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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Traffic. 2011 Nov 9;13(1):108–119. doi: 10.1111/j.1600-0854.2011.01300.x

The enlarged lysosomes in beigej cells result from decreased lysosome fission and not increased lysosome fusion

Nina Durchfort 1,2, Shane Verhoef 1, Michael B Vaughn 3, Rishna Shrestha 1, Dieter Adam 4, Jerry Kaplan 1, Diane McVey Ward 1,5
PMCID: PMC3237799  NIHMSID: NIHMS330361  PMID: 21985295

Abstract

Chediak-Higashi Syndrome is an autosomal recessive disorder that affects vesicle morphology. The Chs1/Lyst protein is a member of the BEACH family of proteins. The absence of Chs1/Lyst gives rise to enlarged lysosomes. Lysosome size is regulated by a balance between vesicle fusion and fission and can be reversibly altered by acidifying the cytoplasm using Acetate Ringer’s or by incubating with the drug vacuolin-1. We took advantage of these procedures to determine rates of lysosome fusion and fission in the presence or absence of Chs1/Lyst. Here we show by microscopy, flow cytometry and in vitro fusion that the absence of the Chs1/Lyst protein does not increase the rate of lysosome fusion. Rather, our data indicate that loss of this protein decreases the rate of lysosome fission. We further show that overexpression of the Chs1/Lyst protein gives rise to a faster rate of lysosome fission. These results indicate that Chs1/Lyst regulates lysosome size by affecting fission.

Keywords: lysosome, fusion, fission, beige, chediak-higashi syndrome

The lysosome is considered the terminal point in the endocytic pathway, as molecules are delivered to the lysosome for degradation (for review see (1)). Fusion of endosomes with lysosomes leads to the intermixing of vesicular contents with lysosomal hydrolases. The addition of vesicular contents and membrane would be expected to increase lysosome size. The steady state median size of lysosomes is constant, however, indicating that lysosomal surface area is regulated by lysosomal membrane efflux. It has been suggested that endocytic membrane influx is transient and that once endosomal contents are transferred to lysosomes, endosomes fission off, a process referred to as “kiss-and-run” (2). Constant fusion and fission of membrane along the endocytic pathway results in remodeling of organelles, which may eventually become lysosomes, a process termed maturation (3). Lysosomes are also capable of homotypic fusion (4–13) yet the number of lysosomes is constant. Therefore, lysosomes must be capable of fission or some other form of membrane recycling, although the machinery required for lysosome fission has not been identified.

Malregulation of fusion or fission will result in alterations in lysosome size. This is a hallmark of Chediak Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by enlarged lysosomes or lysosome-related organelles present within all cells (14–17). The mutated gene responsible for CHS (LYST) and the orthologous mouse disorder beige (Lyst) were identified almost 15 years ago (18, 19). The identification of Chs1/Lyst defined a family of proteins termed the BEige And CHediak (BEACH) family (15, 20). The BEACH family protein LvsA in Dictyostelium discoideum regulates the size of the contractile vacuole and is involved in cytokinesis (21–23). Neurobeachin has been characterized to be involved in central synapse formation (24). FAN, the smallest member of the BEACH family, is thought to be an adapter protein linking TNFa signaling to neutral sphingomyelinase. Bph1, the only Saccharomyces cerevisiae homologue is involved in vesicle trafficking, but loss of Bph1 does not affect vacuole morphology (25). The functions of many members of the BEACH family are still unclear.

Previously, we suggested that loss of Chs1/Lyst resulted in decreased lysosomal fission based upon the observation that overexpression of Lyst resulted in smaller than wild type lysosomes (26). Fusion of wild type and beigej cells led to complementation of the large lysosome size (27). We noted, however, that complementation of large beigej lysosomes required their fusion with wild type lysosomes, suggesting that the wild type lysosome provided a factor promoting decreased lysosome size. Studies on the D. discoideum homologue LvsB suggested that LvsB was either a negative regulator of lysosome fusion (28, 29) or a positive regulator of “post-lysosome” fission (30). In this study we examine whether the loss of Chs1/Lyst changes the rate of lysosome fusion or lysosome fission by measuring the rate of reformation of lysosomes following treatments that increase or decrease lysosome size. We show that the loss of Chs1/Lyst does not affect lysosome fusion rates but affects the rate of lysosome fission and that it is the defect in fission that gives rise to enlarged lysosomes associated with the loss of Chs1/Lyst.

