Biochemical Society Transactions (200 I) Volume 29, part 4
Relationship between endosomes and lysosomes
J. P. Luzio*', B. M. Mullock", P. R. Pryor*, M. R. Lindsay*, D. E. Jarnest and R. C. Piper1
*Department of Clinical Biochemistry and Wellcome Trust Centre for Molecular Mechanisms in Disease, University
of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, U.K., +Institute of Molecular and Cellular
Biology, University of Queensland, Brisbane, QLD 4068, Australia, and 1Department of Physiology and Biophysics,
University of Iowa, Iowa City, IA 52242, U.S.A.
Abstract
that are optimally active at an acid pH. They are
usually regarded as the terminal degradation compartment of the endocytic pathway [l], and also
play an important role in phagocytosis, autophagy,
crinophagy and proteolysis of some cytosolic
proteins that are transported across the lysosomal
membrane. Recently the view that lysosomes are
simply a 'garbage disposal unit' has been challenged, both by a better understanding of how
endocytosed material is delivered to them and also
by the identification, in many cell types, of
lysosomes that secrete their contents after fusion
with the plasma membrane [2,3].
Lysosomes were discovered half a century
ago as a result of the association of a number of
hydrolases with an organelle of defined centrifugal
properties. These hydrolases demonstrated structure-linked latency, which was dependent on the
integrity of the surrounding membrane (reviewed
in [4,5]). From the earliest days it was recognized
that lysosomes demonstrated heterogeneous morphology, containing dense deposits and membrane
whorls [6]. In mammalian cells they characteristically appear as organelles of
0.5 pm diameter, often with electron-dense cores. They can
make up 0.5-5% of the cell volume and are
concentrated near the microtubule organizing
centre [7]. The current definition of mammalian
lysosomes includes not only the presence of lysosoma1 acid hydrolases and lysosomal integral
membrane glycoproteins, but also, to distinguish
them from endosomes, the absence of the two
mannose 6-phosphate receptors (MPRs) and recycling cell surface receptors.
Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late
endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing
assay using late endosomes and lysosomes from rat
liver. There is evidence from electron microscopy
studies that the same process occurs in intact cells.
The fusion process results in the formation of
hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has
opened up three areas of investigation: (i) the
mechanism of direct fusion of late endosomes and
lysosomes, (ii) the mechanism of re-formation
of lysosomes from the hybrid organelle, and (iii)
the function of the hybrid organelle. Fusion has
analogies with homotypic vacuole fusion in yeast.
It requires syntaxin 7 as part of the functional
trans-SNARE [SNAP receptor, where SNAP is
soluble N-ethylmaleimide-sensitivefactor (NSF)
attachment protein] complex and the release of
lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle
occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular
traffic. Lysosomes may thus be regarded as a type
of secretory granule, storing acid hydrolases in
between fusion events with late endosomes. The
hybrid organelle is predicted to function as a 'cell
stomach', acting as a major site of hydrolysis of
endocytosed macromolecules.
-
Lysosomes
Lysosomes are intracellular membrane-bound
organelles containing many hydrolytic enzymes
Endosomes
Key words: endocytic pathway, fusion, late-endosomelysosome
hybrid organelle, SNARES,tethering proteins.
Abbreviations used: MPR mannose 6-phosphate receptor; NSF,
N-ethylmaleimide-sensitivefactor: SNAP, soluble NSF attachment
protein: SNARE, SNAP receptor: V-SNARE, vesicle SNARE; tSNARE, target organelle SNARE; VAMP, vesicle-associatedmembtane protein.
'To whom correspondence should be addressed (e-mail
][email protected]).
The identification of endosomal compartments on
the major endocytic pathway from the clathrincoated pit at the plasma membrane to the lysosome
was achieved in the 1980s using a combination of
microscopy, subcellular fractionation and kinetic
analysis of the passage of endocytosed molecules
through different compartments. Endosomal compartments in mammalian cells are morphologically
heterogeneous, and constitute a pleiomorphic
0 200 I Biochemical Society
4 76
The Interface of Receptor Signalling and Trafficking
lysosomes could not be a sufficient explanation of
endocytic delivery to lysosomes or of lysosome
biogenesis.
