Department of Pathology, Division of Cell Biology and Immunology, University of Utah Health Science Center, Salt Lake City, Utah 84132
Lysosomes are dynamic structures capable of
fusing with endosomes as well as other lysosomes. We
examined the biochemical requirements for homotypic
lysosome fusion in vitro using lysosomes obtained from
rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures
the formation of a biotinylated HRP-avidin conjugate,
in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis.
We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP and an
N-ethylmaleimide (NEM)-sensitive factor from cytosol.
The NEM-sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutant NSF
inhibited fusion. Fusion in vitro was extensive; up to
30% of purified macrophage lysosomes were capable of
self-fusion. Addition of GTPs to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of
rabs. Fusion was also inhibited by the heterotrimeric G
protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a G
i activator, inhibited in vitro lysosome fusion whereas cholera toxin, a
G
s activator did not inhibit the fusion reaction. Addition of agents that either promoted or disrupted microtubule function had little effect on either the extent or
rate of lysosome fusion. The high value of homotypic
fusion was supported by in vivo experiments examining
lysosome fusion in heterokaryons formed between cells containing fluorescently labeled lysosomes. In both
macrophages and J774E cells, almost complete mixing
of the lysosome labels was observed within 1-3 h of UV
sendai-mediated cell fusion. These studies provide a
model system for identifying the components required
for lysosome fusion.
ORGANELLES within the endocytic pathway are dynamic structures, resulting from the fusion and
fission of newly internalized vesicles with preexisting structures. The early portion of the endocytic pathway exists in a "steady state." In the absence of continued
membrane internalization the early endocytic apparatus virtually disappears and the constituent membrane and
contents are then directed to the cell surface and the lysosome (Tsuruhara et al., 1990 Although there is abundant evidence that lysosomes can
fuse, the mechanisms that regulate fusion, however, are
unknown. A diverse series of GTP-binding proteins, ADP
ribosylation factors (ARFs) (Balch et al., 1992 In an effort to discern the factors responsible for lysosomal fusion we have devised an in vitro homotypic lysosome fusion assay. In this communication we report on the
biochemical characteristics of the assay system, its specificity and the minimal requirements for lysosome fusion in
vitro. We demonstrate that lysosomes derived from macrophages have a high capacity for homotypic fusion. Whereas fusion appears to require a heterotrimeric G protein and
rab(s), it is independent of microtubules.
Cells
Rabbit alveolar macrophages were obtained by bronchial lavage (Myrvik
et al., 1961 Materials
Unless otherwise noted, all reagents were obtained from Sigma Chemical
Co. Human holo-transferrin was iodinated as described previously (Ward
et al., 1982 Subcellular Fractionation
Cells were incubated in 1 mg/ml b-HRP in HMEM/BSA at 37°C for 45 min, washed extensively, and then chased for an additional 90-120 min.
Cells were then incubated in 10 In Vitro Fusion Reaction
Cells were incubated with either 0.5 mg/ml b-HRP or avidin at 37°C for
45-60 min in Hanks' minimal essential medium (HMEM). Cells were
washed extensively and chased for an additional 90-120 min. Cells were
washed and homogenized in HB, and then a PNS was obtained as described above. (Typically the vesicle protein concentration was 1-2 mg/ml
with a cytosol protein concentration ranging from 2-4 mg/ml). PNSs and/
or lysosomes (isolated as described) were combined at 4°C in the presence
of excess (20-100 µg/ml) b-insulin. Fusion reactions were performed in
250 mM sucrose, 2 mM DTT, 1 mM MgCl2, 0.5 mM EGTA, 50 mM KCl,
and 20 mM Hepes, pH 7.2 (fusion buffer), plus or minus an ATP regenerating system as described previously (Colombo et al., 1992a Lysosome Isolation
Lysosomes were isolated by fractionating over Percoll gradients (Ward et al.,
1990a Microscopy Studies
J774E cells or rabbit alveolar macrophages were incubated in HMEM
containing either 1 mg/ml fluorescein-dextran (10,000 mol wt) or 0.5 mg/ml
Texas red-dextran (10,000 mol wt) (Molecular Probes, Inc., Eugene, OR)
at 37°C for 30 min. Cells were washed and incubated in HMEM at 37°C
for an additional 90 min. The two cell populations were then washed,
mixed, and fused using UV sendai fusion as per Perou and Kaplan (1993b) Preparation of Microtubules
Phosphocellulose-purified bovine brain tubulin was a gift from Dr. D. Gard (Department of Biology, University of Utah, Salt Lake City, UT). Microtubules were prepared by serially adjusting 0.66 mg/ml tubulin in 80 mM
Pipes, pH 6.8, 1 mM EGTA, 1 mM MgCl2, and 1 mM GTP to 0.2 µM, 2.0 µM,
and 10 µM taxol with incubations at 4°C for 5 min at each step. This solution was added to reaction mixtures containing 10 µM taxol to achieve a
final concentration of 0.033 mg/ml tubulin. The presence of polymerized
