1 National Center for Biological Sciences, Tata Institute of Fundamental
Research, GKVK, Bangalore 560 065, India
2 Department of Biological Sciences, Tata Institute of Fundamental Research,
Homi Bhaba Road, Bombay 400 005, India
Authors for correspondence (e-mail:
ksk{at}tifr.res.in;
mayor{at}ncbs.res.in)
Accepted 28 April 2003
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Summary |
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Key words: Fluid phase, GPI-anchored proteins, Scavenger receptor, Endocytosis, Cell culture, shibire
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Introduction |
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Not surprisingly, essential mediators of synaptic endocytosis like
-adaptin (Gonzalez-Gaitan and
Jackle, 1997
) and the shibire (shi) gene product
Drosophila Dynamin (dDyn), which is the only reported
Drosophila homolog of vertebrate Dyn
(Kosaka and Ikeda, 1983a
;
van der Bliek and Meyerowitz,
1991
), are not restricted to the nervous system
(Chen et al., 1992
;
Dornan et al., 1997
). dDyn is
known to regulate endocytosis in a variety of fly tissues
(Kosaka and Ikeda, 1983a
;
Kosaka and Ikeda, 1983b
;
Tsuruhara et al., 1990
).
Zygotic null mutations at the shi locus are lethal and the mutants
show neural hyperplasia (Poodry,
1990
). Electronmicroscopy (EM) studies that addressed the
phenotypes of temperature-sensitive alleles at the shibire locus
(shits) in larval garland cells showed an accumulation of
`coated pits' at the plasma membrane and reduced uptake of fluid-phase tracers
like horse radish peroxidase (HRP) (Kosaka
and Ikeda, 1983b
). These studies suggested that dDyn is required
for all pathways of endocytosis in the cell types examined.
By contrast, over-expression of an analogous temperature-sensitive mutant
of Dyn (Dynts) in HeLa cells led to an initial reduction of
fluid-phase uptake at 39°C that subsequently recovered
(Damke et al., 1995); the
internalization of human transferrin was completely inhibited at this
temperature. These results argued for pathways of fluid-phase uptake in
mammalian cells that are induced or upregulated when Dyn function is
perturbed. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) (reviewed
by Chatterjee and Mayor, 2001
)
and the D2 dopamine receptor (Vickery and
von Zastrow, 1999
) continue to be internalized into mammalian
cells upon expression of dominant-negative Dyn isoforms. GPI-APs are
endocytosed in a Dyn-independent manner into distinct endocytic compartments
that contain a majority of the internalized fluid phase
(Sabharanjak et al., 2002
).
These studies suggest that mammalian cells exhibit constitutive
Dyn-independent pathways of endocytosis for both membrane and fluid-phase
markers. However, mammals have three distinct genes encoding Dyn and as many
as 25 splice variants (Cao et al.,
1998
), leaving open the possibility that alternate forms of Dyn
might be involved in these endocytic events
(McNiven et al., 2000
). By
contrast, in Drosophila dDyn, a multi-domain protein (see later) is
encoded by a single locus that has six splice variants
(Staples and Ramaswami, 1999
;
van der Bliek and Meyerowitz,
1991
). The availability of shits alleles that
map to domains conserved across all the splice variants makes
Drosophila an attractive system to dissect the involvement of dDyn in
different endocytic processes.
Drosophila macrophages and hemocytes are a part of the innate
immune system of the animal (Lanot et al.,
2001; Tepass et al.,
1994
) and are capable of internalizing a variety of ligands by a
scavenger receptor-mediated (dSR) pathway
(Abrams et al., 1992
). These
cells are also phagocytic and have been shown to engulf both apoptotic cells
and microbes (Franc et al.,
1999
); cells of this lineage in other metazoa are known to have
multiple pathways of endocytosis (Gold et
al., 1999
; Racoosin and
Swanson, 1992
).
Here, we establish a methodology to reproducibly obtain primary cultures of macrophages and hemocytes from wild-type and mutant Drosophila embryos and larvae, respectively. The larval hemocytes have an endogenous anionic-ligand binding receptor (ALBR) with a similar ligand-binding specificity as dSR. In this system, we probed the existence of multiple endocytic pathways. Specifically, we have asked whether cells from embryonic and larval stages of wild-type and temperature-sensitive shibire animals are capable of dDyn-dependent and -independent endocytosis. We find that, first, the endocytic phenotype of ALBR-mediated endocytosis in cells from temperature-sensitive shibire mutants parallels the behavior of mutant flies; ALBR-mediated internalization is reversibly blocked by raising the temperature. Second, at the restrictive temperature, cell-surface, ALBR-bound ligands remain completely accessible to relatively large molecules (70 kDa proteins); upon shifting to low temperatures, the arrested structures are internalized in a surge, restoring `endocytic competence'. This is suggestive of a block in a late step of endocytosis in shits mutants. By contrast, fluid-phase and GPI-AP endocytosis, which occurs via distinct endosomal structures, remains unaffected at the restrictive temperature in the shits mutants, providing evidence for a constitutive dDyn-independent pathway.
