View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central PHAGOSOME-LYSOSOME FUSION Characterization of Intracellular Membrane Fusion in Mouse Macrophages MARGARET C . KIELIAN and ZANVIL A . COHN From the Department of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021 ABSTRACT Several approaches have been used to study the determinants of phagosome- lysosome fusion in intact mouse macrophages . Lysosomes were labeled with the fluorescent vital dye acridine orange and the rate and extent of their fusion with yeast-containing phagosomes was monitored by fluorescence microscopy . Fusion was also assayed by electron microscopy, using horseradish peroxidase or thorium dioxide as a marker for secondary lysosomes . Good agreement was found with results obtained from vitally stained cells, thin-section samples with an enzymatic marker, and thorium dioxide-labeled samples evaluated by stereology . The rate of fusion as assayed by fluorescence was not affected by the number of particles ingested, serum concentration, or prior uptake of digestible or nondi- gestible substances. With this assay it was possible to observe the rate of fusion separate from and uninfluenced by the phagocytic rate . Both the rate and extent of fusion were dramatically increased after several days in culture and similar changes were found by use of the EM assays . Fusion was strongly affected by incubation temperature, having a QIo of 2 .5 . No detectable fusion occurred below 15 ° C, and this inhibition was rapidly reversed when cells were returned to 37°C . Intracellular membrane fusions serve to link dis- criminate fusions do not occur. For this reason, crete, discontinuous cellular compartments, lead- some form of membrane-membrane recognition is ing to the mixing, export, or intake of exogenous likely . Other than a descriptive analysis of the and biosynthesized macromolecules . Major vec- ultrastructural and freeze-fracture patterns of fu- torial pathways include the centrifugal flow of sions, little insight into the involved mechanism is secretory proteins from their point of synthesis in available in eukaryotic systems . the rough endoplasmic reticulum (RER) to their One of the obstacles to gaining information exocytosis at the plasma membrane . The reverse about the fusion of phagosomes with lysosomes flow of endocytic vesicles into the inner organelles (P-L fusion) has been the lack of a rapid, quanti- ofthe vacuolar apparatus represents another route, tative assay for the rate and extent of fusion . For one recently coupled to the recycling of interior- this purpose we have examined a sensitive vital ized plasma membrane (18, 27) . Although mem- dye method used by Hart and Young (12) and branes of apparently different origins fuse, the applied it to the study of the highly endocytic process is restricted within these limits and indis- mouse peritoneal macrophage. In this report we 754 J . CELL BIOLOGY © The Rockefeller University Press - 0021-9525/80/06/0754/12 $1 .00 Volume 85 June 1980 754-765 establish conditions for the reproducible assess- 5% FCS/MEM was added, the cultures were rapidly warmed to ment of P-L fusion and compare conditions at 37°C, and relatively synchronous ingestion of the bound yeast resulted . The usual number of yeast particles bound and ingested both the light and EM level . In addition, we have per cell was 2 . Cells were examined by fluorescence microscopy determined the effects of particle number, com- at various time points after an initial 10-min ingestion period. position, and size, ambient temperature, and pro- The presence of orange-stained intracellularyeast was considered longed in vitro cultivation on the fusion process . positive for P-L fusion . AO Quantitation MATERIALS AND METHODS Coverslip cultures were pulsed with 5 14g/ml AO in FCS/ Cell Culture MEM for 20 min, washed, and extracted in 95% ethanol . Fluo- rescence of samples was compared to standards in the same Primary cultures of peritoneal macrophages were prepared solvent, using an MPF-44 fluorometer (Perkin-Elmer Corp ., from resident cells of female or male Nelson-Collins strain mice Norwalk, Conn.) with an excitation wavelength of 490 nm and (22-30 g) (5) . Cells were routinely cultured in 15-20% fetal calf wavelength of 520 Values were related to the serum (FCS; heat inactivated at 56°C for 30 min) in Dulbecco's emission rim . amount of cell protein on parallel coverslips, using the fluores- modified Eagle'smedium (MEM) containing 100 U/ml penicillin (3) with bovine serum albumin as a stand- and 100 pg/mlstreptomycin and 0.25 Ag/ml amphotericin B, and camine protein assay Acridine standards in solvent with or without unlabeled cells were given fresh medium at least every other day . Cells were ard . plated at densities of 4 x l0' peritoneal exudate cells per 12-mm were comparable . glass coverslip forfluorescence