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Keywords:

  • cytometry;
  • mast cells;
  • secretory lysosomes;
  • granule;
  • organelle sorting;
  • mast cell protease;
  • exocytosis;
  • subcellular fractionation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITTERATURE CITED

Background

Mast cells are specialized secretory cells of the immune system. Through exocytosis of their secretory lysosomes and secretory granules, mast cells release biologically active substances such as histamine and proteases. Mast cell secretory granules have been studied extensively but much less attention has been given to secretory lysosomes. Studies on mast cell secretory lysosomes are limited by the lack of selective markers and the difficulty to isolate this organelle from conventional lysosomes. Our goal was to develop better tools to study secretory lysosomes.

Methods

We engineered a rat mast cell line over expressing a rat mast cell protease (RMCP) tagged with a red fluorescent protein (RMCP-DsRed). We used single organelle flow analysis (SOFA) to detect fluorescently labeled secretory lysosomes. The labeled organelles were then sorted using the fluorescence-assisted organelle sorting (FAOS) method.

Results

We show that the RMCP-DsRed fusion protein selectively localizes to the lysosomal compartment and is exocytosed upon activation, confirming its localization in secretory lysosomes. Lysosomal fractions from cells expressing the RMCP-DsRed fusion were analyzed by SOFA and a specific population of secretory lysosome was identified. Finally, we sorted secretory lysosomes and showed that the sorted material had a higher specific activity for the compartment marker hexosaminidase than a sample obtained by conventional methods.

Conclusions

Our work further demonstrates the usefulness of flow cytometry to study cellular organelles, and provides new tools to better understand the physiology of secretory lysosomes. Cytometry Part A 55A:94–101, 2003. © 2003 Wiley-Liss, Inc.

Mast cells are multifunctional effector cells involved in many inflammatory and pathophysiological processes (1). They are found adjacent to blood or lymphatic vessels but are most prominent beneath the epithelial surfaces of the skin or mucosae of the gastrointestinal and respiratory tracts (2). Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E (IgE), FcϵRI, induces mast cells to release mediators such as histamine and proteases (3). A few minutes following activation, mast cells release secretory vesicles by a process known as compound exocytosis which involves granule-to-plasma membrane and granule-to-granule fusion. This process occurs in other cells of hematopoietic origin, endocrine cells and exocrine cells (4). As opposed to endocrine and exocrine cells, hematopoietic cells use organelles termed secretory lysosomes for storage and release of secretory products (5, 6). Secretory lysosomes share properties with both lysosomes and secretory granules. Like lysosomes, secretory lysosomes are acidic and contain proteases and glycosidases. However, they are distinguished from conventional lysosomes by their ability to undergo regulated secretion. Secretory lysosomes have diverse types of structures including dense cores or multilaminar whereas specialized secretory lysosomes such as melanosomes have unique structures. Secretory lysosomes are present in many different cell types of hematopoietic origin such as cytotoxic T cells, mast cells, neutrophils, eosinophils and osteoclasts. Despite their physiological importance, little is known about their biosynthesis, their regulation and their proteome.

In mast cells, few proteins were shown to localize selectively to secretory lysosomes (7). Proteases such as tryptases, chymases, and carboxypeptidases are the major protein constituents released from activated mast cells (8). Immunohistochemical localization indicates that proteases such as cathepsin D are stored in lysosomes and secreted upon mast cell stimulation (9). However, there is no evidence for localization of mast cell proteases in secretory lysosomes.

Single organelle flow analysis (SOFA) allows for rapid analysis of organelles by flow cytometry (10). Using this technique, vesicles can be analyzed for more than one parameter at a time and co-localization studies can be executed on a single organelle. SOFA also provides quantitative information and the possibility for statistical analysis of the data. Endosomes were amongst the first organelles to be analyzed by SOFA (11). In these experiments, labeling of compartments involved in fluid phase endocytosis was achieved by incubating the cells with fluorescein isothiocyanate and dextran. Crude cell extracts were then analyzed by flow cytometry. These studies demonstrated the presence of three kinetically distinct compartments involved in fluid-phase endocytosis. Other studies have shown that isolated mitochondria can be utilized to study membrane potential by using fluorescent staining with Rh-123 and flow cytometry (12).