Three alternative hypotheses have been proposed to explain content mixing between late
endosomes and lysosomes (reviewed in [3]). T h e
first assumes vesicular transfer between the two
organelles, largely by analogy with the many
membrane traffic steps on both the secretory and
endocytic pathways where such a mechanism
operates. T h e second hypothesis is termed ‘kiss
and run’, and proposes that late endosomes and
lysosomes are undergoing continuous repeated
transient fusion and fission. T h e third hypothesis
is direct fusion between late endosomes and
lysosomes, with subsequent recovery of lysosomes
for re-use. This hypothesis, which could be regarded as an extension of kiss and run, where some
kisses result in fusion, was first discussed by
Griffiths [lS]. He pointed out that, morphologically, lysosomes resemble electron-dense secretory granules, speculated that their function is
to store mature lysosomal enzymes, and proposed
that they fuse periodically with late endosomes in
a regulated fashion to form a compartment, ‘the
cell stomach’, where degradation occurs. He also
proposed that lysosomes subsequently bud off this
compartment and are re-used as required. While
no evidence has emerged supporting vesicular
transport between late endosomes and lysosomes,
there is clear evidence for direct fusion of these
organelles. In the rat liver cell-free system, the
immunoprecipitable product of content mixing
between late endosomes and lysosomes was shown
to be present in a membrane-bound organelle that
was intermediate in density between late endosomes and lysosomes [171. This organelle, which
we have termed a late-endosome-lysosome hybrid
organelle, was shown by electron microscopy to
contain markers derived from both lysosomes and
late endosomes, and to be less electron dense than
lysosomes [17]. In our view this hybrid organelle
has the characteristics of the ‘cell stomach’ [ l 81.
T h e evidence that direct fusion can occur
between late endosomes and lysosomes has been
backed up by experiments showing that an organelle fraction, with the centrifugation and morphological characteristics of the late-endosomelysosome hybrid organelle, can be isolated from
rat liver, and also by electron microscopic experiments on cultured cells. These include a study on
cultured HEP-2 cells by Futter et al. [19] which
showed that transfer of complexes of epidermal
growth factor and its receptor to a late endocytic
smooth membrane system consisting of tubular
and vesicular elements, with many of the latter
containing intra-organelle vesicles and often being
described as multivesicular bodies [S-lo]. Endocytosed macromolecules are delivered first to early
endosomes and then to late endosomes, with the
time of delivery to each endosomal compartment
varying between cell types, and between cells in
culture and those in living tissue [ l l ] . Early
endosomes are loaded within 1-5 min of uptake,
are tubular with varicosities, and many are located
peripherally within the cell close to the plasma
membrane. Late endosomes become loaded
4-30min after uptake, are more spherical and
have the appearance of multivesicular bodies.
They are mostly juxtanuclear, being concentrated
near the microtubule organizing centre. Early and
late endosomes are characterized by different
lumenal pHs [121, different protein compositions
[13] and association with different small GTPases
of the Rab family, with Rab5 on early endosomes
and Rab7 on late endosomes (reviewed in [14]).
T h e early endosome is the major sorting
compartment of the endocytic pathway, where
many ligands dissociate from their receptors in the
acid p H of the lumen and from which many of the
receptors recycle to the cell surface. T h e membrane traffic pathway from early to late endosomes
was the subject of some dispute a few years ago,
with the proposal of maturation and vesicular
transport models. Both models provide for an
intermediate between early and late endosomes,
differing mainly in whether this is, in essence,
what is left after removal of components of early
endosomes that are to be recycled or delivered
elsewhere, or a newly formed endocytic carrier
vesicle [15,16].