microtubules was confirmed by immunofluorescence using a monoclonal
anti- Demonstration of In Vitro Homotypic Lysosome Fusion
The fusion assay relies on the formation of a b-HRP-avidin
complex within lysosomes, with the subsequent capture
of the complex by anti-avidin antibodies as originally described by Braell (1992) Using the protocol described in Materials and Methods
we determined that the pulse-chase procedure resulted
in the localization of >95.0% of the cell-associated ligand
within lysosomes as assessed by Percoll gradients. When
PNSs were mixed and incubated in vitro, there was a time-
and temperature-dependent formation of avidin-b-HRP complexes. Approximately 10-20% of the initial b-HRP
activity was found associated with avidin in PNSs incubated at 37°C for 60 min; whereas at 0°C, minimal avidin-
b-HRP complex formation was observed (Fig. 1). Similar
results were obtained using rabbit alveolar macrophages
(data not shown). Since the presence of b-insulin blocked
the formation of adventitious avidin-b-HRP complexes
released as a result of vesicle breakage, formation of avidin-b-HRP complexes could only result from vesicle fusion. The contents of an alveolar macrophage fusion assay
were fractionated on Percoll gradients before addition of
detergent. Avidin-b-HRP complexes cosedimented with
the lysosomal marker hexoseaminidase indicating that the complex was a result of fusion and not binding of free
ligands (Fig. 2). The data indicate that the products of the
in vitro fusion may result in a slight change in the buoyant
density of lysosomes, the avidin-b-HRP peak and hexoseaminidase peaks do not completely coincide. One explanation of this result is that the fusion products may be larger
and consequently be less dense.
Specificity of Lysosome Fusion
PNSs contain many vesicle populations. It is possible that
the ligand may not be solely restricted to lysosomes, and
that complex formation results from other vesicles, e.g.,
endosomes fusing with each other or with lysosomes. To
address this point, lysosomes purified from Percoll gradients were used in the fusion assay. PNSs from either avidin- or b-HRP-loaded J774E cells were layered over 23%
Percoll and centrifuged to separate lysosomes from endosome/Golgi vesicles. Previously, we demonstrated that Golgi as well as late endosomes are fairly coincident with
early endosomes, but are well separated from lysosomes
on Percoll gradients (Ajioka and Kaplan, 1987
The specificity of the fusion reaction was further demonstrated using rabbit alveolar macrophages. Percoll gradient
fractionation of cellular homogenates showed a single buoyant density peak of lysosomes that was well separated from
both early and late endosomes (Ward et al., 1990a
Homotypic Lysosome Fusion In Vivo
To demonstrate that the high degree of fusion was not due
to an in vitro artifact, we assessed the degree and rate of
lysosome fusion using an in vivo experiment. Cells, containing different fluorescently labeled lysosomes, were
fused together and lysosome fusion assessed by the coincidence of the two fluorescent dyes. Cells, labeled separately
with either fluorescein-dextran or Texas red dextran, were
fused using UV sendai virus as described (Ferris et al., 1987
Biochemical Requirements of Homotypic Fusion
In vitro fusion was dependent upon cytosol, but the origin
of the cytosol was irrelevant. Similar results were obtained
using cytosol from cultured cells, isolated rabbit macrophages, or mouse liver homogenates (data not shown). Fusion in vitro was ATP dependent, as depletion of endogenous ATP by addition of glucose and hexokinase resulted
in the complete inhibition of lysosome fusion (Table I).
Treatment of cytosol with NEM resulted in the complete
loss of fusion activity, suggesting that homotypic lysosome
fusion, like almost all other fusion events, was dependent
on an NEM-sensitive factor that may be NSF (Block et al.,
1988 Table I.
Biochemical Requirements for In Vitro
Lysosome-Lysosome Fusion
; Ward et al., 1995
). Whereas
the lysosome retains its identity in the absence of continued membrane internalization, it is a dynamic organelle
capable of undergoing changes both in morphology and
size (Kornfeld and Mellman, 1989
; Storrie and Desjardins, 1996
). Studies have indicated that lysosomes can undergo
self, or homotypic fusion. Acidification of macrophage cytosol results in the fragmentation of lysosomes into vesicles that are removed from their normal perinuclear location to a more peripheral location (Heuser, 1989
; Perou
and Kaplan, 1993a
). Upon normalization of cytosolic pH,
these fragmented lysosomes return to their normal perinuclear location and re-fuse to generate a population of similar size to that found in untreated cells. Perhaps the most
dramatic example of homotypic lysosome fusion results
from studies in which cells are fused to form a heterokaryon. Lysosomes from each partner (which can be identified through either content markers or species-specific lysosomal membrane proteins [e.g., Lamp-1,2]) within the
heterokaryons show complete intermixing of contents and
membrane (Deng and Storrie, 1988
; Deng et al., 1991
; Ferris et al., 1987
; Perou and Kaplan, 1993b
).
