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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Streszczenie

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Wprowadzenie

Regulated exocytosis is the primary mechanism by which readily made cargo is released from a secretory cell in response to a specific trigger. The cargo is pre-formed and sequestered into secretory vesicles, in which it is stored until a trigger relays the signal for the release of the SGs' content. Different types of signals may result in different modes of regulated exocytosis, or different modes of regulated exocytosis may occur simultaneously. Three main modes of regulated exocytosis are known: full exocytosis, which involves full fusion of a single secretory granule with the plasma membrane; kiss-and-run exocytosis, which involves transient fusion of the secretory granule with the plasma membrane followed by its recycling; and compound exocytosis, which is characterized by homotypic fusion of several SGs prior (i.e., multigranular exocytosis) or sequential (i.e., sequential exocytosis) to fusion with the plasma membrane1. Compound exocytosis is considered the most extensive mode of cargo release2, as it allows for the fast secretion of cargo, including that from SGs that are located distal from the plasma membrane. Compound exocytosis has been documented in both exocrine and endocrine cells3,4,5,6,7,8,9, as well as in immune cells. In immune cells, such as eosinophils10,11,12 and neutrophils13, compound exocytosis allows the fast and robust release of mediators that are required to kill invading pathogens such as bacteria or parasites. Mast cells (MCs) deploy compound exocytosis for the efficient release of pre-stored inflammatory mediators during innate immune responses, anaphylaxis, and other allergic reactions14,15,16,17. Since the different modes of exocytosis may occur simultaneously18,19, it has become a challenge to distinguish between them in real time or to identify their respective fusion machineries, hence elucidating their underlying mechanisms.

Here we present a method, based on live cell imaging of cell loaded FITC-dextran, that allows real time tracking of exocytic events and distinguishing between their different modes. In particular, our method allows exclusive monitoring of compound exocytosis.

FITC-dextran is a conjugate of the pH-sensitive fluorophore FITC with the glucan polysaccharide dextran. Fluorescently labeled dextrans have been shown to enter the cell by micropinocytosis20,21 and macropinocytosis22,23. As endocytic compartments mature into lysosomes, it has been shown that FITC-dextran accumulates in the lysosome with no apparent degradation. However, since FITC is a highly pH-sensitive fluorophore24, and the lysosome lumen is acidic, FITC-dextran fluorescence quenches upon reaching the lysosome24. Thus, establishing dextrans as lysosome targeted cargo, taken together with the pH sensitivity of FITC, have laid the foundation for the use of FITC-dextran in studies of lysosome exocytosis25,26,27,28,29.

In several cell types, including MCs, neutrophils, eosinophils, cytotoxic T cells, melanosomes, and others, the SGs display lysosomal features and are classified as lysosome-related organelles (LROs) or secretory lysosomes30,31. Since LROs have an acidic luminal pH, FITC-dextran can be used to visualize their exocytosis, as a result of higher pH associated with the exteriorization of the LROs. Indeed, FITC-dextran has been used to monitor exocytosis in MCs18,32,33. In this method, FITC-dextran is added to the cell culture, taken up by the cells by pinocytosis and sorted into the SGs. As it is in lysosomes, FITC fluorescence is quenched in the SGs when they are within the cell. However, upon SG fusion with the plasma membrane and consequent exposure to the external milieu, the FITC-dextran regains its fluorescence as the SG pH rises, allowing the simple tracking of exocytic events by live cell microscopy. Here, we adjusted this method to enable unique tracking of compound exocytosis.

Two other methods have been used previously to track compound exocytosis. Electron microscopy was the first method to characterize exocytic structures that suggested the occurrence of different modes of exocytosis. In particular, observations of "secretory tunnels" in pancreatic acinar cells34 and MCs35,36,37 gave rise to the hypothesis of compound exocytosis. However, while the high resolution of electron microscopy has the power to reveal fused vesicles, it cannot track the dynamics of their fusion and hence can't define whether they correspond to SG fusion during compound exocytosis or fusion of recaptured granules following their endocytosis. This obstacle is overcome in other methods that can measure exocytosis in living cells, such as patch clamp measurements of the plasma membrane capacitance11,13,38,39 or amperometry40 of the media. However, patch clamping requires a special set-up and may not be suitable for all cell types. Amperometry measurements are able to track exocytosis only if the cargo is released in very close proximity to the electrode. Therefore, using live cell imaging offers an advantage over these methods, as it not only allows for real time tracking of exocytosis, but it also allows quick and simple acquisition of data from the whole cell.