Delivery of endocytosed
macromolecules to lysosomes
Investigation of the delivery of endocytosed
macromolecules from late endosomes to lysosomes
has been hindered by the problems inherent in the
fact that many such macromolecules are digested
rapidly once they reach the degradative environment in late endocytic compartments. Nevertheless, it has been possible to establish a cell-free
system in which to study content mixing between
late endosomes and lysosomes derived from rat
hepatocytes using a slowly digested ligand delivered to the lysosome [11,17]. T h e demonstration of content mixing between the organelles
itself showed that maturation of late endosomes to
477
0 2001 Biochemical Society
Biochemical Society Transactions (200 I) Volume 29, part 4
degradative compartment involved maturation of
a late endosomal multivesicular body that fused
directly with MPR-negative lysosomes. Our own
experiments with cultured N R K cells have also
provided data consistent with the existence of the
late-endosome-lysosome hybrid organelle in vivo
1201.
complex which includes a protein phosphatase
[27] and/or a direct interaction with VO, the
membrane-integral sector of the vacuolar H+ATPase [28]. Peters et al. [28] have proposed the
formation of VO trans-complexes acting as proteolipid-lined channels at the fusion site.
Although the SNARE hypothesis proposed
specific pairing between v- and t-SNARES, an
alternative classification, based on sequence alignments of the coiled-coil domains and structural
features observed in the crystal structure of the
heterotrimeric synaptic fusion complex [29], has
been proposed. This separates SNAREs into
Q(G1n)-SNARES and R(Arg)-SNARES, with
four-helix bundles composed of three Q-SNARE
helices and one R-SNARE helix being formed
after SNARE complex assembly [30]. Currently, a
model of SNARE architecture in which an intracellular t-SNARE consists of a heavy chain and
two light chains (three Q-SNARES), with the
cognate V-SNARE being a single transmembrane
protein, seems most likely [31]. It is most probable
that the overall fidelity of a membrane traffic
fusion event may not simply reside in the fidelity
of SNARE complex-formation, but in SNARE
complex-formation in concert with other protein
interactions [32,33]. T h u s it is possible to imagine
that SNARE complex assembly adds an additional
layer of specificity to the fusion event after tethering, as well as being a necessary precursor of it,
and/or that it acts as a proof-reading step to ensure
that only correctly tethered membranes are fused.
Heterotypic fusion between late endosomes
and lysosomes to form hybrid organelles in the rat
liver cell-free system is dependent on ATP, cytosol
and temperature, and requires the presence of
NSF, SNAPs and a small GTPase of the Rab
family [17]. It can also be inhibited by a fast-acting
calcium chelator and is calmodulin dependent
[34]. Our approach to identifying specific protein
components of the late-endosome-lysosome
fusion machinery has been to search for mammalian homologues of the proteins encoded by the
yeast VPSIVAM genes known to act late in the
pathway to the yeast vacuole. T h u s antibodies to
syntaxin 7, the closest mammalian homologue
to the yeast vacuole t-SNARE Vam3p, inhibited
fusion [35], whereas antibodies to syntaxin 13, a
SNARE required for homotypic early endosome
fusion, did not. Syntaxin 7 has also been implicated in traffic late in the pathway, because
overexpression of the cytoplasmic domain inhibits
delivery of endocytosed markers to late endocytic
compartments [36]. Permeabilized cell assays in
Protein components of the fusion
machinery
Information about the molecular machinery involved in membrane fusion events occurring in the
endocytic pathway has come from biochemical
analysis of cell-free systems that reconstitute steps
in the pathway, and by studying mammalian
homologues of proteins encoded by vps (vacuolar
protein sorting) and vam (vacuolar morphology)
mutants in the budding yeast Saccharomyces cerevisiae. In this organism the vacuole is functionally
equivalent to the lysosome, as both are acidic
compartments containing a variety of hydrolytic
enzymes involved in the degradation of macromolecules [21,22].
Cell-free systems have shown that, throughout the endocytic pathway, fusion events are
consistent with the action of the common cytosolic
fusion machinery, requiring N S F (N-ethylmaleimide-sensitive factor) and SNAPs (soluble N S F
attachment proteins), that is used at many other
sites within the cell. This machinery functions
according to the tenets of the SNARE (SNAP
receptor) hypothesis, which proposed pairing between specific v- (vesicle) and t- (target organelle)
SNAREs [23] to form a functional trans-SNARE
complex. There is a growing body of evidence that
specific tethering processes occur prior to transSNARE complex-formation, where tethering is
defined as involving links that extend over distances of > 25 nm from a given membrane surface,
in contrast with docking, which holds membranes
within a bilayer’s distance ( < 5-10 nm) of one
another [24]. There is also evidence that, after
trans-SNARE complex-formation, calcium release from the lumen of the docked organelles is
required for membrane fusion to occur, and that
the released calcium mediates fusion via a calmodulin-dependent event(s) (reviewed in [25]).