; Lenhard et
al., 1992
), rabs (Novick and Grennwald, 1993; Zerial and
Stenmark, 1993
; Feng et al., 1995
), and trimeric G proteins
(Bomsel and Mostov, 1992
; Colombo et al., 1992b
; Leyte
et al., 1992
; Pimplikar and Simons, 1993
; Vitale et al., 1993
;
Beron et al., 1995
; Nurnberg and Ahnert-Hilger, 1996
),
have been implicated in regulating fusion events in both the secretory and endocytic pathways (for review see
Rothman, 1994
; Novick and Garret, 1994
; Aridor and
Balch, 1996
; Rothman and Wieland, 1996
). No such proteins have been identified to be either associated with lysosomes or involved in defining lysosome size and shape
(Ali et al., 1989
). The only known protein that affects lysosome size is the Chediak/beige protein (Perou et al., 1996
),
which when absent or defective results in abnormally large lysosomes. Recently, the Chediak/beige protein has been
identified (Barbosa et al., 1996
; Nagle et al., 1996
; Perou
et al., 1996
), although the deduced sequence of the protein
yields no clues as to its function.
Materials and Methods
) and maintained as previously described (Kaplan, 1980
). J774E
cells were grown in
MEM (GIBCO BRL, Gaithersburg, MD) containing
10 µg/ml 2-amino-6-mercaptopurine (Sigma Chemical Co., St. Louis, MO)
supplemented with 10% FBS (HyClone Laboratories Inc., Logan, UT).
).
Macroglobulin was isolated and trypsinized as previously
described (Kaplan and Nielson, 1979
). Biotinylated horseradish peroxidase (b-HRP)1, avidin D, and anti-avidin were obtained from Vector Labs,
Inc. (Burlingame, CA). Mastoparan and mastoparan derivatives were obtained from Peninsula Labs (Belmont, CA). Recombinant NSF and dominant-negative NSF (D1E-Q, Glu329 to Gln) were obtained from Dr. S.W.
Whiteheart (University of Kentucky, Lexington, KY). Canine guanine nucleotide-dissociation inhibitor (GDI) and bovine GDI were generous gifts
from Dr. A. Wandinger-Ness (Northwestern University, Evanston, IL).
ELISA plates were obtained from Corning Glass Works (Corning, NY)
and prepared with a 1:200 dilution of anti-avidin as described by Braell
(1992)
. Cytosol was obtained from either J774E, alveolar macrophages, or
rat liver. Filtered cytosol was prepared using spin columns (Bio-Rad Laboratories, Hercules, CA).
8 M 125I-transferrin (125I-Tf[Fe]2) in
HMEM/BSA for 45 min at 37°C. Cells were washed extensively, and then
homogenized in homogenization buffer (HB) (250 mM sucrose, 20 mM
Hepes, 0.5 mM EGTA, pH 7.2, KOH) at 35-50 x 106 cells/ml using a ball-bearing homogenizer to ~50-75% break up. Homogenates were centrifuged at 800 g for 5 min at 4°C to obtain a postnuclear supernatant (PNS).
PNSs were centrifuged for 3 min at 16,000 g to yield crude cytosols and
vesicle pellets. Vesicle pellets were resuspended in a small volume of HB
and fractionated over 23% Percoll (J774 E cells) or 27% Percoll (alveolar
macrophages) as described previously (Ward et al., 1990a
,b). Gradients
were fractionated and HRP activity was assayed on an ELISA plate using 0.8 mg/ml O-phenylenediamine and 0.02% H2O2 in citrate-phosphate buffer, pH 5.0. The reaction was stopped by addition of 6N HCl and read
at 490 nm on a kinetic microplate reader (Molecular Devices, Sunnyvale,
CA).
-Galactosidase activity was assayed using methylumbelliferyl-
galactoside (Sigma Chemical Co.) as a substrate. 125I-Tf activity was measured on an auto-gamma 5780 counter (Packard Instrument Co., Meriden, CT).
) using O-phenylenediamine. Reactions were stopped by placing samples at 0°C and solubilizing in 0.05% Triton X-100, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mg/ml heparin, 20-100 µg/ml biotinylated insulin. Samples were centrifuged at 16,000 g, for 3 min, and the supernatants assayed for avidin- b-HRP complex formation using precoated anti-avidin ELISA plates as
described (Braell, 1992
). In vitro fusion was expressed as a percentage of
the maximum avidin-b-HRP complex formation in samples solubilized in
the absence of biotinylated insulin. All experiments were performed a
minimum of three times.