The tracking of FITC-dextran by live cell microscopy also offers some advantages to other live cell imaging-based methods. For example, a widely used method is total internal reflection fluorescence microscopy (TIRFM) of cells loaded with a fluorescent SG probe or expressing a fluorescent protein-tagged SG cargo or membrane protein26,41,42,43. The strength of this method lies in its ability to monitor exclusively events that occur close to the plasma membrane (herein referred to as footprint), hence exocytic events. However, this is also the drawback of this method because only the cell fraction that is adjacent to the coverglass and close to the microscope lens can be imaged44. Whether such footprints indeed represent the entire cell membrane surface is still debatable45,46,47. In this regard, using a pH-sensitive dye such as FITC-dextran and a standard fluorescence microscope or a confocal microscope with an open pinhole allows imaging of the whole cell, thus capturing the total exocytic events that occur in that cell.

Additional pH-sensitive reporters that are used to study regulated exocytosis by whole cell imaging or TIRFM include SG cargo or SG membrane protein fused to phlourin, a pH-sensitive GFP variant. Examples include NPY-phlourin-, β-hexoseaminidase-phlourin, and synapto-phluorin48,49,50,51,52. While expression of these probes may represent more closely the endogenous composition of the SGs, it entails transfection of the cells, and may therefore be less suitable for cells that are difficult to transfect. Therefore, when studying cells that are difficult to transfect or under experimental conditions that require multiple genomic manipulations, the use of a compound that can be simply supplemented into the cell culture medium, such as FITC-dextran, is advantageous. FITC-dextran also offers an advantage over acridine orange (AO), another pH-sensitive dye that has been used for the tracking of exocytosis by live cell microscopy53,54,55,56,57,58. AO has been shown to induce photolysis of vesicles that result in false flashes, which do not correspond to actual secretion processes27. In contrast, FITC-dextran reflects better secretion events, probably due to its low photo-induced production of reactive oxygen27.

Notably, an alternative approach for studying exocytosis is by tracking the influx of a dye, from the external medium into the SG through the fusion pore that opens during this process. In this case, the dye is added to the external medium alongside the secretory trigger. Then, when the fusion pore opens, the dye diffuses into the SG59,60. A clear advantage of this method is that it also offers the ability to estimate the fusion pore size, by the use of dyes of variable size. For example, dextrans of different molecular weight (MW), conjugated to different fluorophores, can be used as extracellular dyes whereby the maximum size of dextran that can penetrate the SG would correspond to the size of the fusion pore59,61,62,63,64. In addition, this approach does not require the use of a pH-sensitive probe. However, a significant disadvantage is that the signal to noise ratio is very low, since a large amount of dye is present in the media during acquisition of images, resulting in high background.

Overall, the use of FITC-dextran as a marker for exocytosis overcomes several drawbacks in previously reported methods, such as signal to noise ratio, toxicity, dynamic tracking and complexity.

Here we describe the use of FITC-dextran to monitor compound exocytosis in the RBL-2H3 mast cell line (herein referred to as RBL, primarily established by Eccleston et al.65 and further cloned by Barsumian et al.66), in response to immunoglobulin E (IgE)/antigen (Ag) activation.

Protokół

1. Preparations

  1. Preparation of RBL culture media
    1. Mix 500 mL of low glucose Dulbecco's modified Eagle's medium (DMEM) with 56 mL of fetal bovine serum (FBS), 5.5 mL of penicillin-streptomycin-nystatin solution, 5.5 mL of L-Glutamine 200 mM solution. This results in low glucose DMEM supplemented with 10% FBS, 100 µg/mL streptomycin, 100 units/mL penicillin, 12 units/mL nystatin, and 2 mM L-glutamine.
    2. Filter the media by using a top-vacuum filter of 0.22 µm pore size and store at 4 °C.
  2. Maintenance of RBL cells.
    1. Grow RBL cells to a maximum confluency of 90% in a 10 cm dish. If the cell culture is healthy, the cells should have a spindle shape with occasional protrusions.
    2. For cell splitting, detach the cells from the dish by aspirating the media and replacing with 2 mL of trypsine/EDTA solution B. Incubate for 5-10 minutes in a humidified atmosphere of 5% CO2 at 37 °C.
    3. Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media, using a pipette, and split the cells in a 1:2 - 1:10 ratio.
  3. Preparation of 20x Tyrode's buffer
    1. Prepare a stock 20x solution of 54 mM KCl, 20 mM MgCl2, 2.74 M NaCl, and 8 mM NaH2PO4 in double distilled water (DDW). Mix well and store at 4 °C.