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Materials and Methods |
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Fly stocks
UAS-GFP-Rab5 (Entchev et al.,
2000), UAS-GFP-GPI (Greco et
al., 2001
), UAS-GFP-Clc (Chang
et al., 2002
) and Collagen-Gal4
(Asha et al., 2003
) were
obtained from M. Gonzalez-Gaitan, S. Eaton, I. Mellman and C. Dearolf,
respectively. Other fly strains were obtained as previously described
(Krishnan et al., 1996
).
Cell culture
Stage 11 and 12 embryos were collected from flies freshly transferred to
sucrose-agar bottles. Cell cultures were derived by homogenizing 40-80 embryos
in a Potter-Elvehjem Tissue Grinder (2 ml; Wheaton, Millville, NJ) using a
loose-fitting pestle in complete medium [Schneider's Incomplete Medium
(Gibco-BRL, Gaithersberg, MD) supplemented with 10% non-heat-inactivated FBS
(Gibco-BRL), 1 µg/ml bovine pancreatic insulin, penicillin, streptomycin
and L-glutamine]. The resulting cell suspension was transferred to 35 mm
cover-slip bottom dishes (Sabharanjak et
al., 2002), and maintained in an incubator at 21°C.
Macrophages were identified by their characteristic morphology (Eschalier,
1997) in culture after a period of 2-4 days. Complete medium was `aged' for a
period of 24-36 hours at 4°C prior to use.
Hemocytes from the third larval instar were obtained as described
previously (Lanot et al.,
2001) with modifications
(Sriram et al., 2003
). Male
flies carrying UAS-GFP-protein transgenes were crossed with virgin
shits;CollagenGal4 flies and the progeny male larvae
expressing GFP-tagged proteins were used to obtain hemocytes expressing the
appropriate transgene in the mutant background. The cells were used for
experiments 2 hours after dissection, unless otherwise mentioned.
Probes for endocytosis and immunodetection
mBSA was prepared as described earlier
(Haberland and Fogelman,
1985). Fluorescent conjugates were made according to the
instructions provided by the manufacturer with minor modifications.
Biotinylated-Cy3-mBSA (B-Cy3mBSA) was made by conjugation of BSA with
BiotinXXSSE at a molar ratio of 1:5 according to the instructions provided by
the manufacturer, prior to conjugation to Cy3 and subsequent maleylation. FITC
or Lissamine Rhodamine conjugated to Dextran (10 kDa; F-Dex, LR-Dex,
respectively) was used as a fluid-phase tracer.
Immunofluorescence detection of antibodies was carried out as described
(Sriram et al., 2003). To
enhance contrast in detecting membrane-bound forms of ectopically expressed
GFP-Rab5 or GFP-Clc, cells were fixed for shorter time periods and
permeabilized to remove cytosolic fluorescence.
Uptake assays
For uptake experiments, cells were incubated with endocytic probes in
Schneider's Incomplete Medium supplemented with BSA (1.5 mg/ml) at room
temperature (21-24°C), unless otherwise indicated, and extensively washed
in the same medium. ALBR probes Cy3-mBSA, Cy5-mBSA and Cy3-BiotinXX-BSA were
used at 200-800 ng/ml (0.8-3.3 nM). At these concentrations, the binding and
internalization of labeled mBSA was completely competed by excess unlabeled
mBSA, fucoidan or lipopolysaccharide (LPS) (0.8 mg/ml). F-Dex and LR-Dex,
which are markers for bulk fluid uptake, were used at 1-2 mg/ml, unless
otherwise specified.
For uptake assays at 31°C, the dishes were secured onto an aluminium block immersed in a water bath. The dishes were then covered and probes were added through a hole in the dish lid. The water bath and dishes inside were kept covered except when the probe was added or the cells were washed. In all uptake experiments at 31°C, cells were first incubated at this temperature for a period of 5 minutes prior to being labeled.
To visualize the specific endocytic uptake of GFP-GPI, cells were pre-incubated with Fl-anti-GFP (2-4 µg/ml, 0°C) for 20 minutes followed by incubation of cells at room temperature for 45 seconds or 5 minutes. PI-PLC treatment was carried out (0.3 mg/ml; 30 seconds, room temperature) to quantitatively remove cell-surface GFP-GPI and bound Fl-anti-GFP prior to fixation and imaging.
