The goal of the present protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time analyses of the secretory granule's morphology.
Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs’ mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules (SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.
Mast Cells (MC) are immune cells that are best known for their involvement in allergic and inflammatory reactions such as arthritis, asthma, eosinophilic esophagitis, chronic dermatitis and anaphylactic shock 1,2 as well as other pathologies including coronary artery disease 3,5 and cancer 3,4. In addition, MCs play important roles in innate and adaptive immunity, both in host defense against bacteria and parasites and by suppression of immune responses, for example inducing allograft tolerance 5,6 .
MCs originate from the bone marrow, developing from CD34+/CD117+ pluripotent progenitor cells 7. Committed bone marrow MC progenitors are released into the bloodstream and migrate into the peripheral tissues localizing predominantly within connective tissues and epithelial surfaces 8. Maturation and terminal differentiation are eventually achieved under the influence of cytokines within the surrounding milieu 8,9 .
MCs can be activated by an allergen (antigen, Ag), whose encounter resulted in the generation of immunoglobulin E (IgE) type antibodies. Binding of such IgE to the MC’s FcεRI receptors, followed by cross-linking of cell bound IgE upon re-exposure to the same Ag, results in FcεRI aggregation and initiation of a signaling cascade that culminates in cell degranulation [reviewed in 10,11]. MCs are also activated, independently of IgE, by neuropeptides 5,12, toxins 13 , bacterial and viral antigens 14,15, a number of positively charged peptides collectively referred to as basic secretagogues, immune cells and cytokines 5,13,12,16,17. MCs are also activated by many of their own released mediators, which further amplify the inflammatory response.
MCs are packed with secretory granules (SGs) that contain immunoregulatory mediators, including vasoactive amines, such as histamine and serotonin (in rodents), proteoglycans, proteases, such as chymase and tryptase, vascular endothelial growth factor and several cytokines and chemokines 8,9. These mediators are “ready to go” and once MCs are activated by an appropriate stimulus, these mediators are released from the cells by regulated exocytosis (degranulation) in a matter of seconds to minutes 18,19. This initial event is followed by the de novo synthesis and release of a large array of biologically potent substances, including arachidonic acid metabolites, multiple cytokines and chemokines 20,21,22. Release of newly synthesized products occurs independently of SG release. Collectively, these mediators initiate early and late phase inflammatory and allergic responses. Therefore, understanding the mechanisms accounting for MC activation and degranulation are both of theoretical and clinical importance.
The difficulty to genetically manipulate primary and cultured MCs has hampered the attempts to elucidate the mechanisms underlying MC degranulation, which remained poorly resolved. To overcome this problem we developed a reporter based assay by co-transfecting the mucosal mast cell line, rat basophilic leukemia (RBL)-2H3 (herein referred to as RBL) or bone marrow derived MCs (BMMCs) 30 with a gene of interest and Neuropeptide Y (NPY) fused to monomeric RFP (mRFP), as a SG reporter.
NPY was previously shown to recapitulate the behavior of endogenous SG markers in other systems. Moreover, because mRFP fluorescence is pH insensitive, expression of NPY-mRFP allows visualization of the acidic SGs as well as quantitative assessment of exocytosis by using 96-well plates and a fluorescence plate reader. We have shown that NPY-mRFP is delivered to the acidic SGs of RBL cells and BMMCs and is released from the cells in a regulated fashion alongside the endogenous SG cargo (i.e., β-hexosaminidase and serotonin) 30,32 . This protocol provides a high-resolution imaging-based methodology that allows screening genes of interest for their phenotypic and functional impact on SG characteristics and degranulation in RBL cells 32. Specifically, this protocol allows real time tracking of MC SGs and quantification of their area or volume size, their number, kinetics of assembly, their movement along the cell cytoskeleton and their ultimate fusion with the plasma membrane under different conditions. For example, sensitizing the cells with DNP-specific IgE and triggering the cells with a multivalent Ag (DNP conjugated serum albumin) under different perturbations (i.e., knockdown of genes of interest, over expression of wt or mutant genes, or pharmacological manipulations) and comparing to control cells.
