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https://www.abbs.info ISSN 0582-9879 |
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Short Communication |
Dynamic Imaging of Single Secretory Granule in Cultured PC12 Cells
WU Zheng-Xing, XIA Sheng, XU
Liang, BAI Li, XU Tao*
( Institute
of Biophysics and Biochemistry, Huazhong University of Science and Technology, Wuhan
430074, China )
Abstract Fluorescent labeling and dynamic imaging of secretory vesicles are
new powerful means to study the mechanisms of protein and membrane trafficking.
The large dense-core granules in PC12 cells were labeled by transfecting EGFP-hpNPY
chimera and imaged with epi-fluorescence and evanescent field (EF) fluorescence
simultaneously. Under epi-fluorescence illumination, the cells exhibited nearly
uniform fluorescence, however, EF-fluorescence excitation revealed distinct
fluorescent spots corresponding to GFP-labeled granules. The trafficking,
docking and fusing with plasma membrane of individual granule in cultured PC12
cells were observed directly by EF fluorescence imaging.
Key words TIRFM; GFP; granule;
PC12 cells; exocytosis
Vesicular
trafficking, exocytosis and endo-cytosis are main tracks for spatially and
temporally ordered transport of membrane and proteins in cell, and form the
basis of different cellular activities， such as the release of neural transmitters in neurons, secretion of
hormones in endocrine cells, insertion of proteins into plasma membrane, and so
on. In recent years, the study on the mechanism of vesicular trafficking and
exocytosis has undoubtedly turned to be a hot spot and frontier in biophysics
and neuroscience.
Exocytosis can
be studied by capacitance measurement of the cellular surface and amperometric
detection of the released molecules. However, these two methods can’t provide
information on vesicle location, translocation, tethering or docking. Although
confocal fluorescence microscope has higher spatial resolution compared with
conventional epi-fluorescence microscope, it has a lower efficiency of
fluorescence collection, higher toxicity to cell, stronger photo-bleaching and
lower temporal resolution. Multi-photon fluorescence microscope has the
advantage of intrinsic three-dimensional spatial resolution and no interference
of out-of-focus fluorescence, but the spatial resolution limits its application
in studying dynamics of subcellular structure of 200 nm or less in cell.
In total
internal reflection fluorescence microscope (TIRFM), only a thin layer close to
the cover glass-solution interface is excited by the so-called evanescent field
(EF). The EF decays exponentially, and its penetration depth (where the
intensity declines e-fold) is only thirty to a few hundred nanometers,
corresponding to the thickness of ultra-thin sample section of electronic
microscope. The influence of out-of-focus background fluorescence is reduced
dramatically, which endues TIRFM great spatial sensitivity. What’s more, TIRFM
is extremely sensitive to the objects’ vertical movement, as objects will
brighten when they approach the cover glass and be dim when they retreat. In
depth discrimination, TIRFM is up to tenfold better than confocal microscope.
Moreover, TIRFM has a weak fluorescence bleaching and low light toxicity. All
these advantages make TIRFM naturally a good tool to observe and study the
events of granules chronically including trafficking, docking, and fusion with
plasma membrane[1－6].
In this study, a
prismless TIRF microscope was constructed and used to monitor the docking,
undocking and fusion of single granule labeled by enhanced green fluorescent
protein-human pro-neuropeptide Y (EGFP-hpNPY) in cultured PC12 cells.
1 Materials
and Methods
1.1 Materials
PC12 cells were
bought from the Storage Center for Typical Species of Wuhan University. Plasmid
EGFP-hpNPY was provided by Prof. Wolhard Almers. DMEM and serum were purchased
from Gibco. Unless otherwise specified, other chemicals used were all from
Sigma.
1.2 Methods
1.2.1 Cell
preparation and granule labeling PC12
cells were cultured in DMEM medium supplemented with 5% fetal bovine serum and
10% horse serum. The plasmid vector encoding human pro-neuropeptide-Y fused
into the N-terminal of EGFP was amplified in DH5αF′E. coli. The bacteria were screened by 50 mg/L kanamycin. Then plasmid was
extracted and purified by method of alkali splitting. PC12 cells were
transfected with the plasmid by electroporation in 4 mm chamber at 360 V and
975 μF. After two
days of recovery, the cells were cultured and screened in selective DMEM medium
containing 400 mg/L G418. For experiment, PC12 cells were plated on a round
cover glass (refractive index n488=1.80, thickness=0.17 mm,
Olympus) mounted on an observation chamber and incubated in bath solution
containing (in mmol/L): 135 NaCl, 2 KCl, 5 CaCl2, 2 MgCl2,
20 Glucose, 10 Na-HEPES, pH 7.2.
1.2.2 Setup
of TIRFM system We
constructed the TIRFM setup based on the prismless and through-the-lens system.
