electron microscopy images
Chromaffin cell pellets were fixed with 2.5% glutaraldehyde in 0.2 M
cacodylate buffer at pH 7.0 for 2 h at 4 ˚C. Then pellets were washed with
a solution of 2.5% (w/v) sucrose in 0.2 M cacodylate buffer overnight and
then post-fixed using 1% osmium tetroxide in 0.2 M cacodylate buffer
over 2 h. After extensive washing, the pellets were stained with 2%
aqueous uranyl acetate for 2 h. Pellets were washed again, and
dehydrated through an ethanol series (30%, 50%, 70%, 80%, 96% and
99%, 15 min each) and propylene oxide for 30 min at room temperature.
Finally, cell pellets were embedded in epoxy resin. Ultrathin sections
(70 nm) were obtained using a Leica UC6 ultramicrotome and transferred
onto copper grids (200 mesh). After staining with uranyl acetate for
5 min and lead citrate for 1 min, the ultrathin sections were analyzed
with a JEOL 1011 a 80-kV transmission electron microscope using a
Gatan BioScam mod 792 digital camera to capture the images.
Graphics were obtained with IgorPro, Graphpad Prism (GraphPad
software, San Diego, CA) and Adobe Photoshop 7.0. A non-parametric
Mann–Whitney test for paired samples or a one-way ANOVA Kruskal–
Wallis test was used to establish statistical significance (differences
were considered significant when P,0.05). Data are expressed as
mean6s.e.m. obtained from experiments performed in a number (n) of
individual cells from at least three different cultures.
Competing interests
The authors declare no competing interests.
Author contributions
J.V. performed most of the experiments and analysed data. S.V. performed and
analysed the experiments concerning the motion of organelles. Y.G.-M. performed
and analysed electron microscopy experiments. V.G.-M. and M.d.M.F. assisted in
the over-expression experiments and chromaffin cell cultures. G.E.-R. assisted in
the confocal fluorescence experiments. J.G.-S. helped in the design and writing of
the paper. L.M.G. designed the experiments and wrote the paper.
Funding
This study was supported by grants from the Spanish Ministerio de Economia y
Competitividad [grant numbers BFU2011-25095 to L.M.G., and BFU201017379BFI to J.G.S.].
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.160242/-/DC1
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