This requirement for a calcium/calmodulinmediated late event in a SNARE-dependent fusion
process was first shown in a homotypic yeast
vacuole cell-free fusion assay [26]. In vacuole
fusion, the calcium/calmodulin dependence has
been proposed to be mediated via a protein
0 200 I Biochemical Society
478
The Interface of Receptor Signalling and Trafficking
intraorganelle p H and high Ca2' [45,46], plays an
important role in the formation of the mature
secretory granule. T h u s it seems possible that
the re-formation of lysosomes from lateendosome-lysosome hybrid organelles occurs by
a process analogous to the formation of regulated
secretory granules from the trans-Golgi network
[W.
Overall, recent data support a dynamic model
of lysosome function, in which lysosomes are
constantly fusing with late endosomes and being
re-formed from the resultant hybrid organelles.
which antibodies to specific SNAREs have been
found to inhibit degradation of endocytosed epidermal growth factor have implicated both
VAMP7 (vesicle-associated membrane protein 7 ;
TI-VAMP) and syntaxin 8 in the pathway from
early endosomes to lysosomes [37-391. In cell-free
assays, homotypic late-endosome fusion has been
shown to be inhibited by antibodies to syntaxin 7,
syntaxin 8, Vtilb and VAMP8 (endobrevin). In
contrast, antibodies to VAMP8 have not been
found to inhibit heterotypic late-endosomelysosome fusion, suggesting the possibility that an
alternative V-SNARE is required. T h e complexity
of the data on which SNAREs are required for
which fusion events in the endocytic pathway may
be explained if combinatorial interactions are
allowed, e.g. between one V-SNARE and more
than one t-SNARE, and/or between one tSNARE and more than one V-SNARE.A further
contribution to the complexity may be that different heterotrimeric t-SNARES have some common individual polypeptide chains.
T o date, no specific tethering proteins have
been identified for late-endosomelysosome fusion, unlike the situation for homotypic earlyendosome fusion, where recruitment of tethering
proteins, including early-endosomal antigen 1
(EEAl), is dependent on the presence of Rab5 and
of PtdIns 3-phosphate patches formed as a result
of PtdIns 3-kinase activity [40,41]. Fine striations,
consistent with the existence of tethers, have been
observed between adjacent late endosomes and
lysosomes in several morphological studies on
cultured cells [19,20,42]. T h e inhibitory effects of
GDP-dissociation inhibitor and wortmannin on
late-endosomelysosome fusion [ 171 suggest a
tethering mechanism similar to that used by early
endosomes, i.e. dependent on both a Rab and
PtdIns 3-phosphate patches. By analogy with
yeast homotypic vacuole fusion, the mammalian
homologues of the yeast proteins Vpsl l p , Vpsl6p,
Vpsl8p, Vps33p, Vps39p and Vps4lp, which
together act as a complex [43,44], are clearly
candidates to be involved in this process.
Since the fusion of late endosomes with
lysosomes to form hybrid structures results in
consumption of the starting organelles, it is apparent that a recovery system involving membrane
retrieval and a lysosomal content recondensation
process occurs in vivo [3,17,20]. Another mammalian cell organelle in which condensation of
content has been well described is the regulated
dense-core secretory granule. In this case it is clear
that protein aggregation, under conditions of low
Experimental work in our laboratories referred to above was
funded by grants from the Medical Research Council t o J. P. L.. and
by a Human Frontien Science Program Grant t o RCP.. J.P.L.
and D.E.J.
References
I
2
3
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
479
Komfeld, S. and Mellman, I. ( I 989) Annu. Rev. Cell Biol. 5,
483-525
Stinchcombe, J. C. and Griffrths. G. M. ( I 999) J. Cell Biol.
