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ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(4): 381-386                                    CN 31-1300/Q

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 ( ±s, 41 cells).


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]