), pooling of fractions corresponding to HRP and lysosomal enzyme
activity (typically 3-7 of a 20-fraction gradient). The Percoll was then removed by high speed centrifugation (220,000 g for 2 h).
.
After specified times cells were visualized using an inverted fluorescent
microscope (Nikon Inc., Torrance, CA) with a x100 oil immersion objective (Nikon Inc.). Images were acquired using Oncor image analysis software as previously described (Ward et al., 1995
).
-tubulin (ICN Biomedicals, Inc., Costa Mesa, CA) and a Texas red-
conjugated goat anti-mouse secondary (Molecular Probes Inc.). For in vitro studies on microtubules, cytosol was treated with microtubule depolymerizing agents before the in vitro fusion reaction. Protein determinations
were performed as described (Lowry et al., 1951
).
Results
. One population of cells was incubated with b-HRP, whereas the other cells were incubated
with avidin. Cells were exposed to the ligands and subsequently chased in ligand-free media such that the ligands
were localized in the lysosome. PNSs or purified lysosomes
were mixed together in the presence of b-insulin to capture any released avidin, and samples were incubated at
different temperatures and conditions. At the end of the
incubation samples were treated with detergent, avidin-
b-HRP complex captured by immobilized antibody, and
the amount of complex was determined by measuring HRP
activity. Both b-HRP and avidin are mannose-terminal glycoproteins, and are internalized via the mannose-terminal
glycoprotein receptor. Experiments were performed using
rabbit alveolar macrophages or a clone of the murine macrophage cell line, J774E, as these cell types express high
levels of mannose-terminal glycoprotein receptors.
Fig. 1.
Time and temperature dependent in vitro lysosome-lysosome fusion. J774E
cells were incubated at 37°C
for 45-60 min with either 0.5 mg/ml avidin or 0.5 mg/ml
b-HRP. Cells were washed
extensively and chased for an
additional 90-120 min, and
then homogenized. The homogenate was centrifuged at
800 g, for 5 min to obtain a PNS. Typically the vesicle
concentration was 1-2 mg/ml
with a cytosol protein concentration ranging from 2-4
mg/ml. PNSs from cells labeled with either b-HRP or avidin were combined in the presence
of excess b-insulin (20-100 µg/ml), 1 mM MgCl2, 2 mM DTT, 50-75
mM KCl, ± an ATP-regenerating system at 0°C or 37°C for specified times. At the end of the incubation period, vesicles were solubilized in a Triton X-100-containing buffer with excess b-insulin
and assayed as described in Materials and Methods. The percent
in vitro fusion was calculated using the maximum avidin-b-HRP complex formation in the absence of b-insulin as the denominator.
[View Larger Version of this Image (12K GIF file)]
Fig. 2.
Percoll Gradients
of in vitro lysosome-lysosome fusion. In vitro fusion
was performed as described
in Fig. 1 using alveolar macrophages. At the end of the
fusion reaction, the PNSs
were loaded onto 27% Percoll gradients and subcellular
fractionation performed as
described in Materials and
Methods. Alveolar macrophages yield a single peak of
lysosomal activity (Ward et
al., 1990). Fractions were assayed for hexoseaminidase activity as well as avidin-b-HRP complex formation in the presence and absence of excess b-insulin.
The data are expressed as a percentage of the total activity.
[View Larger Version of this Image (22K GIF file)]
; Ward et al.,
1990a
,b). As demonstrated in Fig. 3 A, lysosomes in J774E
cells show a biphasic buoyant density in which ~50% of
the lysosomes, as defined by
-galactosidase or hexoseaminidase activity (data not shown), were well separated from
125I-Tf-containing endosomes. Lysosomes in many cell types
show heterogeneous buoyant densities (Kjeken et al., 1995
;
Cuervo et al., 1997
). Alternatively, the less dense peak of
hexoseaminidase may also represent a late endosomal population. Studies are currently underway to determine the
fusogenic nature of different vesicle populations along the
endocytic pathway. To avoid the possibility of endosomal contamination, the dense fractions of lysosomal activity
(fractions 3-7), were pooled and used for a fusion assay in
the presence or absence of an ATP-regenerating system
(Fig. 3 B). The degree of fusion seen using this enriched lysosome preparation was equal to or greater than that seen
using the corresponding PNS. In many experiments the
degree of fusion was as high as 30% of the input b-HRP.
Fig. 3.
Lysosome isolation and in vitro fusion. J774E cells were
incubated with either b-HRP or avidin as described in Fig. 1. Cells were then incubated with 125I-Tf(Fe)2 at 37°C for 30 min. PNSs
were obtained and centrifuged at 10,000 g, for 30 min. Pellets were
resuspended in homogenization buffer and layered over 23% Percoll density gradients. Samples were centrifuged at 59,000 g for 27 min and fractionated into 20 samples. A is a representative gradient showing endosomes (125I-TF) and lysosomes (HRP as well
as -Gal activity). Fractions 3-7 were pooled and centrifuged at
220,000 g for 90 min to remove Percoll. The lysosomal pellet was
obtained and washed once in HB before resuspension in either
fresh or frozen cytosol. In vitro fusion was performed on PNS as
well as lysosome pellets and the percent fusion determined (B) as
described in Materials and Methods.