The components included IX-70 invert microscope with 1.65 NA objective lens
(100×, Apo O HR
Olympus), Polychrome IV system for epi-fluorescence illumination (TILL
Photonics GmbH, Germany), argon ion laser system (Melles Griot Inc, USA),
KineFLEX kinematic singlemode fiber and fiber coupler (Point Source Co, UK),
SensiCam super VGA CCD camera and frame grabber (PCO Co, Germany), piezo
Z-drive and E-622 LVPZT amplifier/servo control unit (Physik Instrument,
Waldbronn, Germany), GFP filter set (Chroma, Brattelboro, VT, USA), IBM-PC
compatible computer.
Fluorescent
light was filtered by GFP filter set and collected through the lateral port of
the inverted microscope. Immersion oil (refractive index n488=1.81,
Cargille Laboratories Inc, NJ, USA) was used to bridge the optical contact
between the objective and the cover glass. Images were recorded by scientific
cooled CCD camera which was controlled by Polychrome IV, frame grabber and
image software TILL vision 4.0 (TILL photonics). The digital images from the
camera were grabbed by the frame grabber and stored in hard disk. Exposure time
was adjusted according to the fluorescence intensity of images. Time-lapsed
images were obtained in frequency of 2 to 10 Hz. Both single and time-lapsed images
were viewed, processed and analyzed by TILL vision 4.0 and Photoshop 7.0
software. The dynamics of image data were analyzed using Igor Pro 4.03
(WaveMetrics Inc, Oregon, USA).
1.2.3 Calibration
of the image system 200
μL 20% bovine
serum albumin (BSA) suspension containing 175 nm fluorescent beads (Ex/Em=505
nm/515 nm, PS-Speck Microscope Point Source Kit, Cat. No. P-7220) was added on
the cover glass. After the beads were dried, they were fixed on different
levels from the cover glass. Using piezoelectric drivers, with the primary
focal plane set at the upper surface of the cover glass (0 μm), the object lens was moved from
(－2) μm to (+2) μm with a step length of 50 nm. At
each vertical plane, an image was taken with epi-fluorescence and EF-fluorescence.
The fluorescence intensities of the beads at different vertical plane were used
to calibrate the TIRFM system.
2 Results
2.1 Depth
calibration of EF
When a parallel
beam of light in a medium of high refractive index (n1)
strikes an interface with a medium of lower refractive index (n2),
it suffers total internal reflection. If the angle of incidence, α(θi), exceeds the so-called critical
angle (θi.c.), totol internal reflection generates an EF in the medium of lower
refractive index. EF decayed exponentially with distance from the dielectric
interface. The exponential decay constant d of EF intensity called “penetration depth” can be calculated as the following
function.
In which λ0 was the wavelength of the incident light. Calibration of
penetration depth of EF was needed for calculating the distance of granules to
plasma membrane, and it was also an essential step in construction of TIRFM.
From the
fluorescent images of 175 nm beads we got the curve of fluorescence intensity
against vertical distance. The curves fitted well with Gaussian function. The
maximum fluorescence intensity of Gaussian fit represented the fluorescence
intensity of beads at their focal plane. Fig.1(A) and Fig.1(B) displayed the
fluorescence intensity curves from epi-fluorescence and EF-fluorescence
illumination, respectively. Under epi-fluorescence illumination, the beads at
different distance from cover glass were almost equally bright [Fig.1(A)].
While in EF-fluorescence, the remote bead was dimmer, reflecting the decaying
EF at different distance [Fig.1(B)]. To normalize the variation of beads
fluorescence, peaks of fluorescence intensity ratio
(EF-fluorescence/epi-fluorescence) of beads were plotted against their vertical
distances in Fig.1(C). The data could be well described by an exponential
function with a decline constant of τ= 218 nm which represented the penetration depth of EF. By changing
the distance of laser beam in respect to the light axis of condenser, different
incident angles of excitation light and hence different penetration depth of
evanescent field could also be achieved [Fig.1(D)].
Fig.1 Illustration
of evanescent field illumination and calibration of the penetration depth
(A) Fluorescence intensity
curves of 175 nm beads at different levels under epi-fluorescence illumination.
(B) Fluorescence intensity curves of 175 nm beads at different levels under
EF-fluorescence illumination. The data points were fitted with a Gaussian
function (continuous traces). (C) The peak fluorescence intensities derived
from the Gaussian fits under EF illumination were divided by the peak fluorescence
intensities of epi-illumination. The ratios displayed a single exponential
decay versus their vertical distances from the coverglass. (D) Exponential
fitted curves of fluorescence intensity ratios as a function of vertical distances
at three different incident angles.