Results

The absence of the Chs1/Lyst protein does not result in increased lysosome fusion

We measured the change in lysosome size following perturbations to determine if the loss of Chs1/Lyst affected lysosome fusion or fission. To determine if the enlarged lysosome phenotype in beigej cells was due to increased lysosome fusion we took advantage of a treatment, Acetate Ringer’s, used previously to fragment lysosomes (31, 32) (Figure 1a). Bone marrow-derived macrophages incubated in Acetate Ringer’s buffer fragment their lysosomes and move them to the periphery of the cells. Removal of the Acetate Ringer’s solution results in lysosome movement to a perinuclear region and refusion. If the enlarged lysosome phenotype is due to increased lysosome fusion, then the t1/2 to maximum size or fusion rate would be shorter in beigej cells compared to wild type cells. Wild type and beigej bone marrow-derived macrophages were loaded with fluorescent dextran to mark lysosomes. Lysosomes from wild type and beigej fragmented upon Acetate Ringer’s incubation (Figure 1b) and recovered when placed back in growth medium (Figure 1c). The size of fragmented beigej lysosomes following Acetate Ringer’s was larger than that of C57BL/6 lysosomes. After removal of Acetate Ringer’s, C57BL/6 and beigej lysosomes regained their original size. The t1/2 for recovery or fusion rate for C57BL/6 lysosomes was 13.4 min (+/− 1.6 SEM) compared to 15.2 min (+/− 2.3 SEM) for beigej lysosomes, as determined by flow cytometric analysis (Figure 1c, left panel). The time to achieve final size trended toward being greater for beigej lysosomes but was not statistically significant suggesting that fusion rates were not altered. The initial rate of lysosome recovery was also similar between wild type and beigej strains (Fig. 1c, right panel), although the final size of lysosomes was different. Previously, Perou and Kaplan showed that depolymerizing microtubules with nocodozole resulted in an inability of lysosomes to move to the periphery upon Acetate Ringer’s incubation (30). They also demonstrated that addition of nocodozole during the “recovery” phase, that is movement of lysosomes back to a perinuclear area and refusion, required intact microtubules. We repeated these experiments in hopes of determining if lysosomes size changes in the absence of intact microtubules could be observed/measured using flow cytometry. Unfortunately, lysosomes aggregated significantly upon nocodozole treatment and remained aggregated upon homogenization so that we were unable to determine if lysosome sizes were altered without intact microtubules (data not shown). Previous studies have demonstrated a need for microtubules for lysosome exchange, suggesting that they are required for fusion and possibly fission (7).

Figure 1. Acetate Ringer’s treatment results in fragmentation of lysosomes in wild type and beigej bone marrow-derived macrophages.

Figure 1

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a. Model for fragmented vacuole recovery in C57BL/6 versus beigej macrophages. Acetate Ringer’s causes lysosome fragmentation. Once Acetate Ringer’s is removed, cells regain their respective lysosome size with time. b. Bone marrow-derived macrophages from C57BL/6 or beigej mice were incubated with Alexa 488 dextran (mw 10,000) at 37°C overnight followed by a two h chase to allow all dextran to localize to lysosomes. Cells were incubated with Acetate Ringer’s for 20 min at 37°C, washed 3 times at 0°C. Epifluorescence images were captured using an Olympus BX51 upright microscope with a 60X 1.4NA objective and Pictureframer software (Olympus). c. Cells as in b were treated with Acetate Ringer’s to allow lysosomes to fragment and then placed back in normal growth media for the indicated times to allow lysosomes to refuse. Relative lysosome size during recovery was determined by flow cytometry (left panel) and the initial fold change in lysosome size over time plotted (right panel). Error bars represent the standard error of the mean. All experiments were performed a minimum of three times. Bar = 10 μm.