Cells were fixed with 2.5% paraformaldehyde in medium 1 (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 6.9). Prior to imaging (even in fixed cells), endosomal pH was neutralized by the addition of 10 µM Nigericin in high-potassium buffer (120 mM KCl, 5 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, pH 7.4) or with freshly prepared ammonium chloride (20 mM in medium 1), and finally imaged in medium 1.
Assays for surface accessibility of mBSA
Qualitative assay: after endocytosis of B-Cy3mBSA by ALBRs, to detect
surface-accessible receptors, cells were washed and cooled rapidly on ice and
further incubated with Cy5-SA for an additional 15 minutes to probe for
surface accessibility of B-Cy3-mBSA, and then fixed and imaged on a wide-field
microscope. Cy5-SA-labeled cells were imaged and the Cy5-fluorescence was
completely photobleached prior to imaging Cy3 to eliminate the possibility of
quenching B-Cy3mBSA fluorescence by bound Cy5-SA.
Quantitative assay: to quantify the total accessible pool of receptors, cells were labeled with B-Cy3mBSA on ice for 15 minutes, washed and then labeled with Cy5-SA for 15 minutes, and taken for imaging. These assays used medium 1 as incubation buffer since Schnieder's medium contains free biotin that competed for the binding of Cy5-SA to B-Cy3mBSA. To measure the amount accessible at any other temperature, cells were labeled with B-Cy3mBSA for a period of 5 minutes at the given temperature, washed rapidly and labeled with Cy5-SA for an additional 15 minutes at the same temperature. In experiments designed to assay the reversibility of the temperature-sensitive endocytic defect, after incubation with B-Cy3mBSA at high temperatures, cells were rapidly transferred to ice prior to the addition of Cy5-SA at the same temperature. The ratio of Cy5 to Cy3 fluorescence represents the fraction of the B-Cy3mBSA probe accessible to Cy5-SA. The ratios of Cy5 to Cy3 fluorescence at a given temperature were normalized to the ratio obtained for total accessible pool. To estimate the extent of nonspecific binding of Cy5-SA, in each experiment cells were labeled with B-Cy3mBSA for 15 minutes on ice and subsequently incubated with biocytin-treated (2 µM) Cy5-SA. The nonspecific value never exceeded 10% of the fraction of the total accessible pool in a given experiment.
Fluorescence imaging, quantification and processing
Quantitative digital imaging and confocal microscopy was carried out as
described previously (Sabharanjak et al.,
2002). Fluorescence images were processed using MetaMorph
software. Images were pseudo-coloured using Adobe Photoshop and composites
were assembled using the same software.
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Results |
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In this study, we have mainly used larval hemocytes since they are better suited for the study of endocytic processes because large numbers of ALBR-expressing cells can be obtained more reproducibly. They also exhibit considerably less auto-fluorescence than their culture-derived embryonic counterparts. Nevertheless, qualitatively similar results have been obtained in both types of cell cultures from different genetic backgrounds (Supplementary figure S2).
Pathways of receptor-mediated and fluid-phase endocytosis in
hemocytes
To study the pathways of receptor-mediated and fluid-phase endocytosis in
larval hemocytes from wild-type (CS) animals, Cy3mBSA was used to label the
ALBR-mediated pathway whereas FITC or Lissamine Rhodamine conjugated to
Dextran (10 kDa; F-Dex, LR-Dex, respectively) were used as fluid-phase
tracers. When cells are pulsed with Cy3mBSA and F-Dex for 5 minutes and chased
for 2 minutes both probes extensively colocalize in endosomes
(Fig. 2A-C). These endosomes
are positive for the endosomal marker Rab7
(Fig. 2D-F) and are
multivesicular (Sriram et al.,
2003), indicating that F-Dex and Cy3-mBSA are rapidly delivered to
late endosomes in these hemocytes. These rates are similar to those observed
in mammalian macrophages in culture wherein multivesicular endosomes
containing fluid-phase cargo are visualized as early as 5-8 minutes after a
pulse of BSA-gold in mammalian macrophages
(Rabinowitz et al., 1992
).
Consistent with the passage through an early endosomal compartment, endosomal
intermediates formed in a short pulse of 45 seconds of Cy3mBSA and F-Dex do
not colocalize with Rab7 (Fig.