2.2 Epi-fluorescence
and EF-fluorescence imaging of green fluorescent beads
To further
confirm the TIRFM imaging, fluorescent beads of 6 μm were mixed with those of 175 nm
in diameter. Places where 6 μm fluorescent beads surrounded by 175 nm ones were found under the
microscope. Then the selected areas were imaged by epi-fluorescence and
EF-fluorescence. Under epi-fluorescence illumination [Fig.2(A)], the
fluorescence of large beads was emitted and surpassed that of the surrounding
small beads. Thus, the small beads would be undistinguishable. However, under
EF-fluorescence excitation, only a thin layer of the large beads adjacent to
the cover glass was excited and the fluorescence of small beads was clearly
visible [Fig.2(B)]. This result confirmed that the generation of EF in our
TIRFM system and EF was very efficient to remove out of focus fluorescence
excitation.
Fig.2 Comparison
of images of green fluorescent beads under epi-fluorescence and EF-fluorescence
illumination
After fluorescent beads of
175 nm and 6 μm in diameter were fixed on the coverglass,
they were excited by epi-fluorescence and EF-fluorescence illumination with
the focal plane set at the coverglass surface. (A) Epi-fluorescence image;
(B) EF fluorescence image at the same focal position as in (A).
2.3 Study
of the dynamics of single GFP-labeled granule in PC12 Cells
The transfected
PC12 cells were imaged with epi-fluorescence and EF-fluorescence. The focal
plane was placed at the cover glass-cell interface. Under epi-fluorescence
excitation at 488 nm, the fluorescence of the cell was uniform [Fig.3(A)];
whereas, EF-fluorescence excitation resulted in punctuates of fluorescent
spots, representing GFP-labeled granules. Sometimes, there were larger and
brighter fluorescent spots possibly corresponding to 2 to 3 granules aggregated
together. The dim spots represented those granules distant away from plasma
membrane [Fig.3(B)]. The area excited by EF-fluorescence was smaller than
that of epi-fluorescence because the region where the cell adhered to the
cover glass was usually smaller than the cell’s horizontal projection. In
resting PC12 cells, about 15 to 30 granules were seen as fluorescent spots
in the EF fluorescence images, among them there were 2 to 9 bright fluorescent
spots corresponding to the tethered or docked granules. Average densities
of whole and docked granules are (0.236±0.101) granules/μm2 and (0.074±0.042) granules/μm2, respectively ( Fig.3 Comparison
of images of GFP-hpNPY labeled PC12 cells under epi-fluorescence and
EF-fluorescence illumination
With the focal
plane set at the cover glass surface, the GFP-hpNPY transfected PC12 cells
were imaged under epi-fluorescence and EF-fluorescence illumination. (A) Epi-fluorescence
image of a cell; (B) EF fluorescence image of the same cell.
To analyze the
granule motion, resting PC12 cells were imaged under EF-fluorescence excitation
for several minutes at 2－10 Hz. Generally, granules were found to travel much faster when
they were approaching the plasma membrane, while became less mobile when they
were docking at the plasma membrane. It seemed that there were certain factors
to restrict the movement of the granules (data not shown). Fig.4(A) showed the
336th image in a stack of 500. It could be observed that, first granule 1
became gradually brighter, then fluctuated at maximal fluorescence for a while
followed by a gradual decline of the fluorescence. The stack of images of the
granule and its fluorescence as a function of time were plotted in Fig.4(B) and
Fig.4(C), respectively. Since the fluorescence intensity is dependent on the distance
of granules from the cover glass-medium interface, the increase in fluorescence
of granule 1 was thus interpreted as the transportation of granule from the
interior of the cell to the plasma membrane. The granule docked at the plasma
membrane when it was brightest and it could undock from the plasma membrane
manifesting by the gradual dimming of the fluorescence. From Fig.4(B), a small
displacement of the granule in the x-y directions could also be found.
Occasionally, it could be observed that there was a sudden spread of a single
fluorescence spot and subsequent plummet in fluorescence [Fig.4(D), (E)]. This
motion differs from the undocking in two aspects: first, it developed a cloud
of fluorescence within a very short time (usually several hundred ms)[6] ;
second, after the spread of fluorescence, it was followed by a steep large
decrease in fluorescence, in contrast to a gradual dimming of the spot in the
case of granule undocking. The fluorescent single spot, although greatly
reduced in intensity, did not vanish completely, which argued against a full
fusion event. We thus interpreted it as a “kiss and run” event of single
granule. In this kind of event, a granule can form a fusion pore with the
plasma membrane, and most of its fluorescent content can diffuse out to create
a cloud-like spreading of the fluorescence marker. However, the granule will
not completely fuse with the plasma membrane, but go back into the cytosol with
much dimmer fluorescence[7] .