The absence of the Chs1/Lyst gives rise to decreased lysosome fission

We utilized a second approach to perturb lysosome size in cells and then examine recovery from the perturbation. Vacuolin-1 was identified in a screen for molecules that affect endocytic vesicles. Incubation of cells with vacuolin-1 results in swelling of lysosomes (33, 34). Incubation of C57BL/6 or beigej bone marrow-derived macrophages with vacuolin-1 resulted in lysosome swelling with a complete loss of tubules (Figure 2). Removal of vacuolin-1 resulted in the reappearance of tubular lysosomes and small vesicles in wild type cells. We hypothesize that these structures are necessary for lysosome fission. Eight h after vacuolin-1 removal wild type cells had regained their normal lysosome size, however, beigej macrophage lysosomes remained enlarged. Treatment with vacuolin-1 affected our ability to isolate lysosomes for flow cytometric analysis, as all vacuolin-1-treated lysosomes were susceptible to mechanical shearing during ball-bearing homogenization and all lysosomes showed smaller than normal/predicted relative lysosome sizes (data not shown).

Figure 2. Vacuolin-1 increases lysosome size in C57BL/6 and beigej bone marrow-derived macrophages.

Figure 2

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Bone marrow-derived macrophages from C57BL/6 and beigej mice were grown on glass coverslips in macrophage growth medium. Cells were incubated with Alexa 594 dextran (mw 10,000) at 37°C overnight followed by a two h chase to allow all dextran to localize to lysosomes. Cells were incubated at 37°C for one h without or with 5 μM vacuolin-1 and images captured on an epifluorescence microscope. Vacuolin-1 was removed, cells incubated in growth media and at eight h and lysosome morphology analyzed by microscopy (n=6 with over 100 cells/cell type analyzed). Representative images are shown. Bar = 10 μm.

We took advantage of our cultured fibroblast cells lines to further analyze the role of Chs1/Lyst in lysosome fusion/fission. Incubation of wild type, beigej and YAC-complemented beigej fibroblasts (18, 26) with vacuolin-1 resulted in lysosome swelling as measured by microscopy (Figure 3a and b). Similarly, lysosomes in human fibroblast cell lines (HSFW11-wild type and GM02075-CHS patient) increased in size upon incubation with vacuolin-1. Four h after vacuolin-1 removal, wild type cells (C57BL/6 and HSFW11) and beigej, (YAC) appeared to be recovering with between 30 and 40% of cells showing normal lysosomes (Figure 4a versus Figure 3). Fibroblasts from patients with CHS (GM02075) and beigej mice, however, showed fewer recovered cells (10–20%) after vacuolin-1 treatment. To determine the rate of recovery after vacuolin-1 treatment cells were incubated with vacuolin-1 for one h in growth medium, cells washed, growth medium replaced and images captured at the indicated times. The rate of lysosome recovery was faster for wild type cells (C57BL/6) compared to beigej, lysosomes (Figure 4b). Similarly, the rate of recovery was faster for wild type human fibroblasts (HSFW11) compared to Chediak-Higashi syndrome patient fibroblasts (GM02075) (Figure 4c). Notably, cells overexpressing the Lyst protein (YAC-complemented beigej fibroblasts) recovered lysosome morphology faster than wild type (Figure 4b). How vacuolin-1 works is not known, but it is possible that recovery from enlarged lysosomes requires new protein synthesis or new membrane synthesis. To test this hypothesis wild type C57BL/6 fibroblasts treated with vacuolin-1 were placed in vacuolin-1 free medium in the presence or absence of 100 μg/ml cycloheximide to block protein synthesis and recovery at eight h analyzed by microscopy. C57BL/6 cells recovered lysosome size in the presence or absence of cycloheximide, whereas, beigej cell did not recover (Figure 4d). These data suggest that new protein or membrane synthesis was not necessary and that the molecules required to recover lysosome size were already present in the cells.

Figure 3. Vacuolin-1 increases lysosome size inmouse and human fibroblasts.