studies, or at 6-10 x l0"peritoneal cells per 35-mm dish for electron microscopy . Temperature Studies P388D,, a macrophagelike cell line, was obtained from Dr. 3 . After an initial phagocytosis step at 37°C, cultures were Unkeless and maintained in spinner culture in 10% FCS/MEM maintained at temperatures from 15° to 30°C in a water-jacketed (30) . chamber (l7) gassed with 5% C.2/95% air. Temperature was Culture dishes with 16-mm wells were obtained from Costar, controlled by use of a constant-temperature circulating water Data Packaging, Cambridge, Mass . bath and cultures were kept at 2°C in an ice bath . Particle Preparation for Fusion Studies Fluorescence Microscopy Fresh baker's yeast was processed by the method of Lachman AO-labeled cells were prepared by inverting coverslips over and Hobart (15) . This procedure consists of autoclaving, reduc- a drop of ice-cold PBS, blotting, and rimming with nail polish. tion by mercaptoethanol, alkylation with iodoacetamide, and These live specimens were then examined with a Zeiss photo- extensive washing in phosphate-buffered saline (PBS) . The re- microscope Ill, using a mercury lamp adjusted for epi-illumina- sulting preparation could be stored indefinitely in PBS without Mgt. tion, BG-12 filter . and fluorescein dichroic mirror for excitation, Cat ' and plus 0.02% NaN ;, . and a 53 barrier filter . Black and white pictures were taken with To opsonize particles with complement components, 300la1 of Kodak Tri-X film . a 5% suspension of yeast in veronal buffer was mixed with 300 lal of mouse serum (fresh or stored at -70°C), incubated 30 min Microscopy at 37°C, and washed several times in cold veronal buffer. Prep- Electron arations were used within 3 d of opsonization . Monolayers were fixed for 30 min at room temperature in Latex particles of 0.50, 1 .10, 2.02, 3.14, 4.27, 5.7, and 8.0 Jim 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7 .4. After Diam were obtained from Dow Chemical Co ., Indianapolis, Ind ., three saline washes, horseradish peroxidase (HRP) was visualized and Duke Scientific Corp .. Palo Alto, Calif., washed five times in labeled cultures by the Graham and Karnovsky diaminoben- in PBS, and stored at 4°C until used . zidine method (11). Cultures were then postfixed in 1% osmium tetroxide in 0.1 M cacodylate (pH 7.4) for 60 min on ice, and Fluorescence Assay of P-L Fusion stained with 0.25% uranyl acetate in 0.1 M sodium acetate buffer for 30 min at room temperature. Specimens were dehydrated in on 12-mm glass coverslips were labeled Monolayer cultures graded alcohols and embedded in Epon . Thin sections were for 20 min at ° C in 15% FCS/MEM by adding acridine orange 37 examined in a Siemens Elmiskop 11 with or without uranyl of 5 lag/ml . A stock (AO) stock solution to a final concentration acetate and lead citrate staining . solution of 100 pg AO/ml PBS could be stored in the dark at Scanning electron microscopy was kindly performed by Dr . 4°C for 3-4 wk . Each coverslip was then placed in a 16-mm- Gilla Kaplan . Monolayer cultures on 12-mm coverslips were Diam well of plastic dish containing I prewarmed a culture ml of fixed in 2 .5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 . medium, and incubated for 10 min at 37 ° C to reduce background Specimens were dehydrated in alcohol, transferred to amyl ace- fluorescence and decrease photodamage. The medium was then tate and critical point dried (Sorvall critical point drying system ; aspirated and 1 ml of a cold 0.004% (vol/vol) suspension of DuPont Co., Sorvall Biomedical Div ., Norwalk, Conn .) in liquid opsonized yeast was added per well. The yeast suspension was CO,. The cells were gold coated (Edwards 5150 sputter coater, centrifuged onto the cell monolayer by placing the culture dish Grand Island, N. Y.) and viewed with an ETEC autoscan . in a centrifuge carrier designed for microtiter plates (Cooke Laboratory Products, Alexandria, Va .) and centrifuging in an International centrifuge (International Equipment Co ., Needham EM Evaluation of P-L Fusion Heights, Mass .) at 1,200 rpm for 2 min at 4° C. The yeast particles Two markers for secondary lysosomes were used, HRP and bound to the macrophage complement receptor, and any un- colloidal thorium dioxide (Thorotrast) . Cultures were pulsed with bound particles were removed by washing with cold MEM. Cold 2 mg/ml HRP in medium for 2-3 h, washed four times with MARGARET C . KIELIAN AND ZANVIL A . COHN Phagosome-Lvsosome Fusion 75 5 MEM, and cultured 20-60 min longer in medium without HRP . transferred into the vacuole . To compare rates of Cells were then given yeast or latex particles and fixed I h after P-L fusion in different cell populations, we ana- ingestion . Specimens were processed for EM as described above, and thin sections examined without uranyl acetate and lead lyzed the experimental variables involved .