Fluorescently labeled organelles can be sorted by fluorescence-activated cell sorting (FACS) for analytical or preparative purposes. This procedure, termed fluorescence-activated organelle sorting (FAOS) can be used to sort labeled intracellular organelles from a cell homogenate or following a subcellular fractionation procedure (13). FAOS allows the isolation of highly purified organelle populations. The feasibility of analyzing and sorting single organelles by flow cytometry has been demonstrated previously (14). For example, FAOS was used to analyze and sort apical and basolateral endocytic vesicles from MDCK cells (15). This study led to the identification of syntenin as a protein of the apical early endocytic compartment.

Recent work shows that cells with secretory lysosomes use diverse sorting and secretory pathways (6). To provide better tools to understand these processes, we generated a rat mast cell line stably expressing a fluorescently tagged rat mast cell chymase (RMCP-DsRed). We show that the RMCP-DsRed fusion is mainly localized in secretory lysosomes. We then demonstrate how fluorescently labeled secretory lysosomes from this cell line can be analyzed and highly purified by SOFA and FAOS, respectively.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITTERATURE CITED

Cell Culture

RBL-2H3 cell line (Rat Basophilic Leukemia; ATCC, Manassas, VA) was cultured in OPTI-MEM media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT). The cells were split the day before the experiment into a 60 mm culture dish at a density of 1.5 × 106 cells/dish.

Cloning of RMCP II in the Expression Vector pDsRed1-N1

The RCMP II cDNA (16) was cloned by polymerase chain reaction (PCR). The rat basophilic leukemia (RBL-2H3) cell line was used as a source of RNA. The reverse transcriptase reaction was done using the Superscript amplification system (Invitrogen) according to the manufacturer's instructions. The PCR amplification was performed on first strand cDNA using the following oligos: 5′-TCAGATCTCGAGATGCAGGCCCTACTATTCCTG-3′ and 5′-CTGCAGAATTCGGCTACTTGTATTAATGACTGCAT-3′. The PCR conditions were as follows: 94°C (30 s), 62°C (30 s), and 72°C (50 s). The PCR product was purified and digested with the restriction enzymes XhoI and EcoRI. The fragment was then cloned in the same sites of the pDsRed1-N1 vector from Clontech (Palo Alto, CA) resulting in the fusion of the RMCPII cDNA in-frame with the cDNA encoding the Discosoma sp. red fluorescent protein (DsRed). The identity of the recombinant vector, RMCP-DsRed, was confirmed by DNA sequencing.

Stable Expression of RMCP-DsRed Fusion Protein in RBL-2H3 Cells

RBL-2H3 cells were stably transfected with the expression vector for the DsRed protein alone or the RMCP-DsRed fusion protein. RBL-2H3 cells (8 × 106) were transfected by electroporation on a Gene Pulser (BioRad, Hercules, CA) with 45 μg of vector (17). The conditions for electroporation were 300 V and 960 μF in a total volume of 800 μl. Transfected cells were transferred to OPTI-MEM supplemented with 10% FBS and cultured for 48 h. Positive clones were then selected by the addition of 1 mg/ml of active Geneticin (Gibco BRL, Grand Island, NY). After 10 days of selection, cells positive for expression of DsRed or RMCP-DsRed were sorted by FACS. Individual clones were expanded and analyzed by FACS and confocal microscopy. One clone of each cells expressing DsRed and RMCP-DsRed were retained for this study.

Antigen-Induced Exocytosis

RBL-2H3 cells were stimulated mainly as described by Baram et al (7). Briefly, cells were seeded in 24-well plates at a density of 2 × 105 cells per well and incubated overnight. Cells were then washed twice in culture media and incubated for 2 h at 37°C in the same buffer containing 1 μg/ml of anti-DNP IgE monoclonal antibody (SPE7 clone, Sigma, St. Louis, MO). Sensitized cells were washed twice in Tyrode's buffer (10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% bovine serum albumin) and then stimulated with 100 ng/ml DNP-HSA (Sigma) in Tyrode's buffer for the indicated times. Aliquots from the culture supernatants were analyzed for histamine, red fluorescence and hexosaminidase content. Histamine was quantified using an enzyme-linked immunosorbent assay (ICN, Costa Mesa, CA). The red fluorescence emitted from DsRed was detected on an LJL Bioanalyst (LJL Biosystems, Sunnyvale, CA) set at 530 nm for excitation and 580 nm for emission. Hexosaminidase was assayed as described below.