1. Preparation of RBL Cell Culture Media
2. Culture of RBL Cells
3. Preparation of Transfection Media.
4. Transfection of RBL Cells
5. Measuring NPY-mRFP Exocytosis
6. Time-lapse Microscopy of Exocytosis
7. Image Analyses
Because of the low transfection efficiency of MCs, genetic manipulations are unlikely to leave an impact on readouts of average secretion measured by endogenous SGs mediators. Nevertheless, by establishing complete co-expression of the reporter gene NPY-mRFP and the co-transfected plasmid at the same cells, monitoring of NPY-mRFP results in monitoring exclusively the cell population that expresses the gene of interest. Therefore, the advantage of this assay compared to conventional methods is the ability to selectively monitor genetically manipulated cells (Figure 1).
Indeed, using this assay we have screened genes that regulate vesicular trafficking including soluble NSF [N-ethylmaleimide sensitive fusion] attachment protein receptor (SNARE) proteins and 44 members of the Rab GTPases family. We identified genes that regulate SGsize, number, cargo composition and exocytosis 23.
We identified 30 Rabs that altered (i.e., inhibit or stimulate) exocytosis in RBL cells stimulated by either Ag or by the combination of a Ca2+ ionophore and the phorbol ester TPA (Ion/TPA) 23. Confocal imaging revealed SNAREs and Rabs that caused dramatic phenotypes altering the morphology of the cells or the SGs 30,32. Confocal imaging of these proteins revealed 8 SNAREs and Rabs that caused dramatic phenotypes on the cells or the SG morphology 30,32. Using this protocol we were able to track individual SGs and perform measurements of their size, fluorescence intensity, their movement and exocytosis. For example, we identified Rab5 as a regulator of SG biogenesis 30. Rab5 has been identified as a master regulator of endocytosis and endosomal fusion 24. We have shown that co-expression of constitutively negative (CN) GDP-locked mutants of the endogenously expressed isoforms of Rab5 (Rab5A, Rab5B and Rab5C), or expression of Rab5A/B/C targeting shRNAs, reduced significantly the SGs’ size with a concomitant increase in their numbers 30. Conversely, expression of a GTP-locked, constitutively-active (CA) Rab5A mutant (Rab5A Q79L, herein: “CA Rab5A”), which is known to facilitate homotypic fusion of early endosomes (EEs) 24, resulted in the formation of giant Rab5A-decorated vesicles, which we identified as SGs based on their content of the secretory cargo NPY-mRFP and serotonin, and their capacity to exocytose in a regulated fashion 30.
Figure 2 demonstrates morphometric analyses of the NPY-mRFP–containing SGs. The mean SGs volume in CA Rab5A-expressing cells reached the value of 44 μm3, which was >20-fold larger than the mean volume of the SGs in the control GFP-expressing cells while their number was decreased by 20-fold (Figure 2A). The inverse relationship between the number and size of SGs (Figure 2A)suggested that the Rab5-mediated enlargement of the SGs involves their homotypic fusion. The giant NPY-mRFP containing SGs formed by CA Rab5A allows confocal microscopy imaging of living cells in a high-resolution that enables monitoring the SGs in details that could not be possible otherwise. For example, we detected Rab5A in fusing SGs alongside smaller structures scattered among the giant SGs, most likely corresponding to endosomes (Figure 2B). Additionally, we have shown that Rab5 transiently and preferably associates with newly formed SGs suggesting that the selective and transient association of Rab5A with newly formed SGs is compatible with a fusogenic apparatus in which only newly generated granules have the capacity to fuse with other SGs 30.
Figure 3 shows representative time-lapse images of MC giant SGs that were formed by expression of CA Rab5A and the kinetics of exocytosis in the resolution of a single SG after stimulation. During MC exocytosis, the SGs move towards and fuse with the plasma membrane. As a consequence the secretory cargo is released from the cells into the extracellular space. In this experimental system, NPY-mRFP is released from the cells and found in the cells supernatants. The fluorescence of NPY-mRFP in the cell supernatants can be measured by a fluorescence reader. However, because NPY-mRFP is diluted in the supernatants its fluorescence signal cannot be detected or visualized by fluorescence microscopy. The release of NPY-mRFP from the granules during exocytosis results in a rapid and dramatic decrease in NPY-mRFP fluorescence intensity and shrinkage of the NPY-mRFP-containing SGs and their disappearance.Therefore, live cell imaging of stimulated cells and measurements of NPY-mRFP fluorescence intensity or NPY-mRFP containing SG size provide accurate assessments of the kinetics of exocytosis of individual granules. Figure 3B shows the fluorescence intensities of SGs after stimulating the cells with a Ca2+Ionophore. The SGs undergo exocytosis at different time points (i.e., 2, 3, 6, and 11 min post stimulation), while the fluorescence intensity of a single SG that did not fuse with the plasma membrane remained constant.