147. 1-5
Luzio, J. P., Rous, B. A., Bright, N. A,, Pryor, P. R., Mullock
B. M. and Piper, R C. (2000) J. Cell Sci. I 13, I 5 15- I524
De Duve, C. ( 1983) Eur. J. Biochem. 137, 39 1-397
Bowen, W. E. ( I 998) Trends Cell Biol. 8, 33&333
Holtzmann, E. ( I 989) Lysosomes, Plenum Press, New York
and London
Matteoni, R and Kreis, T. E. ( I 987) J. Cell. Biol. 105,
I 253- I265
Hopkins, C. R. (I 983) Nature (London) 304,684-685
Hopkins, C. R., Gibson, A., Shipman, M. and Miller, K.
( I 990) Nature (London) 346, 335-339
Trowbridge, I. S., Collawn, J. F. and Hopkins, C. R ( I 993)
Annu. Rev. Cell Biol. 9, 129- I 6 I
Mullock B. M.. Perez, J. H., Kuwana. T., Gray, S. R. and
Luzio, J. P. (1994) J. Cell Biol. 126, I 173-1 I82
Schmid, S., Fuchs, R., Kielian, M., Helenius, A. and Mellman,
I. ( 1989) J. Cell Biol. 108, I29 I - I 300
Schmid, S. L., Fuchs, R., Male, P. and Mellman, I. ( I 988) Cell
52,73-83
Rodman,J. S. and Wandinger-Ness, A. (2000) J. Cell Sci.
113, 183-192
Gruenberg, J. and Maxfield, F. R. ( I 995) Cur. Opin. Cell
Biol. 7, 552-563
Gu, F. and Gruenberg, J. ( 1999) FEBS Lett. 452,6 1-66
Mullock B. M.. Bright, N. A., Fearon. C. W.. Gray. S. R and
Luzio, J. P. ( 1998) J. Cell Biol. 140, 59 1-60 I
Grifiths, G. ( 1996) Protoplasma 195, 37-58
Futter, C. E.. Peane, A., Hewlett, L. J. and Hopkins, C. R.
( I 996) J. Cell Biol. 132, I0 I I- I023
Bright. N. A., Reaves, B. J.,Mullock B. M. and Luzio, J. P.
( 1997) J. Cell Sci. I 10, 2027-2040
Klionsky, D.J., Herman. P. K. and Emr, S. D. ( I 990)
Microbiol. Rev. 54, 266-292
Klionsky, D.J. (1997) J. Membr. Biol. 157, 105-1 15
Rothman, J. E. ( I 994) Nature (London) 372, 55-63
Pfeffer, S. R. ( 1999) Nat. Cell Biol. I , E I7-E22
0 2001 Biochemical Society
Biochemical Society Transactions (200 I) Volume 29, part 4
25
26
27
28
29
30
31
32
33
34
35
Ptyor, P. R., Buss, F. and Luzio, J. P. (2000) ELSO Gazette
(http ://www.the-elso-gazette.org/magazines/issue2/reviews/
reviews I -pr.asp)
Peters, C. and Mayer, A. ( I 998) Nature (London) 396,
575-579
Peters, C., Andrews, P. D., Stark M. J. R., Cesaro-Tadic, S.,
Glatz, A., Podtelejnikov. A,, Mann, M. and Mayer, A. ( I 999)
Science 285, 1084- I087
Peters, C., Bayer, M. J., Buhler, S., Andersen, J.S., Mann, M.
and Mayer, A. (200 I) Nature (London) 409, 58 1-588
Sutton, R B., Fasshauer, D., Jahn,R and Brunger, A. T.
( I 998) Nature (London) 395, 347-353
Sutton, R B., Brunger, A. T. and Jahn, R
Fasshauer, D.,
( 1998) Proc. Natl. Acad. Sci. U.S.A. 95, I578 I- I5786
Fukuda, R.. McNew, J. A., Weber, T., Pariati, F., Engel, T.,
Nickel, W., Rothman, J. E. and Sollner, T. H. (2000) Nature
(London) 407, 198-202
Bock J.B. and Scheller, R. H. ( I 999) Proc. Natl. Acad. Sci.
U.S.A. 96, 12227- I2229
Chen. Y. A. and Scheller, R H. (2001) Nat. Rev. Mol. Cell
Biol. 2, 1-9
Pryor, P. R., Mullock B. M., Bright, N. A.. Gray, S. R and
Luzio. J.P. (2000) J. Cell Biol. 149, 1053- I062
Mullock B. M., Smith, C. W., Bright, N. A,. Ihrke, G.,
Lindsay, M.. Parkinson, E. J., Brooks, D. A,, Parton, R. G.,
James, D.E., Luzio, J. P. and Piper, R C. (2000) Mol. Biol.