Hexosaminidase Assay

This protocol was adapted from Schwartz et al. (18). Briefly, the reaction mixtures were prepared in a 96 well plate using 10 μl of culture supernatant and 50 μl of 4 mM p-nitrophenol-β-D-2-acetamido-2-deoxyglucopyranoside (Sigma) in 0.04 M citrate buffer (buffer titrated to pH 4.5 with 0.2 M dibasic sodium phosphate). As a negative control, 10 μl of Tyrode's buffer was used. After mixing, the plate was incubated at 37°C in a humidified incubator for 90 min. The reaction was stopped by adding 150 μl of 0.2 M glycine pH 10.7. The samples were read on a SPECTRAmax 340 plate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 410 nm.

Subcellular Fractionation of Secretory Lysosomes

The fractionation was performed mainly as described by Kjeldsen et al. for neutrophils (19). Briefly, 3 × 107 cells (expressing DsRed or RMCP-DsRed) were detached by trypsinization and washed once with 50 ml of phosphate buffered saline (PBS). The pellet was resuspended in 700 μl of homogenization buffer (HB; 340 mM sucrose, 10 mM Hepes, pH 7.3, 0.3 mM ethylene-diamine-tetraacetic acid) and submitted to 2 freeze/thaw cycles. The homogenate was then sonicated on a Branson sonicator (Sonifier 200) at level 2 and 30% output for 8 pulses. The homogenate was spun at 3000 rpm for 10 min on a bench top microfuge to pellet nuclei. The post nuclear supernatant (PNS) was collected and applied on a two layer Percoll gradient. The Percoll was diluted with HB and gradient densities were 1.05 and 1.12 (1.2 ml/layer). The gradient was layered in 3.2 ml polycarbonate ultracentrifugation tubes (Beckman, Fullerton, CA). After applying the PNS on top of the gradient, the samples were spun at 30,000 rpm for 50 min (TLA 100.4 rotor) in a Beckman Optima TLX ultracentrifuge (Beckman). Fractions of 100 μl were collected starting from the top of the gradient. The fractions or fraction pool were diluted 5-fold in HB prior to SOFA or FAOS analysis.

Confocal Microscopy

Both DsRed and RMCP-DsRed expressing cells were grown overnight in 1 ml chamber slides (Falcon no. 35-4101) at a density of 1 × 105 cells/ml in a humidified CO2 incubator. The cells were fixed with 3.7% paraformaldehyde for 20 min at room temperature. Following washes with PBS and water, coverslips were mounted on the slides.

The RMCP-DsRed cells treated with Lysotracker Green DND-26 (Molecular Probes, Eugene, OR) were grown as above. LysoTracker Green at 100 nM was added to the cells and incubated at 37°C for 45 min. Following incubation, the cells were washed with PBS and fixed as above before mounting a coverslip.

All cells were examined on a Zeiss LSM 410 confocal microscope (Carl Zeiss, Inc., Thornwood, NY) mounted on an Axiovert 135 using a 63 × numerical aperture = 1.4 oil immersion lens. An argon/krypton laser was used with excitation of 488 nm and 568 nm. LysoTracker Green emission was measured with a 515LP filter, DsRed emission with a 560/590BP filter, and the image size was 512 × 512. For each selected sample z stacks were obtained with 0.5-μm steps.

SOFA

The lysosomal fractions of the RMCP-DsRed cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 488 nm argon ion laser and CellQuest software. The threshold was set on forward light scatter. The red fluorescence (DsRed) was measured using the FL2 parameter (585 nm) with log amplification. The DsRed expressing cells were used as the negative control. The FL1 parameter (530 nm) with log amplification was used to establish autofluorescence.