Fig.4 Tracking
the movement of single granule
Time-lapsed images of GFP-hpNPY
transfected PC12 cells were collected at 2 Hz. (A) The 336th image in a stack
of 500; (B) The trafficking, docking and undocking of a single granule [marked
as 1 in (A)]; (C) The movement and fusion event of another granule [marked
as 2 in （A）]; (D) Time course of the fluorescence intensity of granule 1. (E)
Time course of the fluorescence intensity of granule 2.
3 Discussion
Neurotransmitters
and hormones are released through exocytosis of vesicles. The vesicular
exocytosis consists of complex serial events of trafficking, tethering, docking
and fusion with cell membrane. It is controlled and regulated strictly by many
factors. Exocytosis and endocytosis participate in many essential and important
physiological events. Hence, the molecular mechanisms and regulations of
exocytosis and endocytosis have been extensively studied recently[8,9] .
“Seeing is
believing”. Real-time dynamic observation of single granule by TIRFM imaging
inaugurates undoubtedly a new field in the study of vesicular exocytosis and
endocytosis. The measure and calibration of EF is an important step to judge
whether the construction of TIRFM system is successful or not. There are two
calibraion methods reported so far: achieving different vertical positions of
fluorescent beads by adhering beads to a convex lens[10]; or
changing the vertical position of a fluorescent bead by attaching it to the tip
of a glass micropipette controlled by a piezoelectric micromanipulator[1].
In practice, it was found that the possibility to find enough fluorescent beads
distributed on different vertical levels was very low by using convex lens. On
the other hand, it was also difficult to pull a glass microelectrode into a tip
diameter of around 170 nm. In experiments, we found out that fluorescent beads
dried in 20% BSA solution would happen to be fixed at different distances from
the cover glass surface. So these beads at different vertical distances could
be used to calibrate the TIRFM. This method had been proven to be simple and
convenient in the practice.
GFP, especially
its various genetic mutants, were excellent molecular markers. GFP fused
proteins were useful tools for the studies of proteins, second messengers,
signal transduction, vesicles and actin network dynamics, etc.[11,12].
We found that GFP-hpNPY chimera was expressed well in the granules of PC12
cells and could be released by exocytosis. Evidently it was a good fluorescent
marker for dense-core secretory granules in PC12 cells and chromaffin cells,
and could also be used for detailed study of the mechanism of granule
translocation and fusion.
With a large
penetration depth using EF-fluorescence excitation, images of dim and bright
fluorescent spots in PC12 cells could be acquired. The dim fluorescent spots
represented granules deeper within the cell, and the bright ones were counted
as tethered or docked granules. The densities of docked granules were not
significantly different from that of primary cultured bovine chromaffin cells[1].
It was first proposed by Xu et al. [13] from indirect
evidence that the docking process might be reversible. Subsequent evidence
after that also confirmed this postulation[14]. In this study, we
did “see” the docked granules undocking from plasma membrane and moving back
into cytosol.
Although
capacitance and electrochemical techniques let measurement of single granule
fusion and/or the released content possible, they can’t provide information
before membrane fusion or after the endocytotic pinch off, while EF fluorescent
illumination makes it available to acquire such important information as the
vesicle translocation, trafficking, tethering, docking and endocytotic
processes, and so on.
Many molecular
and cellular events had been studied using TIRFM, i.e., regulated secretion of
neuronal transmitters and hormones[6,15] , endocytosis and
constitutive secretion[16, 17]， single molecule of hormone binding to plasma membrane and its
receptor clustering, signal transduction in cell[18, 19]. Recently,
most experiments with EF fluorescence focused on secretory granules, but there
would be other potentials for this method. Most prominent applications would
be: (1) Simultaneous EF-fluorescence imaging of two different proteins tagged
by two different chromophores [for example GFP and DsRED pair, or cyan
fluorescent protein (CFP) and yellow fluorescent protein (YFP) pair], to study
protein-protein interaction employing the fluorescence resonance energy
transfer; (2) studying the temporal and spatial relationship of different
color-labeled cellular structures or proteins using multi-wavelength
excitation, which will help to determine the function and interaction of each
component.
Acknowledgements We thank
Mrs. YE Qin for excellent work in cell culture and assistance. The plasmid
encoding hp-NPY-EGFP was kindly provided by Prof. Wolhard Almers.
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Received:
November 19, 2002 Accepted: December 31, 2002
This work was supported by grants from National Natural Sciences Foundation
of China (No. 30025023, 30130230) and from the Major State Basic Research
Development Program (973 Program) of China (No. G1999054000)
*Corresponding author: Tel, 86-27-87542499; Fax, 86-27-87542499; e-mail, [email protected]