[View Larger Versions of these Images (17 + 27K GIF file)]
,b). Previous studies using macrophages determined that brief exposure of cells to endocytic ligands results in the localization of those ligands strictly to the early endocytic apparatus
(Diaz et al., 1988
; Ward et al., 1990b
). Homotypic in vitro
fusion can readily be demonstrated for purified early endosomes as well as purified lysosomes (Fig. 4). However,
no evidence of fusion between early endosomes and lysosomes was observed, indicating again the highly selective
nature of the fusion process. These results support other
studies that have demonstrated the specificity of homotypic vesicle fusion (Diaz et al., 1988
; Gruenberg et al., 1989
;
Colombo et al., 1992).
Fig. 4.
In vitro endosome-endosome and lysosome-lysosome
in alveolar macrophages. Cells were incubated at 37°C with 0.5 mg/ml b-HRP or avidin for either 5 min (endosome) or 45 min, followed by a 90 minute chase period (lysosome). Cells were homogenized, PNS was obtained and layered over separate 27%
Percoll gradients, and then subcellular fractionation was performed as described in Fig. 3. Endosomes (fractions 11-16) and
lysosomes (fractions 3-7) were isolated and Percoll removed before in vitro vesicle fusion as described in Materials and Methods.
[View Larger Version of this Image (27K GIF file)]
;
Perou and Kaplan, 1993b
). Heterokaryons began showing a small amount of coincidence of fluorescent dextrans as
early 15 min with almost complete coincidence evident at
1-3 h (Fig. 5). Nonfused cells on the same cover slip did
not contain the second dye, eliminating the possibility that
cells released and reaccumulated the fluorescent dye. Multiple fields were scanned and in fused cells the majority
(70-90%) of lysosomes contained both dyes. This result
indicates that homotypic lysosome fusion is not a rare
event but in fact is highly frequent.
Fig. 5.
Lysosome-lysosome fusion in vivo. To determine if lysosome-lysosome fusion occurred in vivo, alveolar macrophages
were loaded with either 1 mg/ml FITC-dextran or 0.5 mg/ml
Texas red-dextran at 37°C for 30 min. Cells were washed and incubated for an additional 90 min in HMEM. Cells were then
fused using UV sendai virus protocols as described in Materials
and Methods. Cells were washed, plated onto glass coverslips,
and then placed back in HMEM at 37°C for various times and examined by fluorescence microscopy.
[View Larger Version of this Image (12K GIF file)]
). Addition of recombinant NSF in the absence of cytosol reconstituted fusion activity. Further, addition of purified NSF completely reconstituted fusion activity in the
presence of NEM-treated cytosol, and did not enhance fusion activity when added to untreated cytosol. Under similar conditions a mutant NSF, unable to hydrolyze ATP
(D1E-Q, Glu329 to Gln), did not promote fusion. Further,
this mutant NSF acted as a dominant negative and inhibited fusion when added back to a complete reaction with
cytosol. These results demonstrate that NSF and its ability
to hydrolyze ATP are required for homotypic lysosome
fusion. The data also suggest that the only soluble protein required is NSF; all other proteins are lysosome bound.
Previous studies on in vivo, as well as in vitro vesicle fusion have demonstrated the role of GTP-binding proteins
(for review see Gruenberg and Maxfield, 1995; Robinson
et al., 1996
). No GTP-binding proteins have been identified that are associated with lysosomes (Ali et al., 1989
).
Studies in yeast have demonstrated the role of yeast Ypt7
in vacuolar inheritance and vacuole fusion in vitro (Conradt et al., 1992
; Haas et al., 1994
, 1995
). The mammalian analogue of Ypt7 is rab7, which has been shown to be associated with late endosomes (Ali et al., 1989
; Aniento et
al., 1993
; Feng et al., 1995
). To determine if a GTP-binding
protein may be involved in lysosome-lysosome fusion, we
added the nonhydrolyzable analogues GTP
s or GDP
s
to the in vitro fusion reactions. Fig. 6 demonstrates that
GTP
s and GDP
s inhibited fusion in a concentration-
dependent manner.