2G,H).
|
To confirm that the punctate structures formed by Cy3mBSA in short internalization times are endosomal, we incubated cells with biotinylated Cy3mBSA (B-Cy3mBSA) for 45 seconds and assayed for the surface accessibility of the biotin-tag by incubation with Cy5-labeled Streptavidin (Cy5-SA) on ice. As a control, we ascertained that, if cells were labeled at 0°C, almost all punctate Cy3mBSA structures are colocalized with Cy5-SA (data not shown). After 45 seconds, a fraction of peripherally distributed B-Cy3mBSA remains accessible to Cy5-SA, indicating cell-surface localization (compare Fig. 3A and B, small arrowheads). At the same time, endocytosed B-Cy3mBSA (defined as Cy5-SA inaccessible structures) is distributed in numerous small endosomal structures (Fig. 3A, small arrows) distinct from endosomes formed by F-Dex present in the incubation medium (Fig. 3C, arrowheads; merge in Fig. 3D). After the 45 seconds pulse, when cells were chased for 5 minutes in the absence of fluorescent probes, both Cy3mBSA and F-Dex colocalize in late endosomes (Supplementary figure S1A-C). Following a 2 minutes pulse, the majority of Cy3mBSA and F-Dex were found to colocalize in late endosomal structures, but compartments containing only F-Dex or Cy3-mBSA are also seen (Supplementary figure S1D-F).
|
The difference in distribution of B-Cy3mBSA- and F-Dex-containing endosomes after a 45 seconds pulse suggested two possibilities. First, that F-Dex and B-Cy3mBSA are internalized via independent pathways. Second, that F-Dex and Cy3mBSA are internalized via a common intermediate wherein the observed segregation reflected differences in their relative rates of internalization. To discriminate between these alternatives, we incubated cells with B-Cy3mBSA alone for 3 minutes to label all early endosomal structures and then pulsed in F-Dex and B-Cy3mBSA together for an additional 45 seconds. Under these conditions, we find that the majority of F-Dex-containing structures remain devoid of B-Cy3mBSA (Fig. 3E-H). After a 5 minutes chase, F-Dex and B-Cy3mBSA are once again extensively colocalized (data not shown).
These experiments suggest that the difference in distribution of Cy3mBSA and F-Dex after brief pulses could be due to the involvement of different endocytic pathways, and that the ALBR-mediated pathway has a poor capacity for fluid-phase uptake. Consistent with this, if cells were pulsed with tenfold higher concentrations of F-Dex (5-8 mg/ml), the fluid-phase marker was detected in the distinct endosomes as well as in Cy3mBSA-labeled endosomes under the same conditions of imaging (data not shown). These results confirm that Cy3mBSA (or B-Cy3mBSA)-labeled early endosomes do not account for a major fraction of the fluid-phase uptake in hemocytes.
The Cy3mBSA structures accessed in 45 seconds are positive for Rab5
(Fig. 4A-C, small arrows)
consistent with their designation as early endosomes
(Zerial and McBride, 2001). At
the same time, early (
45 seconds) endosomes of the fluid-phase pathway do
not stain for Rab5 (Fig. 4D-F,
arrowheads), suggesting that these endosomal compartments are part of a
Rab5-independent pathway.
|
In Chinese Hamster Ovary (CHO) cells, fluid-phase uptake takes place
predominantly via a pathway that selectively incorporates GPI-APs. This
pathway is also Rab5 independent, and independent of clathrin, dynamin and
caveolin (Sabharanjak et al.,
2002). To examine if fluid-phase uptake occurs via a similar
pathway in Drosophila hemocytes, we have followed the endocytosis of
exogenously added fluorescently labeled antibodies (Fl-anti-GFP) against
GFP-GPI transgenically expressed in hemocytes using the UAS-Gal4 system
(Brand and Perrimon, 1993
).
Visualization of endocytosed GFP-GPI was facilitated by the quantitative
removal (data not shown) of cell-surface GFP-GPI (and Fl-anti-GFP) by
treatment with PI-PLC (Materials and Methods). Observation at 45 seconds
post-internalization shows that the peripheral small early endosomes
containing Cy5mBSA as well as the large fluid-filled early endosomes are both
labeled with PI-PLC-resistant Fl-anti-GFP specifically internalized by GFP-GPI
(Fig. 5A,B); cells that do not
express GFP-GPI do not show uptake of detectable amounts of Fl-anti-GFP (data
not shown). These results suggest that, similar to CHO cells, the fluid-phase
uptake is also mediated by a Rab5-negative, GPI-AP-enriched endosomal pathway.
At later times, Fl-anti-GFP (endocytosed via GFP-GPI) is delivered to late
endosomes (Fig. 5C,D). The
delivery of GPI-APs to late endosomes in Drosophila hemocytes is in
contrast to the eventual delivery of GPI-APs to endosomal recycling
compartments in CHO cells. However, it is similar to the route followed by
GPI-APs in Baby Hamster Kidney cells, consistent with the differential sorting
and fate of endocytosed GPI-APs observed in multiple cell types
(Fivaz et al., 2002
;
Sabharanjak et al., 2002
;
Sharma et al., 2002
).
|
Together, these results suggest that hemocytes from Drosophila internalize ALBR-bound ligands and fluid-phase markers into two distinct classes of early endosomes, Rab5-positive and negative, respectively. These later fuse to form Rab7-positive late endosomes.