FAOS

Labeled secretory lysosomes were sorted using a FACS Vantage SE flow cytometer (Becton Dickinson) equipped with an air-cooled argon ion laser and TurboSort Plus option. The 488 nm laser was tuned to 100 mW output and a 70 micron nozzle tip was used with a sheath pressure of 34 psi. The threshold was set on forward light scatter and no ND filter was used. Unlabeled lysosomes were used to adjust the PMT voltages and the sort gate was set using FL1 (530/30BP) and FL2 (575/26BP) parameters. The samples were run at approximately 4,000 to 6,000 events/s with less than 10% abort rate.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITTERATURE CITED

RMCP-DsRed Is Targeted to Secretory Lysosomes

RMCP II selectively localizes to mast cell secretory compartments and is released upon cell activation (16). We stably transfected the RBL-2H3 cells with an expression vector encoding the RMCP II protein tagged with Discosoma sp. red fluorescent protein (RMCP-DsRed). Cells expressing DsRed alone or the RMCP-DsRed fusion show a very different distribution pattern of fluorescence (Fig. 1). DsRed is localized in the cytoplasm whereas the RMCP-DsRed fusion shows a punctuate pattern of fluorescence. This punctuate fluorescence does not co-localize with the endoplasmic reticulum or the Golgi apparatus (data not shown).

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Figure 1. Stable expression of DsRed and RMCP-DsRed in RBL-2H3 cells. A: Confocal image of RBL-2H3 cells expressing the DsRed protein. B: Confocal image of RBL-2H3 cells expressing the RMCP-DsRed fusion protein.

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Lysosomes are acidic organelles that can be selectively labeled with fluorescent probes such as Lysotracker. As shown in Figure 2, a strong co-localization is observed between the lysosomal compartment labeled with Lysotracker and the RMCP-DsRed fusion. This confirms the selective localization of the RMCP-DsRed to lysosomal compartments. The organelles labeled with Lysotracker but not with RMCP-DsRed are likely to be conventional lysosomes (non-secretory).

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Figure 2. RMCP-DsRed fusion localizes in secretory lysosomes. Cells stably expressing the RMCP-DsRed fusion protein were treated with the LysoTracker probe. Left panel shows intracellular localization of lysosomes using the LysoTracker probe. Center panel shows the distribution of the RMCP-DsRed fusion protein. The overlay of LysoTracker and RMCP-DsRed images is shown on the right panel.

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To demonstrate that the RMCP-DsRed protein is part of a secretory compartment, we sensitized the cells with an anti-DNP IgE monoclonal antibody and stimulated them with the corresponding antigen DNP-HSA. This stimulation leads to a rapid exocytosis of secretory granules and lysosomal content such as histamine, proteases and hexosaminidase. Within minutes following antigen addition, the RMCP-DsRed cells release histamine and hexosaminidase (Fig. 3). The activated cells also release the RMCP-DsRed protein (red fluorescence) with similar kinetics; which confirms its localization in secretory lysosomes. The RBL-2H3 cells expressing the DsRed protein alone showed minimal release of red fluorescence (up to a maximum of 7%, 2.5 h after stimulation; data not shown).

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Figure 3. RMCP-DsRed is exocytosed upon activation. RBL-2H3 cells expressing RMCP-DsRed were sensitized with anti-DNP IgE and stimulated with DNP-HSA for the indicated times. Aliquots from the culture supernatants were then analyzed for the presence of secreted RMCP-DsRed (red fluorescence), hexosaminidase and histamine.