Figure 3

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Fibroblasts cell lines C57BL/6, beigej and YAC-complemented beigej as well as human control fibroblasts (HSFW11) and fibroblasts from a patient with Chediak Higashi syndrome (GM02075) were grown on glass coverslips in DMEM with 10% FBS. Cells were incubated with Alexa 594 dextran (mw 10,000) at 37°C overnight followed by a two h chase to allow all dextran to localize to lysosomes. Cells were incubated at 37°C for one h a without or b with 5 μM vacuolin-1 and lysosome morphology analyzed by microscopy (n=5 with over 100 cells/type examined). Bar = 10 μm.

Figure 4. The absence of Chs1/Lyst delays lysosome recovery after vacuolin-1 treatment.

Figure 4

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a. Cells incubated with vacuolin-1 as in Fig. 3 were washed and placed in DMEM with 10% FBS for four h and lysosome morphology examined by fluorescence microscopy. b. and c. Cells treated as in Fig. 3 were placed in growth medium, images captured at defined times and cell recovery determined by contrasting several fields (2–10 fields) of untreated cells to several fields (2–10 fields) of treated cells without recovery and then counting the number of cells at each recovery time that were more like untreated or treated cells. The data are expressed as the percentage of cells showing lysosome recovery. The experiments were performed three times and the data represent the average of 10–20 fields of cells with 2–10 cells per field (40–100 cells/cell type). d. Cells were incubated with or without 100 μg/ml cycloheximide during vacuolin-1 recovery and cell recovery determined at eight h by contrasting untreated cells to treated cells as in b and c. Bar = 10 μm.

The approximate size of lysosomes before vacuolin-1, after vacuolin-1 and during recovery was determined using NIH Image J software (Figure 5). The data confirm that YAC-complemented beigej fibroblasts recovered their lysosome size faster than wild type C57BL/6 or HSFW11 and that the absence of the Chs1/Lyst protein in beigej or GM02075 resulted in a slower rate of recovery of lysosome size. These results support the hypothesis that the Chs1/Lyst protein functions in lysosome fission.

Figure 5. Measurement of lysosome size during vacuolin-1 recovery.

Figure 5

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Fibroblasts were incubated with Alexa 594-dextran overnight at 37°C. Cells were washed and incubated in growth medium for an additional two h. Cells were treated with or without vacuolin-1 for one h at 37°C followed by incubation in growth medium for indicated times. Lysosome morphology was examined by epifluorescence microscopy and images analyzed using Image J software measuring lysosome size (diameter μm) from three separate experiments. The data represent the average of 10–20 fields with two to 10 cells per field. Over 200 vesicles per cell type were measured at each time point. Error bars represent the standard error of the mean.

In vitro lysosome fusion is not increased in the absence of the Chs1/Lyst protein

We hypothesized that if the Chs1/Lyst protein negatively regulates lysosome fusion, then lysosome fusion in vitro might be increased in the absence of the Chs1/Lyst protein, which is predicted to be a cytosolic protein. We utilized our in vitro lysosome fusion assay developed in rabbit alveolar macrophages, which measures the formation of avidin-biotin complexes within the lysosomes (4, 7). As shown previously, in vitro lysosome fusion is dependent upon ATP and cytosol (Figure 6a). The addition of cytosol from beigej cells did not alter the total amount of in vitro lysosome fusion compared to cytosol from wild type cells, nor did the absence of Chs1/Lyst alter the rate of in vitro lysosome fusion (Figure 6b). These results further support the hypothesis that the absence of the Chs1/Lyst does not increase the rate or amount of lysosome fusion.

Figure 6. In vitro lysosome fusion is not affected by the absence Lyst.