Role of dDyn in endocytosis
We next asked if dDyn plays any role in endocytosis of the two types of
probes. For this purpose we obtained cells from shits
alleles, shits1 and shits2. Both these
alleles contain missense mutations (van
der Bliek and Meyerowitz, 1991) conserved across all splice
variants (Staples and Ramaswami,
1999
). The shits2 mutation (G141S) lies in the
GTP binding and hydrolysis domain (G domain) and the
shits1 mutation (G267D) lies at the interface of the G
domain and the `middle' domain (Fig.
6A). shits1 and shits2
flies paralyze within 3 minutes at 27°C and 27.5°C (the restrictive
temperature), respectively, and recover just as rapidly when returned to
21°C (the permissive temperature)
(Krishnan et al., 1996
).
|
Hemocytes from CS, shits1 and shits2 animals were pulsed with Cy3mBSA and F-Dex for 5 minutes at 21°C. At this temperature, the uptake of both probes in shits1 and shits2 cells is indistinguishable from that of CS cells; both Cy3mBSA and F-Dex colocalize in large centrally located endosomes (Fig. 6B-D). In CS cells, following a 5 minutes pulse at 31°C, Cy3mBSA and F-Dex are seen in endosomes indistinguishable from those formed at 21°C (compare Fig. 6B with 6E). However, the distribution of Cy3mBSA is dramatically altered in both mutant shi alleles; Cy3mBSA has a punctate peripheral distribution (Fig. 6F,G; small arrowheads). The distribution of Cy3mBSA in shits1 and shits2 cells at 31°C is comparable to cells labeled on ice (Fig. 6H-J), a condition in which all endocytic activity is inhibited, suggesting an arrest in the internalization of ALBR-ligands.
In contrast to Cy3mBSA, fluid-phase endocytosis appears unaffected; F-Dex is internalized into similar endosomes in cells from the mutant shi animals (Fig. 6F,G; green) when compared with the endocytic structures observed in cells from CS animals (Fig. 6E, green) at the non-permissive temperature (see below for a detailed analysis of fluid-phase and GPI-AP endocytosis in shits mutants). These observations confirm that the inhibition of ALBR internalization at the restrictive temperature is not result of any nonspecific cellular toxicity.
Nature of endocytic defect in shits mutants at
restrictive temperature
The process of formation of the endocytic vesicle minimally comprises three
distinct stages: invagination, closure and fission
(Sever et al., 2000a). At the
synapse, shits mutants exhibit a block at a late stage in
this process (Ramaswami et al.,
1994
). To understand the nature of the endocytic defect in
hemocytes, and to quantify the extent of Cy3mBSA internalization at 31°C,
we devised a quantitative surface-accessibility assay
(Fig. 7A). In this assay, we
determined the accessibility of Biotinylated-Cy3mBSA (B-Cy3mBSA) to
exogenously added Cy5-labeled Streptavidin (Cy5-SA). Thus, the ratio of Cy5-SA
to B-Cy3mBSA fluorescence is a read out of the extent of the accessibility of
B-Cy3mBSA to Cy5-SA added at a given temperature (Materials and Methods).
Maximum accessibility was determined from cells incubated with B-Cy3mBSA on
ice followed by Cy5-SA incubation at the same temperature
(Fig. 7B, shits1 ice). In shits1 cells at
31°C, most of the B-Cy3mBSA was surface accessible; Cy5 to Cy3
fluorescence ratio in a population of shits1 cells labeled
on ice is indistinguishable from that observed when the cells are labeled at
31°C (Fig. 7B,
shits1 31°C). In sharp contrast, only a small fraction
of the ALBR ligand is accessible to Cy5-SA at the permissive temperature of
21°C; Cy5 to Cy3 fluorescence ratio in a population of
shits1 cells at 21°C
(Fig. 7B,
shits1 21°C) resembles that obtained for nonspecific
binding of the Cy5-SA to cells (Fig.
7B, shits1 Noise). Greater than 90% of total
ligand-bound ALBR remained arrested at the cell surface in both
shits1 and shits2 cells at 31°C.