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Analysis of Secretory Lysosomes by SOFA

Protocols to obtain crude lysosomal fractions are well established but these preparations contain contaminating organelles such as mitochondria and ER proteins. Moreover, existing organelle separation techniques do not allow to separate secretory lysosomes from the total lysosomal compartment. We sought to determine if SOFA and FAOS could be used to further analyze and purify fluorescently labeled secretory lysosomes. We fractionated lysosomes from RBL-2H3 cells stably expressing RMCP-DsRed on Percoll gradients. Figure 4 shows an analysis of the different gradient fractions for their histamine, hexosaminidase and red fluorescence content. Hexosaminidase is an enzymatic marker for the lysosomal compartments (18, 20) whereas histamine is known to be present predominantly in dense core granules (7). The red fluorescence was localized mainly with the hexosaminidase fraction. RMCP-DsRed was also found in a denser fraction of the gradient which is enriched in dense core granules as shown by the presence of histamine. The fluorescence from cells expressing DsRed alone was found broadly at the top of the gradient in fractions 1 to 12, consistent with a cytoplasmic localization (data not shown).

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Figure 4. Subcellular fractionation of RMCP-DsRed expressing cells. Fractions collected from a Percoll gradient were tested for the presence of RMCP-DsRed (fluorescence), histamine levels and hexosaminidase activity. Fraction 1 corresponds to the top of the gradient.

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The fractions enriched in fluorescent secretory lysosomes (fractions 10–18) were pooled and analyzed by SOFA as shown in Figure 5. The lysosomal fraction from cells expressing the DsRed protein alone was utilized as a negative control (Fig. 5A). The fluorescently labeled secretory lysosomes can be detected from the lysosomal fraction of cells expressing RMCP-DsRed (Fig. 5B; R1 region). The correction for autofluorescence is an important prerequisite for the accurate analysis of fluorescence data (21). For this reason, we performed a dual parameter analysis (FL1/FL2) of the samples. This analysis allows to exclude a false positive signal caused by autofluorescence. As shown in Figure 5C, a significant signal is observed from particles or organelles in the autofluorescence channel (FL1) for the control DsRed sample. Once the autofluorescence signal is established, the population of fluorescent secretory lysosomes can be selectively identified (Fig. 5D, R2 region).

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Figure 5. SOFA analysis of lysosomal fractions from DsRed and RMCP-DsRed expressing cells. The lysosomal fraction of cells expressing DsRed (A, C) or RMCP-DsRed (B, D) were analyzed by flow cytometry. The red fluorescence was detected in FL2 (585 nm) and compared to particle size (A, B) or the control fluorescence (530 nm; C, D).

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Purification of Secretory Lysosomes by FAOS

To obtain a highly purified population of secretory lysosomes, we sorted the crude lysosomal fraction by the FAOS method. An autofluorescence analysis was initially performed on the lysosomal fraction from the control DsRed cells and the sort gate (R1) was established (Fig. 6A). The R1 red fluorescent organelle sorting gate was then applied to the fluorescent lysosomes present in the lysosomal fraction from cells expressing the RMCP-DsRed fusion protein (Fig. 6B). The gated secretory lysosomes represent 3% of the total number of particles detected in this fraction. Typically, the number of fluorescent secretory lysosomes sorted for analytical purposes ranged from 105 to 106.

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Figure 6. Isolation of secretory lysosomes using FAOS. A: The lysosomal fraction from cells expressing DsRed was analyzed based on emission of red fluorescence (575/26 nm) and control fluorescence (530/30 nm). B: The lysosomal fraction from cells expressing RMCP-DsRed. The fluorescently labeled secretory lysosomes in the R1 gate were sorted.

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Hexosaminidase activity can be used as a marker for secretory lysosomes in the RBL-2H3 cell line (Figs. 3, 4). To determine the level of purification of secretory lysosomes obtained by FAOS, we tested the specific lysosomal activity of the sorted material using hexosaminidase as a marker (Fig. 7). The lysosomal fraction obtained by Percoll gradient showed a 14-fold increase in specific hexosaminidase activity compared to the PNS. The sorted lysosomes showed a 50-fold increase in specific activity compared to the PNS and a 4-fold increase over the lysosomal fraction.