Figure 6

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a. Alveolar macrophages were isolated as described previously (7). Macrophages were incubated with either 0.5 mg/ml biotinylated-horseradish peroxidase or avidin at 37°C for 60 min. Cells were washed extensively and chased for an additional two h. Cells were washed, homogenized and lysosomes isolated as previously described (7). Lysosomes (1–2 mg/ml) were combined at 4°C in the presence of excess biotinylated-insulin with or without an ATP-regenerating system and with or without cytosol isolated from wild type C57BL/6 liver or beigej liver (2–4 mg/ml). Samples were shifted to 37°C for 60 min, fusion stopped by placing samples at 4°C and fusion assayed as previously described (7). In vitro fusion was expressed as the percentage of maximum avidin-b-HRP complex formation. b. Samples as in 5a where shifted to 37°C for time to measure the kinetics of in vitro fusion in the presence or absence of Chs1/Lyst. All experiments were performed in triplicate and error bars represent the standard error of the mean.

beigej macrophages show increased steady state lysosome tubulation compared to wild type macrophages

In an elegant study by Patterson and Lippincott-Schwartz the dynamic nature of lysosomes was observed using live cell microscopy (8). The authors observed a high degree of microtubule-dependent exchange of material between lysosomes that appeared to be mediated by tubules emanating from lysosomes. Lysosome morphology in bone marrow-derived macrophages often shows both vesicular and tubular structures (35, 36). Epifluorescent microscopic examination of lysosomes in wild type or beigej bone marrow-derived macrophages suggested that lysosomes from beigej cells showed higher levels of tubulation (Figure 7a). Quantification of the number of cells with vesicular or tubular lysosomes shows that 45% of wild type cells had tubular lysosomes compared to 85% in beigej bone marrow-derived macrophages (Figure 7b). Wild type cells showed 40–50% mild to medium tubulation with only 2–4% of cells showing extreme tubulation of lysosomes. In contrast, 40–50% of beigej cells showed extreme tubulation of lysosomes. An example with 3D-rendering of extreme tubulation in wild type and beigej is shown (Figure 7c, SVideo1 and SVideo2).

Figure 7. Bone marrow-derived macrophage lysosomes in beigej show increased tubular morphology.

Figure 7

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Wild type and beigej bone marrow-derived macrophages were cultured, plated on glass coverslips and labeled as described in Fig. 2. a. Images were captured on an FV1000 Olympus confocal microscope. Bar = 10 μm. b. The number of cells (greater than 200 cells each) showing vesicular or tubular lysosomes was quantified by four different individuals using the same range and same three planes of focus to determine no tubular lysosomes (N), primarily vesicles with a few tubular lysosomes (Mi), approximately equivalent numbers of vesicular or tubular lysosomes medium (Me) or predominantly tubular lysosomes with few vesicular lysosomes as extreme (E) and the average plus and minus the standard error of the mean of three separate experiments used to determine the percent tubulation. c. Confocal images of extreme tubulation in C57BL/6 and beigej bone marrow-derived macrophages showing xy, xz and yz projections. Bar = 5 μm.

The increased tubulation in beigej lysosomes might reflect decreased vesicle fission as tubules may be a precursor to vesicle fission. A prediction of this is beigej lysosomes would not fission as rapidly as lysosomes from wild type cells. We utilized time-lapse microscopy to quantify lysosome fission events. Measurements of lysosome fission showed that wild type macrophages showed increased rates of fission compared to beigej lysosomes (Figure 8a). The average time required for lysosome fission in wild type cells was 70–75 s whereas the time required for beigej lysosome fission was 135–170 s. A distribution of all quantified fission events is shown in Figure 8b. Lysosome fission in C57BL/6 and beigej macrophages could be observed as early as 10 seconds. The majority of C57BL/6 lysosome fission events occurred in the first 150 s with a few fission events occurring after 150 s. In contrast, several beigej lysosome fission events occurred after 150 s. An example of lysosome fission for C57BL/6 and beigej lysosomes is shown (Figure 8c, SVideo3 and SVideo4). These data demonstrate that the loss of the Chs1/Lyst protein affects the ability of lysosomes to fission and suggest that the Chs1/Lyst protein may not be required for cytoskeletal association, but is required for efficient lysosome fission.

Figure 8. Wild type lysosomes show increased fission rates compared to beigej lysosomes.