At the same temperature,
50% of ligand-bound ALBR remains at the cell
surface in CS cells (Fig. 7C),
compared with the
25% detected at 21°C. These experiments show that
shits1 and shits2 cells quantitatively
arrested ALBR sequestration at 31°C but not at 21°C, confirming that
ligand-bound ALBR is internalized by a dDyn-dependent mechanism. These results
are consistent with studies conducted in mammalian cells overexpressing
Dynts (analogous to shits1) on the
internalization of biotinylated transferrin endocytosed via the transferrin
receptor wherein the coated pits with wide openings also appear to accumulate
at the restrictive temperature (Damke et
al., 1995
).
|
To test whether the reversible nature of the temperature-sensitive
shi mutation (Koenig et al.,
1983; Kosaka and Ikeda,
1983b
) is reproduced in the primary culture cells, we asked if
Cy3mBSA arrested at the cell surface at 31°C in shits
cells (Fig. 7C) is capable of
being internalized into subsequently formed Cy5mBSA-containing endosomes at
the permissible temperature (see scheme in
Fig. 8A). The extensive
colocalization of Cy3mBSA in Cy5mBSA-containing endosomes formed at the
permissive temperature in hemocytes from shits animals
provides evidence for the reversible nature of the arrest of dDyn-mediated
internalization in these cells (Fig.
8B).
|
To gain further insight into the nature of structures containing
surface-accessible receptors that accumulate at the restrictive temperature we
followed the scheme outlined in Fig.
8C. This protocol addresses the rapidity with which
surface-accessible structures that accumulate at the restrictive temperature
in shi mutant cells become inaccessible to large-molecular-weight
molecules (Cy5-SA). After rapid transfer to ice (<5 seconds to equilibrate
to 0°C), we find that a surprisingly significant fraction (65%) of
surface-accessible B-Cy3mBSA pre-bound at 31°C
(Fig. 7C) becomes inaccessible
to Cy5-SA (Fig. 8D). In
comparison with the quantitative accessibility of B-Cy3mBSA at 32°C,
transfer to ice results in a significant reduction in accessible receptors.
This suggests a rapid sequestration of the B-Cy3mBSA in this experimental
paradigm. As observed in a representative figure (right panel in
Fig. 8C), although the majority
(87%) of the B-Cy3mBSA fluorescence remains inaccessible to Cy5-SA, the
B-Cy3mBSA is predominantly peripherally distributed
(Fig. 8C, 32°C,
arrowheads), which is indistinguishable from cells labeled on ice
(Fig. 6H-J), or cells from
shits animals held at the high temperature
(Fig. 6F,G). The distribution
of Cy5-SA-inaccessible ligands in this paradigm is quite different from the
distribution of internalized B-Cy3mBSA in cells incubated at the permissive
temperature (21°C) post to transfer to ice
(Fig. 8C, 21°C). These
results suggest that the temperature-sensitive step in dDyn mutants is at a
late step in the sequestration process mediated by dDyn.
Surface-accessible ligands accumulate in clathrin- and
adaptin-positive structures
Since various Dyn-dependent pathways described in mammalian cells
(McNiven et al., 2000) are
clathrin dependent or independent, we ascertained whether internalization of
ALBR occurs by a clathrin-mediated pathway. For this purpose, we examined the
colocalization of ectopically expressed GFP-clathrin light chain (GFP-Clc)
(Chang et al., 2002
) with
Cy3mBSA-labeled ALBR incubated at 0°C. We find that most of the
surface-localized (and quantitatively surface-accessible;
Fig. 7B) receptors are present
in GFP-Clc-positive punctate structures
(Fig. 9A-C, small arrowheads).
At the restrictive temperature in shits2 cells, the
surface-accessible receptors are also localized in GFP-Clc-positive structures
(Fig. 9D-F, small arrowheads).
Furthermore
-adaptin, a component of the clathrin-recruiting AP-2
complex (Chang et al., 1993
),
also colocalizes with the peripheral, surface-accessible Cy3mBSA puncta in
both shits1 and shits2 cells
(Supplementary figure S3). These data support the conclusion that ALBR ligands
are internalized into primary larval hemocytes by a clathrin- and
dDyn-mediated pathway.
|
Fluid-phase and GPI-AP endocytosis in shits
cells
Unlike the internalization of Cy3mBSA, uptake of F-Dex into
shits1 and shits2 cells at the
restrictive temperature does not appear to be perturbed
(Fig. 6E-G). A quantification
of the amount of F-Dex internalized (Fig.
