Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells
Bethe A. Scalettar1,*,
Patrizia Rosa2,
Elena Taverna2,
Maura Francolini2,
Takashi Tsuboi3,
Susumu Terakawa3,
Schuichi Koizumi4,
John Roder5 and
Andreas Jeromin5
1 Department of Physics, Lewis and Clark College, Portland, OR 97219, USA
2 CNR-Cellular and Molecular Pharmacology Center, Department of Pharmacology,
Via Vanvitelli 32, Milan, Italy
3 Photon Medical Research Center, Hamamatsu University School of Medicine,
Hamamatsu 431-3192, Japan
4 Division of Pharmacology, National Institute of Health Sciences, Tokyo
158-8501, Japan
5 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G
1x5 Canada
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Fig. 1. Western blots (A) showing the distributions of NCS-1-EYFP and endogenous
NCS-1 in postnuclear supernatants (PNS), total membrane fractions (M) and
cytosolic fractions (C) obtained from wild-type and stably transfected PC12
cells. Western blots (B,C) showing the distributions of NCS-1-EYFP, endogenous
NCS-1, synaptophysin (Syn) and SgII in fractions collected after (B) velocity
gradient centrifugation of PNSs and (C) equilibrium gradient centrifugation of
fractions 4-8 from the velocity gradient. The blots also show the distribution
of the Na/K ATPase. Quantitative data (D) showing the distributions of
NCS-1-EYFP, endogenous NCS-1, synaptophysin and SgII in fractions collected
from the equilibrium gradient. Data were obtained using the NCS-1-EYFP band in
the right panel of (C) and the NCS-1, synaptophysin and SgII bands in the left
panel of (C).
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Fig. 2. Deblurred image of a living PC12 cell expressing NCS-1-EYFP. All aspects of
distribution that are visible in this image were also visible in images that
were not deblurred. Bar, 10 µm.
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Fig. 3. Representative dual-color images that demonstrate (A) extensive
colocalization of NCS-1-EYFP and endogenous synaptophysin and (B) minor
colocalization of NCS-1-EYFP and endogenous SgII. NCS-1-EYFP is shown in
green, and synaptophysin and SgII are shown in red. Areas of overlap appear
yellow. Bars, 5 µm.
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Fig. 4. TIRFM image of a growth cone (A) showing the distribution of NCS-1-EYFP on
plasma-membrane-apposed puncta before electrical stimulation. Bar, 2 µm.
(B) Images showing the changes in the appearance of a vesicle that underwent
stimulation-dependent exocytosis. 33 milliseconds after application of the
stimulus, the vesicle fluorescence was widened relative to its initial
fluorescence, and 200 milliseconds after application of the stimulus, the
vesicle essentially was invisible as its associated fluorescence had spread
into the background. Bar, 1 µm. Individual (C) and averaged (D) curves
showing fluorophore movement toward the plasma membrane and spreading of
fluorescence after electrical stimulation. Data obtained from circles and
annuli are represented by closed and open circles, respectively. Fluorescence
is expressed in pixel units (P.U.). Bars, 200 milliseconds and 50
milliseconds.
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Fig. 5. Time-lapse images showing a tubulovesicular organelle containing NCS-1-EYFP
(arrows). Bars, 10 µm.
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Fig. 6. Immunoelectron micrographs showing the morphology of organelles containing
NCS-1 at high resolution in PC12 cells. Most immunoreactivity is likely to
arise from NCS-1-EYFP because endogenous NCS-1 is much less abundant than
NCS-1-EYFP. Immunoreactivity is partially associated with clear vesicles and
tubulovesicular organelles (arrows in B-D) but is absent from SGs (arrowheads
in A and D). M, mitochondrion. Bars, 100 nm.
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Fig. 7. Dual-color images showing that the distribution of vesicles containing (A)
NCS-1-EYFP and (B) synaptophysin-EGFP changes appreciably in 20 seconds. After
20 seconds, overlap (yellow) is associated mostly with vesicles along neurites
(arrows) and with cytosolic NCS-1-EYFP, which generates a diffuse yellow
background in the cell in A. Bars, 10 µm.
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Fig. 8. Trajectories of vesicles in (A) the cell in
Fig. 7A and (B) the growth cone
in Fig. 4A, superimposed on
outlines of the cell and growth cone. Starting vesicle positions are labeled
with a closed circle. Not all of these trajectories were generated
simultaneously. Bars, 10 µm (A) and 2 µm (B).
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Fig. 9. Plots of (A and C) <r2>1/2 versus
t and (B and D) <r2> versus t deduced in
part from the trajectories in Fig.
8A. Letters next to the plots identify the trajectories from which
the plots were derived. Some values were rescaled so that all plots could be
displayed together conveniently. In A, values of
<r2>1/2 were multiplied by 2.5 for
vesicle g. In B, values of <r2> were multiplied by
three and 20 for vesicles a and g, respectively. In C and D,
values for the dashed curves were multiplied by 2.5 and 25, respectively.
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Fig. 10. Confocal images showing the uptake of anti-synaptotagmin antibody by PC12
cells that contain a vector control (Control) and PC12 cells that overexpress
NCS-1 (NCS-1) under basal and UTP-stimulation (UTP) conditions. Images
correspond to 45 minutes of basal uptake (A,B) and 15 minutes of
UTP-stimulated uptake (C,D). Bar, 20 µm.
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© The Company of Biologists Ltd 2002