2. Culture of RBL Cells for Live Cell Microscopy

  1. Preparation of FITC-dextran solution.
    1. Mix 1 mg of FITC-dextran powder (150 K) per 1 mL of culture media (see step 1.1.1). For a full chambered coverglass, prepare 3 mL.
    2. Using a cellulose acetate syringe filter unit with 0.22 µm pore size, filter the dissolved FITC-dextran.
    3. Add mouse IgE to a concentration of 1 µg/mL.
  2. Seeding RBL cells for imaging
    1. The day before imaging, aspirate the media from the culture dish and replace with 2 mL of trypsine/EDTA solution B. Incubate for 5 - 10 minutes in a humidified atmosphere of 5% CO2 at 37 °C. Once the cells have detached, neutralize the trypsin by adding 2 mL of culture media.
    2. Count RBL cells using a hemocytometer and adjust the volume accordingly with culture media to get a cell concentration of 7.5 x 105/mL.
    3. Add 10 µL of cell suspension to a chambered coverglass pre-filled with fresh FITC-dextran supplemented culture media (this results in seeding of 7.5 x 103 cells in a chamber).
    4. Grow RBL cells overnight in a humified atmosphere of 5% CO2 at 37 °C. The cells should remain in a sub-confluent level in order to make sure that the cells are separated and that it is easy to identify each cell individually under the microscope.
  3. Transfection of RBL cells - optional.
    1. For imaging exocytic events in combination with other fluorescently tagged proteins, see transfection protocol for RBL cells in Azouz et al.67

3. Live Cell Microscopy of Exocytosis

  1. Preparation of solutions:
    1. Prepare a final Tyrode's buffer solution by diluting the stock solution in DDW in a 1:20 dilution and supplement with 20 mM Hepes pH 7, 1.8 mM CaCl2, 1 mg/mL BSA, and 5.6 mM glucose.
    2. Freshly prepare a 20x secretagogue reagent in Tyrode's buffer [1 µg/mL dinitrophenyl conjugated to human serum albumin (DNP-HAS (Ag)) in our case, for a 50 ng/mL 1x concentration].
    3. Prepare a 400 mM ammonium chloride solution by dissolving powder in Tyrode's buffer.
  2. Preparation of the cells.
    1. Wash the chambered coverglass 3 times by aspirating the media from the chamber and refilling it with 300 µL of Tyrode's buffer, prewarmed to 37 °C. Finally, replenish the chamber with 300 µL of Tyrode's buffer, prewarmed to 37 °C.
    2. Place the chambered coverglass in the microscope's incubator chamber. Make sure that the chamber is stable.
  3. Setting up the microscope
    1. To choose a region of interest to track, turn on the fluorescent light source (traditionally a mercury lamp) and choose the appropriate fluorescence filter (choose the filter for green fluorophores for viewing FITC-dextran). Once the region of interest is in focus and is in the middle of the field of view, turn off the light source in order to avoid photo-bleaching and toxicity.
      NOTE: Some of the FITC-dextran incorporated in the cells may retain fluorescence. This is because FITC-dextran may also be sorted to non-lysosomal compartments such as endosomes.
    2. Turn on the appropriate lighting for FITC excitation. If using a laser-based microscope, turn on the 488 nm laser. Emission should be gathered around 500 - 550 nm (FITC emission peaks at 510 - 520 nm).
    3. When using a confocal microscope, open the pinhole to the maximum. This will allow using lower laser power to avoid bleaching and toxicity and will ensure the capture of exocytosis events from all planes of the cell.
    4. Calibrate the time interval between image acquisitions.
      1. Different cells may vary in their kinetics of regulated exocytosis68. For specific imaging of compound exocytosis, we recommend a time interval of at least 5 seconds. However, as a rule of thumb, to allow for fast imaging, make sure that the acquisition time of a single frame is fast.
      2. When using a laser-scanning confocal microscope, set the scan direction to bi-directional, do not allow for averaging, and set resolution at 512 x 512 (recommended; the latter two provisions will also help to minimize bleaching and toxicity).
  4. Imaging of exocytosis.
    1. Image cells for desired durations, depending on the type of cell or secretagogue. In RBL cells triggered with IgE/Ag, most exocytic events occur within 15 - 20 min of activation.
    2. For activation of the cells, add 16 µL from the 20x secretagogue solution to the chamber.
  5. Confirming FITC-dextran presence in the cells.
    1. To confirm the presence and localization of FITC-dextran to the SGs, add 16 µL of ammonium chloride solution (be careful not to move the chamber in order not to lose focus) to the chamber gently (this will make a final concentration of 20 mM). This will induce alkalization of the SGs and dequenching of FITC fluorescence, which will occur within seconds.