10A) shows that fluid-phase uptake in shits1
and shits2 cells at 21°C, 31°C and 33°C is
comparable with CS cells at the same temperatures. A common pathway of
endocytosis for both Cy3mBSA and F-Dex would predict that a decrease in the
rate of mBSA internalization should be accompanied by a decrease in the rate
of F-Dex uptake. This does not take place, thus ruling out the possibility of
a common pathway. It is unlikely that the conditions of primary culture medium
components upregulate alternative endocytic pathways in hemocytes, since cells
directly extracted into buffered saline also show dDyn-independent F-Dex
internalization (Supplementary figure S2A-F).
|
To determine whether F-Dex-containing endosomes formed at the higher temperature in shits cells are bona fide endocytic structures, we examined if these endosomes are capable of merging with ALBR-ligand-containing endosomes formed during the recovery of shits cells at 21°C. For this purpose, we first incubated cells with F-Dex (and Cy3mBSA) at 31°C and then shifted the temperature to 21°C in the presence of Cy5mBSA (see schematic, Fig. 8A). After shifting to 21°C, Cy3mBSA, Cy5mBSA and F-Dex colocalize in both shits1 and shits2 cells (Fig. 8B). These results show that the fluid-filled structures formed at restrictive temperatures are capable of merging with ligand-bound ALBR subsequently internalized from the cell surface in shits cells at the permissible temperature.
The endocytosis of GPI-APs via the fluid-filled endosomes in mammalian
cells has been shown to be Dyn independent
(Sabharanjak et al., 2002). To
ascertain the requirment for dDyn in GPI-AP internalization, we examined
GPI-AP endocytosis in cells from shits animals. At the
restrictive temperature, Fl-anti-GFP is taken up into cells from
shits2 in fluid-filled endosomes similar in morphology
(Fig. 10B) and extent as in
cells from wild-type animals at the same temperature (data not shown).
Furthermore, in the same cells, Cy5-mBSA is excluded from the endocytic
pathway at the restrictive temperature
(Fig. 10C). In cells
expressing comparable levels of GFP-GPI, the ratio of internalized Fl-anti-GFP
to the total amount of GFP fluorescence is 0.57±0.37 (15 cells) in wild
type and 0.79±0.27 (20 cells) in the shits2
background. Thus, the shits2 mutation does not prevent the
internalization of GPI-APs. This further reiterates the existence of a
distinct dDyn-independent pathway involved in GPI-AP endocytosis in
Drosophila hemocytes. Taken together, these data show that mBSA
internalization and F-Dex (and GPI-AP) uptake occur by distinct mechanisms;
ALBR-bound mBSA internalization is dDyn-dependent.
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Discussion |
---|
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At the restrictive temperature, the receptors are trapped at the cell
surface in clathrin- and -adaptin-decorated sites, accessible to large
molecules (Cy5-SA). This accessibility is quantitatively similar to that
observed when cells from both wild type and mutants are directly labeled on
ice, indicating a complete blockage of uptake and sequestration. These results
are analogous to observations in mammalian cells overexpressing
Dynts, a homologue of shits1, wherein coated
pits with wide openings accumulated at the restrictive temperature
(Damke et al., 1995
). In
contrast to studies where, in mammalian cells overexpressing Dynts,
the receptors retained avidin accessibility even after transfer to ice
(Damke et al., 1995
), the
receptors in shits cells pre-treated at restrictive
temperature become completely sequestered upon transfer to ice. This
difference is likely to reflect the intrinsic nature of mutations in dDyn in
the absence of interference from copolymerization with endogenous wild-type
Dyn molecules. The rapidity with which receptor sequestration takes place
(within 5 seconds), suggests that the block in dDyn function in the
shits mutants is at a stage where commitment to endocytic
sequestration has already occurred. The sharp transition in the extent of
sequestration induced by shifting to ice after incubation at the restrictive
temperature is suggestive of a structural role for dDyn in sequestration.
Consistent with these results is a previous report where synaptic endocytosis
is blocked in shi mutants at a late stage
(Ramaswami et al., 1994
).
Current models for the role of dynamin in receptor-mediated endocytosis via
clathrin-coated pits fall into two broad classes (reviewed in
Hinshaw, 2000;
Sever et al., 2000a
), one in
which the GTPase activity of the enzyme is directly involved in a
mechano-chemical scission of the invaginated pit
(Marks et al., 2001
), and the
other where Dyn is a regulatory GTPase involved in recruiting molecular
players responsible for the fission process
(Sever et al., 2000b
).
Although our results do not distinguish between these two models, they support
a structural role for dDyn at late stages in the sequestration process without
precluding a regulatory role in the earlier stages of the formation of the
endocytic invagination. Understanding of the stage in the GTP-GDP cycle at
which the shi mutants are blocked will further discriminate between
the two models (Damke et al.,
2001
).