Figure 1: Illustration of the main steps of the protocol. RBL cells are cotransfected with NPY-mRFP and a second gene of interest or corresponding control plasmid and immediately seeded on coverslips, 8-well chamber borosilicate coverglass system or 96-well plates. Next day, Images of the NPY-mRFP-containing SGs in resting and triggered cells are acquired by confocal microscopy (A). In addition, exocytosis of resting and triggered cellsis quantified by measuring the release of NPY-mRFP in a 96 well plate fluorescence reader (B).
Figure 2: Quantification of the SGs’ size. RBL cells were cotransfected with NPY-mRFP and either GFP or GFP-CA Rab5A and seeded on 8-well chamber borosilicate coverglass system. 24 hr later, the 8-well chamber was placed in laser confocal microscope equipped heated chamber (37 °C). Z-stack images of successive images with optical slices 0.7 μm were captured using a 63X oil/1.4 numerical aperture objective. The images were deconvoluted and three dimension images were constructed by the Imaris software. The volume of the SGs and their number were calculated using the Imaris software (A). Part of the NPY-mRFP–containing granules appears to be embedded within Rab5A (arrowheads). Others are naked or bridged with Rab5A (arrows). Scale bar = 5 µm (B). Please click here to view a larger version of this figure.
Figure 3: Measuring the extent and kinetics of exocytosis in the resolution of a single SG in activatedRBL cells. RBL cells were cotransfected with NPY-mRFP and GFP-CA Rab5A and seeded in8-well chamber borosilicate coverglass system. 24 hr later in8-well chamber was placed in a laser confocal microscope equipped with a heated chamber (37 °C) and the cells were triggered with 10 μM Ca2+ Ionophore. Images were acquired every 15 sec using a 63X oil/1.4 numerical aperture objective. Images were deconvoluted and then processed by the Imaris software. Scale bar = 5 µm (A). The mean fluorescence intensity of the NPY-mRFP containing granules was determined by the Imaris software and the data were normalized according to time 0 (time of addition of the Ca2+ Ionophore). The arrows represent the time points at which release of SGcargo wasnoted (B). Please click here to view a larger version of this figure.
We describe an innovative strategy that combines quantification of MCs exocytosis and four (x, y, z, t) dimension quantifications by time-lapsed three-dimensional imaging of the SGs in living cells using a reporter gene for exocytosis. This technique enables screening of families of proteins for their impact on MC function such as monitoring SGs starting as early as their exit from the Golgi through their maturation, acquisition of exocytosis competence and degranulation. The combination of measurements of exocytosis together with morphometric and kinetic characterizations of the SGs provides insights into the mechanisms by which the tested proteins mediate MC functions. Therefore, using this methodology, genetic manipulation of targeted genes can unveil new molecular mechanisms by which the MC SGs store and release their cargo. Notably, NPY can serve as a reporter for exocytosis in other secretory cells such as MCF-7 cells, a cell line derived from a human mammary gland adenocarcinoma 31, chromaffin cells 34 and pancreatic β-cells 35.
Genetic manipulations are the most powerful tools currently available for researching functional networks. Genetic manipulations allow the identification of essential proteins in each network, whose deletion will enforce collapse of the system, as opposed to other proteins, whose deletion will enforce crosstalk between parallel networks or have no effect due to redundancy. However, because of the low transfection efficiency of MCs, when measuring exocytosis of endogenous mediators only a small fraction of cells is expressing the 'manipulating' test DNA. Hence, when measuring exocytosis of endogenous mediators, the contribution of the transfected cells to the total readout is minor. Therefore, attempting to adopt such approach to explore the complex networks associated with regulated exocytosis in MCs was hampered. This technique overcomes the obstacle of the low transfection efficiency and provides a simple way to observe phenotypic alternations of MC SGs caused by over expression, knockdown and mutagenesis of genes of interest.