Wyniki

Figure 1a represents schematically how FITC-dextran may act as a reporter for regulated exocytosis and recapitulate the different modes of exocytic events. First, cells are incubated with FITC-dextran, which is internalized by pinocytosis and sorted to the SGs. Since the SGs of MCs are LROs, their low pH dampens the fluorescence of FITC, shown here as black granules (A-C, I). When cells are triggered by a secretagogue and the SGs fuse with the plasma membrane...

Dyskusje

Here we describe how tracing the fluorescence of FITC-dextran loaded into SGs can be used to specifically capture compound exocytosis events. This was achieved by setting the microscope to acquire an image every 15 seconds, thus ensuring that only long-lasting events will be recorded over time, and therefore excluding short events that would correspond to full exocytosis or kiss-and-run exocytosis. To establish the method, we showed that knockdown of the Rab5 isoforms that are expressed in RBL cells, and are pivotal for ...

Ujawnienia

The authors declare no competing financial interest

Podziękowania

We thank Dr. U. Ashery for the generous gift of cDNA. We thank Drs. G. Mass, L. Mittleman, M. Shaharbani, and Y.Zilberstein from the  Sackler Cellular & Molecular Imaging Center for their invaluable assistance with microscopy. This work was supported by the United States-Israel Binational Science Foundation (grant 2013263 to R. Sagi-Eisenberg, I. Hammel, and S.J.Galli) and grant 933/15 from the Israel Science Foundation, founded by the Israel Academy for Sciences (to R.Sagi-Eisenberg) and NIH grants U19 AI 104209 and R01 AR067145 (to S.J. Galli).

Materiały

NameCompanyCatalog NumberComments
DMEM low glucoseBiological Inductries01-050-1A
Fetal bovine serumGibco12657
Pen-strep-nystatin solutionBiological Inductries03-032-1B
L-Glutamine 200 mM solution Biological Inductries03-020-1A
FITC dextran 150KSigma-Aldrich46946-500MG-F
Trypsin/EDTA Solution BBiological Inductries03-052-1AWarm in 37 °C water bath before use
Top-vacuum filter of 0.22 µm pore size Sigma-AldrichCLS430769-1EA
Cellulose acetate syringe filter unit, 0.22 µm pore sizeSartorius16534K
Chambered coverglass Thermo scientific155411
Corning tissue-culture treated culture dishesSigma-AldrichCLS430167
Bovine serum albuminSigma-AldrichA4503
Anti-DNP monoclonal IgESigma-AldrichD8406
DNP-HSA (Ag)Sigma-AldrichA6661 Avoid direct light exposure
Hepes buffer 1M, pH 7.4Biological Inductries03-025-1B
CaCl2MERK102382
GlucoseBDH Laboratories284515V
Ammonium chlorideMERK1145
PIPES dipotassium saltSigma-Aldrich108321-27-3
Calcium acetate hydrateSigma-Aldrich114460-21-8
Magnesium acetate tetrahydrateSigma-AldrichM5661
Potassium glutamate (L-Glutamic acid potassium salt monohydrate)Sigma-AldrichG1501
NaH2PO4MERK6346
NaClMERK106404
MgCl2MERK105833
KClMERK104936
Electroporator BTXECM830
Confocal microscope, ZeissZeissLSM 5 Pascal, Axiovert 200MUsed in Figure 3. Equipped with an electronic temperature-controlled
airstream incubator
Confocal microscope, ZeissZeissLSM 800, Axio Observer.Z1 /7Used in Figure 2. Equipped with a GaAsP detector an electronic temperature-controlled
airstream incubator
Confocal microscope, LeicaLeicaSP5Used in Figure 1. Equipped with a leica HyD detector and an top-stage incubator (okolab)
RBL-2H3 cellsRBL-2H3 cells were cloned in the lab of Reuben P. Siraganian. See reference 67

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Keywords FITC dextranRegulated ExocytosisMast Cell ExocytosisNeutrophilsEosinophilsTyrode s BufferRBL CellsAmmonium ChlorideSecretagogueFluorescence Microscopy

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