These results reported here also demonstrate that the process of
dDyn-dependent internalization is intrinsically temperature sensitive; >85%
of the receptors are sequestered at permissive temperatures compared with
50-60% at the restrictive temperature in the same time interval in
wild-type flies. Interestingly, this also has a correlate in the temperature
sensitivity of the paralysis of wild-type (CS) flies in our hands; CS flies
paralyze in a manner very similar to shits flies but at
temperatures >40°C. Mutations at the shi locus,
shits1 and shits2, can thus be seen as
enhancers of the intrinsic temperature sensitivity of this process.
By contrast, fluid-phase uptake does not exhibit any temperature
sensitivity in wild-type and shits animals, arguing
strongly for independent regulation. Cells completely lacking any
shibire gene product are likely to be autonomously cell-lethal
(Grant et al., 1998),
precluding an analysis of fluid-phase uptake in cells without any dDyn
expression. Therefore, these data do not completely rule out a role for dDyn
in fluid-phase uptake but they certainly rule out a role similar to the
function of dDyn in receptor-mediated uptake in this endocytic process. Uptake
of fluid occurs by various means (Lamaze
and Schmid, 1995
); however, except for macropinocytosis and the
pathway for the internalization of GPI-APs
(Sabharanjak et al., 2002
),
all other pathways require Dyn function in mammalian cells. Live imaging with
cultured larval hemocytes expressing either GFP-actin or other actin-binding
proteins fused to GFP have revealed that these cells undergo extensive
membrane ruffling (A. Guha and S. Mayor, unpublished). Whereas
macropinocytosis correlates with membrane ruffling
(Racoosin and Swanson, 1992
),
the earliest F-Dex-filled compartments are unlike typical macropinosomes.
Macropinocytosis is usually a triggered process resulting in large,
phase-lucent organelles at the time of formation
(Racoosin and Swanson, 1992
).
Although endosomes labeled in a 5 minutes pulse in larval hemocytes develop
into phase-bright structures, the majority of the earliest F-Dex-filled
endosomes formed in a 45 seconds pulse are not, arguing against the
dDyn-independent pathway resembling macropinocytosis.
Another marker that has been extensively used to study fluid-phase
endocytosis in Drosophila and other cell culture systems is HRP. The
reduced uptake of HRP seen in garland cells from shits1
animals at the restrictive temperature
(Kosaka and Ikeda, 1983b)
would, therefore, appear contrary to the results presented here. However, at
comparable molar concentrations, it is likely that HRP may be taken up via
multiple means, including receptor-mediated pathways. We find that HRP is
internalized via a yeast-mannan-competable pathway in hemocytes
(Sriram et al., 2003
),
consistent with the possibility that HRP uptake is not restricted to the fluid
phase. Furthermore, similar to the distinct cdc42-regulated,
dynamin-independent pinocytic pathway recently described in mammalian cells
(Sabharanjak et al., 2002
),
our results show that the Drosophila hemocytes are also capable of
dDyn-independent GPI-AP endocytosis via the fluid-phase pathway. Another point
of similarity with the fluid-phase pathway recently described in mammalian
cells is the absence of Rab5 on fluid-phase-containing endosomes
(Sabharanjak et al., 2002
).
This suggests that Rab5 defines only a subset of early endosomal compartments,
strengthening the notion that there may be multiple early endosomal systems
that are connected to each other via cell-type-dependent trafficking pathways
(Fivaz et al., 2002
;
Sabharanjak et al., 2002
).
This primary cell culture system will provide an opportunity for analysis of
the molecular players involved in this pathway in a genetically amenable
system, and will allow an exploration of the trafficking consequences of
alterations in this pathway in the whole animal.
In conclusion, we show that Drosophila cells exhibit Dyn
(dDyn)-dependent and -independent pathways of endocytosis. These pathways
converge in a population of Rab7-positive endosomes and subsequently target
endocytosed cargo for degradation in lysosomes
(Sriram et al., 2003). It is
conceivable that several GPI-AP-containing membranes in Drosophila,
including `argosomes' (Greco et al.,
2001
) and modulators of signaling such as heparan sulfate
proteoglycans (Selleck, 2000
)
are endocytosed via the dDyn-independent pathway, distinct from dDyn-dependent
pathways utilized to regulate signaling receptors
(Ceresa and Schmid, 2000
;
Pierce and Lefkowitz, 2001
).
This could provide a mechanism by which cells can independently modulate their
responses to the extracellular environment. At least in the case of Notch and
Delta, the clearance of inactive Notch at the cell surface appears to be
facilitated by a dDyn-independent pathway
(Parks et al., 2000
). The
primary culture system described in this report makes it possible to examine,
at high resolution, endocytic trafficking of molecules internalized by various
pathways in Drosophila and use available genetic tools to dissect
these pathways.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
* These authors contributed equally to this work
![]() |
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