Figure 8

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a. Wild type and beigej macrophages loaded with Alexa 594 dextran as in Fig. 7 were imaged on a Nikon automated Metamorph microscope capturing six different fields with five-to-ten cells per field (n=2 separate experiments). Images were captured every 5 sec for up to ten min. Movies were generated and image analysis performed with NIH Image J software measuring time elapsed to vesicle/tubule fission. Between 50–150 vesiculation events were quantified/cell type. The data are expressed as seconds/fission event in either tubular or vesicular lysosomes. b. The combined data from a are plotted as a histogram with the data expressed as the number of vesiculation events (Y axis) per second (X axis). c. Shown are representative snapshots of lysosome fission in C57BL/6 and beigej over 210 sec. Arrowheads identify vesicle formation and arrows identify incomplete fission events. Bar = 5 μm.

Discussion

Regulation of vesicle size is determined by the rate of fusion versus the rate of fission. The absence of the Chs1/Lyst protein results in enlarged lysosomes, which could be explained by either increased rates of lysosome fusion or decreased rates of fission. Here we provide evidence that Chs1/Lyst regulates lysosome size by regulating lysosome fission. We show that in the absence of Chs1/Lyst, lysosomes do not have increased rates of fusion, rather there is decreased fission and that overexpression of Chs1/Lyst results in an increased rate of lysosome fission.

Vesicle fission has been described for many membrane trafficking events including: endocytosis (37, 38), multivesicular body formation (39), movement of molecules from ER to Golgi (40–42) and back to ER (43), mitochondrial division (44), peroxisome division (45), vacuole inheritance (46) and late endosome-lysosome “kiss-and-run” (2). The idea that mammalian lysosomes fission has been underappreciated and little is known about the mechanism of lysosome membrane recycling. Perhaps the best description of lysosome membrane fission to date is that of Patterson and Lippincott-Schwartz who, through the use of photoactivatable GFP, demonstrated tubular lysosomes and the generation of vesicles pinching off from lysosomes with lysosomes undergoing frequent homotypic fusion and fission events (8). The finding that lysosomes pinch off vesicles which are aligned with microtubules suggest a role for microtubules in lysosome fission (47). Both kinesin and dynein have been shown to be associated with lysosomes (36, 48) and wild type or beigej lysosomes can move to the cell periphery and back to a perinuclear area suggesting that the motors can be recruited in the absence of the Chs1/Lyst protein (31). We hypothesize that Chs1/Lyst is a necessary componenet for efficient lysosome fission. Many vesicle fission events require coat proteins such as clathrin, adaptors (AP-1, AP-2 and AP-3) or COPs, cytoskeletal elements and motor proteins and often a GTPases-like dynamin. There is one report of clathrin being assembled on lysosomes (49); however, the role of clathrin in lysosome recycling is not established. A dynamin-related protein Vps1 has been shown to be important for vacuole tubulation and fission in Schizosaccharomyces pombe (50), but a mammalian homologue of Vps1 has not been identified. F-BAR proteins that assist in membrane tubulation events have also been shown to be important in vesicle fission (51) but a lysosomal F-BAR protein has not been identified.

Our studies support the hypothesis that Chs1/Lyst is important for lysosome membrane fission. Our live cell microscopy does not show aberrant fusion of lysosomes, but shows a reduced amount of lysosome fission. The absence of the Chs1/Lyst protein does not completely block lysosome fission. Microscopic examination and flow cytometry showed that normal as well as enlarged lysosomes are present in the absence of Chs1/Lyst (Perou et al., 1997). This is also supported by heterokaryon cell-cell fusion experiments, where lysosome mixing occurred in beigej-beigej cell fusions, but the average size of the lysosomes did not increase (27). These results suggest that there are multiple pathways to regulate lysosome size.