Cell I I . 3137-3153
36
37
38
39
40
41
42
43
44
45
46
Nakamura, N., Yamamoto, A,, Wada, Y. and Futai, M.
(2000) J. Biol. Chem. 275, 6523-6529
Advani, R. J., Yang, B., Prekeris, R, Lee, K. C., Klumperman,
J. and Scheller, R. H. ( I 999) J. Cell Biol. 146, 765-776
Prekeris. R, Yang, B., Oorschot. V.. Klumperman, J.and
Scheller. R H. ( I 999) Mol. Biol. Cell 10, 389 1-3908
Martinez-Arca, S.. Alberts, P. and Galli, T. (2000) Biol. Cell
92,449453
McBride, H. M., Rybin, V., Murphy, C., Giner, A,, Teasdale,
R and Zerial, M. ( 1999) Cell 98, 377-386
Zerial, M. and McBride, H. (200 I) Nat. Rev. Mol. Cell Biol.
2, 107-1 19
van Deun, B., Holm, P. K., Kayser, L. and Sandvig, K. (I 995)
Eur. J. Cell Biol. 66, 309-323
Seals, D. F., Eitzen, G., Margolis, N., Wickner, W. T. and
Price, A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,
9402-9407
Wurmser, A. E., Sato, T. K. and Emr, S. D.(2000) J. Cell
Biol. I 5 I , 55 1-562
Bauerfeind. R. and Huttner, W. B. ( I 993) Cum Opin. Cell
Biol. 5, 628-635
Tooze, S. A. ( 1998) Biochim. Biophys. Acta I404,23 1-244
Received 5 March 200 I
Internalization of the epidermal growth factor receptor: role in signalling
A. Sorkin'
Department of Pharmacology, University of Colorado Health Science Center, 4200 E. Ninth Avenue, Denver,
CO 80 I I I, U.S.A.
~~
Abstract
method of measuring fluorescence resonance
energy transfer (FRET) on a pixel-by-pixel basis
using E G F R fused to cyan fluorescent protein
(CFP) in pair with Grb2 or Shc fused to yellow
fluorescent protein (YFP). Stimulation by E G F
resulted in the recruitment of Grb2-YFP and
YFP-Shc to cellular compartments that contained
EGFR-CFP, and a large increase in the F R E T
signal. In particular, F R E T measurements indicated that activated EGFR-CFP interacted with
YFP-Shc and Grb2-YFP in membrane ruffles
and endosomes. These results demonstrate that
signalling via EGFRs can occur in the endosomal
compartment. Moreover, in contrast with previous biochemical studies, F R E T experiments
show that a large pool of Grb2 and Shc is
associated with EGFRs for a prolonged period
after E G F stimulation.
T h e interaction of the activated epidermal growth
factor (EGF) receptor (EGFR) with the Src
homology 2 (SH2) domain of Grb2 (growthfactor-receptor-bound protein 2) initiates signalling through Ras and mitogen-activated protein
kinase. Grb2 can bind E G F R directly or through
another SH2-containing protein, Shc. Activation
of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. T o analyse
the spatial and temporal regulation of E G F R interactions with SH2 domains in living cells, we have
combined imaging microscopy with a modified
Key words: EGF receptor, endocytosis, FRET, Grb2, Shc.
Abbreviations used: CFP, cyan fluorescent protein; EGF,epidermal
growth factor; EGFR, EGF receptor; FRET,fluorescence resonance
energy transfer; F R W , corrected FRET: Grb2, growth-factorreceptor-bound protein 2; MAPK, mitogen-activated protein
kinase; PAE, porcine aortic endothelial; PTB, phosphotyrosinebinding; SH2, Src homology 2; YFP, yellow fluorescent protein.
'e-mail [email protected]
0 2001 Biochemical Society
Introduction
Binding of epidermal growth factor (EGF) to its
receptor (EGFR) at the cell surface results in
480