The concentration of GTP analogues that inhibited fusion ranged from 50-200 µM, a range slightly higher than
that required for inhibition of other vesicle fusions (Bomsel and Mostov, 1992; Colombo et al., 1992b; Beron et al.,
1995
). This concentration was similar, however, to that required to inhibit homotypic yeast vacuolar fusion (Conradt et al., 1992
; Haas et al., 1994
, 1995
). The fact that GTP
s
inhibits lysosome-lysosome fusion in vitro suggests that a
GTP-binding protein(s) is involved. GDI prevents the dissociation of GDP from a variety of rab proteins (Sasaki et
al., 1990
) and has been shown to inhibit several in vitro
vesicle fusion reactions (Dirac-Svejstrup et al., 1994
; Elazar
et al., 1994
; Peter et al., 1994
; Acharya et al., 1995
; Haas et
al., 1995
; Ikonen et al., 1995; Turner et al., 1997
). Addition
of GDI inhibits lysosome-lysosome fusion in vitro, whereas
heat-inactivation of GDI before addition to the fusion reaction completely blocked the inhibitory effect (Table II).
These data suggest that a rab protein is required for homotypic lysosome fusion.
Table II. GDI Addition to In Vitro Lysosome-Lysosome Fusion |
Previous studies have also demonstrated the involvement of heterotrimeric G proteins in vesicle fusion (Colombo et al., 1992b; Pimplikar and Simon, 1993; Haas et
al., 1994
). Mastoparan, a heterotrimeric, G protein activator (Fig. 7) inhibited in vitro lysosome fusion, whereas addition of the inactive analogue Mas-17 had no inhibitory effect on in vitro lysosome fusion. To define the heterotrimeric G protein involved in homotypic lysosome fusion,
Cholera toxin (Chtx), a G
s activator and Pertussis toxin
(Ptx), a G
i activator were added to the fusion reaction.
Cholera toxin has been shown to specifically activate G
s
and studies on endosome-endosome fusion have demonstrated the role of a heterotrimeric G
s in early endosome
fusion (Colombo et al., 1992b
). Ptx, on the other hand, has
been shown to specifically activate G
i and studies have
shown a G
i to be involved in regulation of apical transport in epithelial cells (Pimplikar and Simon, 1993). As
demonstrated in Table III Ptx inhibited in vitro lysosome
fusion while Chtx did not inhibit fusion. The inhibitory activity was dependent on the presence of NAD, which activates the toxin.
Table III.
Effect of G |
Role of Microtubules in Lysosome Fusion
Studies on in vitro endosome-endosome fusion and late
endosome-lysosome fusion have demonstrated the need
for polymerized microtubules (Gruenberg et al., 1989; Aniento et al., 1993
; Mullock et al., 1989
, 1994
). It is clear that
microtubules can affect lysosome distribution and shape in
macrophages (Heuser, 1989
; Hollenbeck and Swanson, 1990
;
Knapp and Swanson, 1990
; Lin and Collins, 1992
; Swanson
et al., 1992
; Perou and Kaplan, 1993a
; Oh and Swanson,
1996
). Their role, however, in affecting lysosomal fusion
events is less clear. Incubation of alveolar macrophages with either nocodozole or colchicine resulted in the disruption of microtubules (data not shown) but had minimal
effects on movement of internalized ligands through the
endocytic system to the lysosome (Fig. 8 A). Similarly, microtubule disruption (data not shown), or addition of
taxol-stabilized microtubules did not alter the rate or extent of in vitro lysosome fusion (Fig. 8 B). The presented experiment was performed using a microtubule concentration of 0.033 mg/ml, similar to previous studies (Gruenberg
et al., 1989
). A fivefold increase or decrease in microtubule concentration had no effect on the extent or kinetics
of homotypic lysosome fusion (data not shown). Based on
these results we conclude that neither fusion of endocytic
vesicles with lysosomes nor homotypic lysosome fusion in
alveolar macrophages is dependent upon microtubules.
A variety of studies have defined molecules responsible for
both internalization of plasma membrane vesicles, homotypic endosome fusion, and fusion of Golgi vesicles to late
endosomes (Lombardi et al., 1993; Pfeffer, 1994
; Gruenberg and Maxfield, 1995
; Robinson et al., 1996
). Similar
studies have been carried out in the secretory pathway
(Rothman and Wieland, 1996
). These studies have used in vitro assays as well as molecular biology techniques to define the machinery for vesicle recognition, fusion, and fission. Such experiments have been buttressed by genetic
studies in yeast. In many respects, the machinery for vesicle transport in yeast is homologous with those in mammalian cells, facilitating the identification of putative genes
and gene products (Riezman, 1993
; Stack and Emr, 1993
).
The biochemical requirements for yeast vacuolar fusion in
vitro, have recently been defined. Vacuolar fusion is involved in vacuolar inheritance (Conradt et al., 1992
, 1994
; Haas et al., 1994
, 1995
; Mayer et al., 1996
). Less is known,
however, about the mechanisms that regulate lysosome fusion and fission in mammalian cells.