The mechanism by which Chs1/Lyst regulates lysosome fission is not known. The function of Chs1/Lyst is not informed by the amino acid sequence. The sequence contains a long alpha-helical domain along with a highly conserved carboxyl terminus. This most conserved region, the BEACH domain, has been crystallized for the neurobeachin protein, a BEACH family protein (52, 53). Neurobeachin is a protein kinase A-anchoring protein that plays a role in neuromuscular synaptic transmission and has been found to be peripherally associated with tubulovesicular endomembranes near the trans Golgi network (54). For vesicles to divide, membranes need to grow and bend and this is usually achieved by proteins that deform the membrane. It is possible that the Chs1/Lyst protein also associates with membranes to assist in tubulation/deformation and vesicle fission. Studies in Dictyostelium have shown the Chs1/Lyst homolog LvsB associates with lysosomes (28, 29), however, Chs1/Lyst has not been shown to associate with lysosomes. A link between Chs1/Lyst and protein kinase C activity has been suggested (55–58) and protein kinase C delta has been found to localize to secretory lysosomes (59, 60). How changes in protein kinase C activity affect lysosome fission is not understood, but it may be that the Chs1/Lyst protein acts as an anchoring protein similar to neurobeachin, bringing molecules required for membrane fission specifically to the lysosome.

Lysosome fusion like endosomal fusion events can be reproduced in a test tube, which is dimensionless (7, 61–64). In contrast, fission events might not occur in the absence of a force vector to provide tension. In particular, if membrane loss is due to fission rather than budding, a system to provide force is required. Studies by Wolkoff and colleagues have reconstituted endosomal fission events using microtubules and the motor proteins kinesin and dynein (65–67). Interestingly, they demonstrated a role for the zeta form of protein kinase C in regulating early endosomal fission but not late endosome fission (68). In vitro systems might provide a mechanism to tease apart the biochemical requirements for lysosome fission and for the role of Chs1/Lyst in the regulation of lysosome fission.

Materials and Methods

Cells and media

Bone marrow cells were isolated from femurs of 6 to 8 week-old C57BL/6 and beigej mice and seeded on 10-cm dishes. Macrophages were cultured in RPMI medium (Invitrogen, Carlsbad, CA), 2 mM L-glutamine, 20 μM supplemented with 20% fetal bovine serum (FBS) and 30% L-cell conditioned medium. C57BL/6, beigej, wild type human and LYST mutant (GM02075) fibroblasts were maintained in Dulbecco’s MEM with 10% FBS. Macrophages were incubated in Acetate Ringer’s medium (80 mM NaCl, 70 mM Sodium acetate, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM Hepes, 10 mM glucose and 0.5 mg/ml bovine serum albumin (BSA)) for 20 min at 37°C (31, 32). Rabbit alveolar macrophages were isolated as previously described (4).

Microscopy

Cells were plated onto 1 mm glass coverslips in the respective growth medium, incubated at 37°C with 5 μg/ml Alexa 488 or Alexa 594 dextran (mw 10,000) (Invitrogen, Carlsbad, CA) overnight followed by a two h chase to allow all dextran to localize to lysosomes. Quantifications of lysosome size changes were done on epifluorescent images using greater than 20 fields per cell type with 2–10 cells per field. Images were quantified by four separate individuals and an average of that analysis is presented in graph form. Measurements of lysosome diameter were performed using NIH Image J software. Confocal images of Alexa 594-dextran labeled lysosomes were captured on an Olympus FV1000-XY microscope using Metamorph software (Molecular Devices, Sunnyvale, CA) for capture for analysis. Lysosome fission analyses were performed on bone marrow-derived macrophages that had been incubated with Alexa 594 dextran to mark lysosomes. Cells were imaged every five sec for up to ten min using a Nikon automated Metamorph workstation and movies generated. Lysosome fission events in six time-lapse movies per cell type were measured by counting the number of frames (five seconds/frame) between each fission/vesiculation event. The average time to observe a fission event was calculated and the data represent the standard error of the mean.

Flow Cytometry

Cells incubated with Alexa 488 dextran were placed in 0.25 M sucrose/10 mM tris-HCl pH 7.2/0.5 mM EDTA. Cells were homogenized using a ball-bearing homogenizer, homogenates centrifuged at 500 × g for 5 min and the PNS analyzed for fluorescence lysosomes as described previously (26).

In vitro lysosome fusion

Rabbit alveolar macrophages were isolated, incubated with either biotin-horseradish peroxidase (HRP) or avidin, lysosomes isolated and in vitro fusion performed as previously described (7).