This technique can be extended for investigating the hierarchy and mode of interaction of functional networks using transient triple-transfection. The results will be analyzed to determine which gene can rescue a certain phenotype; cause a more dramatic phenotype or have no effect. Based on such combinatorial transfections, exocytosis networks can be constructed. For example, using triple-transfection we were able to identify the SNARE protein VAMP8 as a downstream mediator in the process of Rab5-dependent SG fusion 25.
This method permits visualization and quantification of exocytosis in the resolution of single SGs. Indeed we were able to show that SGs display differential responses to stimuli. The reason as to why certain SGs fused with the plasma membrane while others did not or the reason for the differential kinetics of MC SG fusion remain to be determined. Future studies using this experimental approach have the potential to answer these questions. For example, quantification of different SNAREs or Rabs on individual SGs and correlation with exocytosis competence and the kinetics of exocytosis in the resolution of a single SG should reveal novel regulators of exocytosis.
The main limitation of this technique is the genetic manipulation because overexpression of a protein might affect cell function or morphology. Therefore, it is essential to validate the results with complementary approaches. For example, when overexpression of a constitutively active mutant inhibits exocytosis, the effect of a constitutive negative mutant or knock down of the gene should be tested as well.
Using this technique, we identified novel regulators of MC functions, which affect MC exocytosis or alter the SG morphology. The combination of exocytosis measurements together with SGs morphometric characterization provides deeper insights into the function and mechanisms of action of the tested proteins.
The authors have nothing to disclose.
We thank Dr. U. Ashery for the gift of NPY-mRFP cDNA. We thank Drs. M. J. Kofron, L. Mittleman, M. Shaharbani, and Y. Zilberstein for invaluable assistance with microscopy and image analyses. We also thank Dr. Joseph Orly for critical reading of this manuscript. This work was supported by a grant from the Israel Science Foundation, founded by the Israel Academy for Sciences (1139/12 to R.S-E.).
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
DMEM | Sigma-Aldrich | D6046-500ML | Warm in 37 °C water bath before use |
Fetal Bovine Serum | GE health care Life sciences | SH30071.01 | |
Penicillin-Streptomycin | Life technologies | ||
Cellulose acetate membrane, pore size 0.22 μm | Sigma-Aldrich | CLS430769-1EA | |
Corning tissue-culture treated culture dishes | Sigma-Aldrich | CLS430167 | |
Trypsin/EDTA Solution (TE) | Life technologies | R001100 | Warm in 37 °C water bath before use |
PIPES dipotassium salt | Sigma-Aldrich | 108321-27-3 | |
Calcium acetate hydrate | Sigma-Aldrich | 114460-21-8 | |
Magnesium acetate tetrahydrate | Sigma-Aldrich | M5661 | |
L-Glutamic acid potassium salt monohydrate (Potassium glutamate) | Sigma-Aldrich | G1501 | |
4 mm electroporation cuvettes | cell projects | EP-104 | |
GENE PULSER WITH PULSE CONTROLLER & CAPACITANCE | Bio rad | ||
Chambered coverglass | Thermo scientific | 155411 | |
24 well, flat bottom | Sigma-Aldrich | CLS3524 | |
Corning 96 well plates | Sigma-Aldrich | CLS3367 or CLS390 | |
96 well plate fluorescence reader- Infinite 200 | Tecan | ||
Calcium ionophore A23187 | Sigma-Aldrich | C7522 | Avoid from direct light exposure |
12-O-tetradecanoyl-13-acetate (TPA) | Calbiochem | P3766 | |
anti-DNP monoclonal IgE | Sigma-Aldrich | D8406 | |
DNP-BSA/ DNP-HAS | Sigma-Aldrich | A6661 | Avoid from direct light exposure |
Triton-x-100 | Sigma-Aldrich | T8787 | |
Confocal fluorescent microscope: | |||
Zeiss LSM 510 | |||
Leica | SP5 | ||
Nikon A1 inverted | |||
Imaris software | BITLANE | ||
Microsoft exel or Prism or other analyses software | |||
Other reagent: | |||
Magnesium Chloride | MERK | 5833 | |
Sodium chloride | MERK | 6404 | |
Calcium chloride | MERK | 2382 | |
Bovine serum albumin | Sigma-Aldrich | A4503 | |
Glucose | BDH Laboratories | 284515V | |
Monosodium phosphate | MERK | 5345 | |
Sterile water |