Lysosomes are known to obtain membrane and vacuolar contents from endocytosis and de novo-synthesized
enzymes from the Golgi apparatus through the late endosome via vesicle fusion events. Membrane fusion was suggested by DeDuve (1963) to explain the relationship between lysosomes, phagosomes, digestive vacuoles, and
residual bodies. In general, however, it has not been well appreciated that lysosomes exhibit extensive rounds of self-
or homotypic fusion. Studies by Oates and Touster (1980)
demonstrated fusion of lysosomes (termed phagolysosomes)
in vitro in homogenates obtained from Acanthamoeba castellanii. Storrie and colleagues first observed lysosome fusion in mammalian cells using an assay based on somatic
cell fusion (Ferris et al., 1987
; Deng and Storrie, 1988
). Their
studies demonstrated that both the contents and membranes of lysosomes underwent mixing. Perou and Kaplan
(1993b)
took advantage of the cell fusion approach to devise an assay for the Chediak/beige gene product. Cultured
Chediak cells when fused with wild-type cells undergo lysosomal mixing and the fused lysosomes then showed the
phenotype of the normal parent. This observation formed
the basis of a complementation assay that resulted in the
identification of the Chediak/beige gene (Nagle et al., 1996
;
Perou et al., 1996
). These studies also demonstrate that lysosomal mixing occurs and further, that mechanisms exist
which regulate the size of lysosomes.
The studies presented here extend these in vivo observations. The in vivo experiments, as demonstrated in Fig. 5, showed that lysosomal fusion is both extensive and rapid. Intermixing of the contents of lysosomes began as early as 15 min of heterokaryon formation where maximum fusion was observed in 90-120 min. It is possible that the observed mixing in vivo may be explained either by unidirectional movement between lysosomes, or by bidirectional movement between late endosomes and lysosomes where late endosomes may also be fusing. The in vitro assay, however, recapitulates the rapidity and extent of lysosomal fusion seen in vivo. Using PNSs as a source of lysosomes, we observed a maximum extent of fusion ranging from 7 to 20% of input b-HRP. When purified lysosomes were used the degree of fusion reached as high as 30% of input b-HRP. Since our system does not detect self-fusion of HRP vesicles or self-fusion of avidin vesicles, we feel that we might be underestimating the actual degree of fusion. There are two explanations why the degree of fusion is higher in the purified lysosome preparation. (a) Lysosomes may aggregate through the isolation procedure and thus be in closer physical proximity; and (b) the purification of lysosomes results in lowered levels of other membranous systems (Golgi, ER, early and late endosomes) that compete for soluble fusion proteins and/or nucleotides. These explanations are not mutually exclusive.
Lysosome fusion relies on cytosolic factors and nucleotides as do most but not all fusion systems. The source of
cytosol was irrelevant. Liver, alveolar macrophage, and
cultured cell J774E cytosol supported fusion to the same
extent. NSF is required for most fusion systems (for review
see Rothman and Wieland, 1996) and may well be rate
limiting in an in vitro system due to competition by different vesicle systems. Addition of wild-type NSF to the in
vitro lysosome-lysosome fusion reaction, in the absence of cytosol, resulted in reconstitution of fusion activity. Addition of an NSF analogue, which is unable to hydrolyze
ATP, did not result in lysosome-lysosome fusion. This
NSF analogue also acts as a dominant-negative component
that inhibited in vitro fusion in the presence of cytosol.
The fact that recombinant NSF could replace cytosol in
the fusion suggests that all other required macromolecular
factors are membrane bound.
Whereas the source of cytosol was irrelevant, what appeared to be relevant was the source of lysosomes and the
physiological state of the cells before fractionation. The
cultured line J774E gave variable results, with different degrees of fusion depending on the culture conditions. PNSs
obtained from cells cultured in 5% serum were consistently less fusogenic then were lysosomes obtained from
cells in 10% serum (data not shown). (This applied to in
vitro endosome fusion as well.) Lysosomes from alveolar macrophages gave higher degrees of fusion than lysosomes
from cultured mouse bone marrow macrophages (data not
shown). The reasons for the differences in fusion are unknown. Cell-type differences in lysosomal fusion can, however, be seen in vivo. In alveolar macrophage heterokaryons, lysosome fusion occurs within 1-3 h, while a similar
degree of fusion takes 4-6 h in cultured human or murine
fibroblasts (Perou and Kaplan, 1993b). The kinetics of lysosomal fusion using PNS from J774 cells appeared to show a slight lag (compare with Fig. 1), whereas in alveolar macrophages no lag was observed using either PNS or purified
lysosomes (compare with Fig. 8). These data further demonstrate that there is cell-type difference in fusion in vivo
as well as in vitro.