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Figure 7. Specific hexosaminidase activity from sorted secretory lysosomes. An aliquot from the PNS, the Percoll gradient fraction containing the lysosomal fraction and the sorted secretory lysosomes were analyzed for hexosaminidase activity. The specific hexosaminidase activity in each purification step is shown.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITTERATURE CITED

We show herein that the RMCP-DsRed fusion protein is localized predominantly to secretory lysosomes and that the labeled lysosomes can be analyzed and purified by flow cytometry. Several genetic diseases such as Chediak-Higashi syndrome, Hermansky-Pudlak syndromes, and Griscelli's syndrome are associated with defects in secretory lysosomes (6). Moreover, mast cell secretory lysosomes and dense core granules play a crucial role in chronic inflammation and allergic diseases. Our strategy offers new tools to study this important organelle.

Our results demonstrate that both secretory lysosomes and dense core granules are exocytosed upon antigen stimulation of the RBL-2H3 cells (Figs. 3, 4). The dense core granules contain most of the cellular histamine with a minor amount in the secretory lysosomes. Conversely, the RMCP-DsRed fusion protein is detected mainly in secretory lysosomes. These results are in agreement with a recent model suggesting the presence of cross-talk and fusion events between secretory lysosomes and granules (7). A better understanding of the relationship between these two organelles is of great interest and multiparameter SOFA analysis could be used in the future to better understand the biogenesis of secretory lysosomes, their maturation and their relationship to dense core granules.

Confocal microscopy has greatly enhanced our knowledge of subcellular organization, organelle physiology, protein localization and transport. However, confocal microscopy techniques are still limited in their ability to quantify these processes. More recent technologies such as laser-scanning cytometry have attributes of both flow and image cytometry (22); but the level of image resolution compared to confocal analysis is still a limitation. Our work shows that fluorescently labeled organelles can be localized at the cellular level at high resolution by confocal microscopy and analyzed by SOFA in parallel. It should be noted that we also used SOFA to directly detect populations of fluorescent secretory lysosomes from a post nuclear supernatant, thus avoiding any prior cellular fractionation (unpublished observation).

The lysosomes sorted by FAOS showed a 50-fold increase in hexosaminidase specific activity compared to the PNS and a 4-fold increase over the lysosomal fraction. The 4-fold enrichment over the lysosomal fraction might appear modest given that 3% of the total particles present were gated for sorting. However, it should be noted that all the secretory lysosomes might not strongly express RMCP-DsRED (see Fig. 3) but will likely express the endogenous hexosaminidase marker. Moreover, the lysosomal fraction contains fragments of lysosomes that are not gated but are associated with hexosaminidase activity. Thus, the sorted population, with an overall 50-fold increase in hexosaminidase specific activity, constitutes a highly purified population of secretory lysosomes.

In the field of proteomics, there has been a long-standing interest in the analysis of sub-cellular fractions and organelles. Fractionation allows for a reduction of the proteome complexity of the sample compared to a single step analysis. More importantly, the establishment of subcellular proteomes provides reference maps that are useful tools for researchers working on a given organelle or cellular compartment (23–25). Fialka et al. recently developed FAOS to sort apical and basolateral endocytic vesicles and to compare their proteome by two-dimensional gel analysis (15). Our results show that FAOS allows one to obtain a highly purified preparation of secretory lysosomes. These purified lysosomes could then be analyzed to establish their proteome or could be used in a differential protein analysis. For example, lysosomes from activated versus unactivated mast cells could be sorted and their proteome compared. One technical limitation of FAOS is that it may require large quantities of unsorted material for preparative proteomic work. This requirement will obviously depend on the abundance of the labeled organelle in the pre-sort material. However, recent advances in proteomic technologies allow for a direct analysis of proteins or peptides without the requirement for gel separation and extraction. Such technologies were used successfully for the profiling of microsomal proteins (26). These highly sensitive techniques require much less starting material and may offer a good complement to FAOS.

In conclusion, our study confirms the usefulness of flow cytometry to study cellular organelles and provides an additional approach to study secretory lysosomes and their physiological roles.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITTERATURE CITED

We thank Lukas Huber (University of Innsbruck, Austria) and Peter Steinlein (IMP, Vienna, Austria) for help and discussion on the FAOS technology. We gratefully acknowledge the use of the confocal microscope with training and technical assistance provided by Jason Kirk and Susan Krueger at the Center for Biomedical Imaging Technology at the University of Connecticut Health Center.

LITTERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITTERATURE CITED