Other procedures

Vacuolin-1 was obtained from (Sigma-Aldrich, St. Louis, MO) and used as previously described (33, 34). Briefly, cells preincubated with Alexa 488 or 594-dextran to label lysosomes were incubated with 5 μM vacuolin-1 at 37°C for one h in growth medium. Cells were then washed and imaged at specified times. Cycloheximide was obtained from Sigma-Aldrich (St. Louis, MO) and used at 100 μg/ml.

Supplementary Material

Supp Movie S3. Supplemental Movie 3. C57BL/6 bone marrow-derived macrophage lysosomes show movement and vesicle fission.

Images from Fig. 8 were assembled into a time-lapse movie.

Download video file (9.6MB, avi)
Supp Movie S4. Supplemental Movie 4. beigej bone marrow-derived macrophage lysosomes show decreased movement and delayed vesicle fission.

Images from Fig. 8 were assembled into a time-lapse movie.

Download video file (5.3MB, avi)
Supp Video S1. Supplemental Movie 1. 3D rendering of extreme tubulation in C57BL/6 bone marrow-derived macrophage lysosomes.

Confocal images from Fig. 7c where analyzed using Volocity software and 3D images generated.

Download video file (23.3MB, mov)
Supp Video S2. Supplemental Movie 2. 3D rendering of extreme tubulation in beigej bone marrow-derived macrophage lysosomes.

Confocal images from Fig. 7c where analyzed using Volocity software and 3D images generated.

Download video file (22.6MB, mov)

Table 1.

Measurement of Relative Fibroblast Lysosome Size using Flow Cytometry

Cell Line FSC-H Mean +/− SD
C57BL/6 20.91 +/− 2.1
beigej 31.23 +/− 5.3
HSFW11 22.63 +/− 1.2
GM02075 38.61 +/− 7.2
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Fibroblasts were incubated with 5 μg/ml Alexa 488-conjugated dextran (10,000 mw) at 37°C for 12 h. Cells were washed and incubated for an additional two h in dextran-free growth medium to allow accumulation of dextran in lysosomes. Cells were washed in PBS, resuspended in 2.0 ml STE and homogenized using a ball-bearing homogenizer. Homogenates were centrifuged at 500 × g for five min to remove nuclei and intact cells. Post-nuclear supernatants were used to measure relative lysosome size (FSC-H) based upon fluorescent dextran as previously described (26).

Acknowledgments

The authors would like to thank the members of the Kaplan lab for critically reading the manuscript and the Imaging Core at the University of Utah for assistance in analyzing lysosome morphology. Specifically, the authors want to acknowledge the assistance of Dr. Chris Rodesch in live cell microscopy and vesicle formation/fission analysis. This work is funded by NIH grant HL026922 to JK.

Abbreviations

BEACH

Beige and Chediak

BSA

bovine serum albumin

CHS

Chediak Higashi Syndrome

FBS

fetal bovine serum

PBS

phosphate buffered saline

PNS

post nuclear supernatant

Footnotes

Competing Interests

The authors declare no competing financial interests.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Movie S3. Supplemental Movie 3. C57BL/6 bone marrow-derived macrophage lysosomes show movement and vesicle fission.

Images from Fig. 8 were assembled into a time-lapse movie.

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Supp Movie S4. Supplemental Movie 4. beigej bone marrow-derived macrophage lysosomes show decreased movement and delayed vesicle fission.

Images from Fig. 8 were assembled into a time-lapse movie.

Download video file (5.3MB, avi)
Supp Video S1. Supplemental Movie 1. 3D rendering of extreme tubulation in C57BL/6 bone marrow-derived macrophage lysosomes.

Confocal images from Fig. 7c where analyzed using Volocity software and 3D images generated.

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Supp Video S2. Supplemental Movie 2. 3D rendering of extreme tubulation in beigej bone marrow-derived macrophage lysosomes.

Confocal images from Fig. 7c where analyzed using Volocity software and 3D images generated.

Download video file (22.6MB, mov)

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