Multiple studies have identified critical roles for GTP-binding proteins in vesicle trafficking pathways in the secretory and endocytic pathways (Novick and Brennwald,
1993; Pfeffer, 1994
; Gruenberg and Maxfield, 1995
; Rothman and Wieland, 1996
). Studies have localized rabs to a
number of different endocytic vesicles. For example, rab4
and rab5 are found on early endosomes, rab7 on late endosomes, and rab9 on trans-Golgi elements (Zerial and Stenmark, 1993
). Recently Turner et al. (1997)
demonstrated
that a rab protein is involved in homotypic ER fusion. To
date no rabs, ARFs, or trimeric G proteins have been identified being associated with lysosomes. Our data suggests
that such molecules must play a role in homotypic lysosome fusion. Addition of the nonhydrolyzable GTP
s analogue, GDI, or mastoparan, an activator of heterotrimeric G proteins (Higashijima et al., 1988
, 1990
) inhibits homotypic lysosome fusion. We also observed that treatment of
lysosomes with Ptx inhibited fusion. Other studies have
demonstrated the involvement of heterotrimeric G proteins in vesicle fusion (Colombo et al., 1992b
; Ktistakis et al.,
1992
; Pimplikar and Simons, 1993
; Haas et al., 1994
). Since
Ptx requires ARF activity to catalyze incorporation of the
target trimeric G protein, this result suggests that an ARF
activity must also be membrane bound.
What is the need for extensive homotypic lysosome fusion? One reasonable explanation is that fusion events redistribute luminal contents among the population of lysosomes. Fusion of lysosomes with a late endocytic vesicle
results in the contents of that vesicle being subject to the
action of lysosomal hydrolases. In many respects, particularly as a result of phagocytosis or macropinocytosis, a single lysosome may fuse with a phagocytic vacuole or a large endocytic vesicle (Racoosin and Swanson, 1993; Oh and
Swanson, 1996
). The lysosome then obtains material as a
bolus. Multiple rounds of lysosomal fusion and fission may
guarantee that the amount of substrate in a given lysosome does not overwhelm the amount of lysosomal enzymes available for degradation. Thus, lysosomal fusion
may ensure that substrates are always exposed to conditions in which there is an excess of degradative enzymes.
The data in Fig. 2, on alveolar macrophages, gives an indication that in vitro fusion may result in a slight change in buoyant density of lysosomes. The avidin-b-HRP peak and hexoseaminidase peaks do not completely coincide. One explanation of this result is that the fusion products may be larger and consequently less dense.
Whereas our current in vitro assay does not demonstrate it, membrane economics suggest that there must be
a corresponding fission event. This is demonstrated by the
heterokaryon studies in which extensive lysosome fusion
occurs, yet the resulting size distribution reflects the original population (Deng and Storrie, 1988; Deng et al., 1991
).
Fusion between Chediak and normal cells results in the normal cell phenotype predominating (Perou and Kaplan,
1993b
). Even when fusion occurs between Chediak cells, the resulting lysosome size is not a multiple of the original lysosome but reflects the size distribution of the parental
cells. The inescapable conclusion is that once fused, lysosomal fission must occur. Lysosome fusion and fission may
not only redistribute the contents of vesicles within cells
but generate new lysosomes. Studies on vacuolar inheritance in yeast have shown that fission is required for specific vesicles to be inherited by daughter cells (Warren and
Wickner, 1996
). Studies are currently underway to determine if lysosome fission is also occurring in vitro and what
components are required for lysosome fission.
Lysosomes, as terminal organelles in the endocytic pathway, are continually obtaining solute and membrane from
both the endocytic apparatus and the Golgi apparatus.
Clearly, increased membrane influx must be balanced by a
corresponding loss of membrane (Duncan and Pratten,
1977). There are several possible fates for maintaining membrane homeostasis: recycling to the cell surface, increased degradation (i.e., as seen in multivesicular bodies),
and membrane fission. Our data suggests that membrane
fission may be a way of dealing with this excess membrane,
particularly in conjunction with fusion events. A dynamic
fusion/fission system would allow for the generation of
new vesicles of a defined size class, providing a uniform
size population. Further studies are underway to identify the biochemical requirements for lysosome fusion as well
as fission.
Received for publication 10 February 1997 and in revised form 20 July 1997.
Address all correspondence to J. Kaplan, Department of Pathology, Division of Cell Biology and Immunology, University of Utah Health Science Center, Salt Lake City, UT 84132. Tel.: (801) 581-7427. Fax: (801) 581-4517. E-mail: kaplan{at}bioscience.bilogy.utah.edu
ARF, ADP ribosylation factor;
b-HRP, biotinylated horseradish peroxidase;
b-insulin, biotinylated insulin;
Chtx, Cholera toxin;
GDI, guanine nucleotide-dissociation inhibitor;
HB, homogenization buffer;
HMEM, Hanks' minimal essential medium;
125I-Tf(Fe)2, 125I-transferrin;
M125I-T,
macroglobulin-125I-trypsin;
PNS, postnuclear
supernatant;
Ptx, Pertussis toxin.
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