(Received for publication, November 30, 1995)
From the
Mutations that result in defective -integrin
focal adhesion formation were analyzed for effects on bacterial
internalization. Mutations in the cytoplasmic domain of the
chain that disrupt the sequence NPIY resulted in integrins
deficient in bacterial uptake. Other mutations in the
chain that reduced cytoskeletal association showed enhanced
bacterial uptake. Replacement of the NPIY sequence of the
subunit by the endocytosis internalization sequence PPGY resulted
in integrin receptors highly proficient in bacterial internalization,
yet severely defective in focal contact localization. Electron
microscopy indicated that coated structures associated specifically
with bacteria-binding
-integrins, with an apparent
recruitment of coated pits from ventral cell surfaces to apical
surfaces corresponding to nascent bacterial phagosomes. Clathrin
inhibition studies indicated a role for the adaptor molecule AP2 as
well as clathrin in integrin-mediated bacterial internalization. These
results indicate that association of
-integrins with
the cytoskeleton at focal contacts interferes with integrin-mediated
bacterial internalization. Also, although actin polymerization is
required for bacterial uptake, clathrin is probably involved in
bacterial uptake promoted by
-integrins.
Integrins are heterodimeric receptors involved in numerous
cellular processes including migration, differentiation, and adhesion
to extracellular matrix and cell-surface
proteins(1, 2, 3) . Members of this family
are heterodimeric transmembrane proteins that are involved
in both inside-out and outside-in
signaling(2, 4, 5) . Ligand specificity of
each receptor is determined by the particular
and
chains
found in the heterodimer. The unique cytoplasmic domains of the
different subunits allow diversity and regulation of receptor
function(6) . For example, the cytoplasmic domain of the
subunit interacts with the cytoskeleton by binding to
actin-binding proteins such as talin (7) and
-actinin(8) . Mutational analysis as well as peptide
inhibition studies have identified regions of the cytoplasmic domain of
the
chain important for this cytoskeletal
association(9, 10, 11) . The
chain
cytoplasmic domain also plays a prominent role in transduction of
signals originating either from outside or inside the
cell(12, 13, 14) . The cytoplasmic domain of
the
subunit appears to regulate ligand affinity (15) and
participates in processes such as gel contraction (16) .
Integrins also promote the internalization of various
microorganisms. In the macrophage, the integrin receptor CR3 (or
) can mediate internalization of
complement-coated particles(17) . In normally non-phagocytic
cells, viruses and bacteria have been shown to be internalized by
integrins containing
,
, and
chains(18, 19) . The uptake of the
enteroinvasive bacterium Yersinia pseudotuberculosis by
-integrins has been studied in detail. Uptake of this
microorganism occurs via the bacterial surface protein
invasin(20) , which binds multiple
-integrins(18) . Invasin-mediated bacterial
uptake is accompanied by a local rearrangement of the actin network (21) and is inhibited by drugs that antagonize actin
polymerization(22) . This has led to speculation that direct
association of integrins with the cytoskeleton is required during
internalization.
By using various anti-integrin antibodies to coat
the Gram-positive bacterium Staphylococcus aureus, we have
been able to mimic the phenotype of invasin-expressing bacteria with
respect to uptake. We have shown that integrin-mediated internalization
following bacterial attachment to the cell is primarily dependent on
the affinity of the bacterial ligand for the
integrin(23, 24) . In this study, we use this
technique to study the role of the cytoplasmic domain of the integrin
subunit. We identify a region of the
subunit cytoplasmic domain that is critical for internalization
and show a potentially antagonistic relationship between cytoskeletal
association of the cytoplasmic domain and bacterial uptake.
The tannic acid post-fixation, which has been
described as an efficient technique to enhance clathrin-coated membrane
visualization, also leads to intense staining of bacterial membrane
components, ()rendering difficult the interpretation of
results. To visualize coated membrane structures, the samples were
processed for transmission electron microscopy as described
previously(28) . The extent of membrane was quantitated from
micrographs enlarged at least 40,000 times using the
Bioquant
system IV (R & M Biometrics, Inc.,
Nashville, TN). The incidence of coated pits was determined by scoring
coated pits from micrographs over 50 randomly selected cell sections.
The total number of coated pits on the apical or ventral membrane was
related to the total length of the apical or ventral membrane,
respectively. The relative percentage of coated membranes over the
ventral plasma membrane could not be unambiguously determined because
of numerous cell areas of dense cytoplasmic background.
The chicken integrin subunit expressed in
mammalian cells is reported to associate with the endogenous
chains, forming heterodimers functional in adhesion and cytoskeletal
association(3, 9) . After transfection with the
chicken
clone, the expression levels of the
chicken/human hybrid integrins on the surface of various transfected
clones of HEp-2 cells were determined by an ELISA-based assay (see
``Materials and Methods''). From immunoprecipitation
experiments, the levels of chicken
/human hybrid
integrins in the various transfectants were generally <25% of the
endogenous levels of human integrin
chains (data not
shown). Transfectants expressing various derivatives of the chicken
chain bearing the F768A, P786A, or N785I substitution
in the
cytoplasmic domain (Table 1) were also
analyzed by immunoprecipitation using the CSAT mAb. No obvious
qualitative or quantitative differences in association with the
endogenous
subunits could be observed among the different mutants
when compared with the wild-type chicken
chain (data
not shown). The values obtained by scanning the intensity of the band
corresponding to the chicken
chain were in good
agreement with the values obtained in the ELISA-based assay, and these
ELISA-based determinations were used to quantitate the expression
levels of chicken/human hybrid receptors at the surface of the various
transfectants (Table 1).
Transfectants expressing the
wild-type chicken chain internalized significant
levels of S. aureus coated with the anti-chicken
-integrin mAb CSAT (Table 1,
c-wild type, uptake/expression equal to 4.1) when
compared with nontransfected HEp-2 cells. Therefore, the chicken
/human hybrids could be used to analyze the effect on
bacterial uptake of mutations located in the cytoplasmic domain of the
integrin
chain.
Figure 1:
Effect of mutations in the
integrin chain cytoplasmic domain on bacterial uptake
and focal contact localization. The amino acid residues of the integrin
chain cytoplasmic tail are numbered according to
Tamkun et al.(62) . Amino acid substitutions are
depicted with arrows. Deletions and the PPGY substitution are
represented as boxes. Except for the PPGY mutation, data
concerning focal contact localization are derived from Reszka et
al.(10) . Bacterial uptake: -, receptor deficient in
bacterial uptake; +, wild-type levels of internalization;
, internalization levels superior to
wild-type levels. Focal contact localization: -, receptor
deficient in focal contact localization; +, intermediate levels of
localization;
, wild-type levels of
focal contact localization.
Transfectants were
cloned, and the efficiency of bacterial internalization as well as the
levels of expression of chicken/human hybrid integrins were normalized
relative to nontransfected HEp-2 cells. Several substitutions in the
cytoplasmic region cyto-2 (NPIY) (Fig. 1) (10) resulted
in no detectable levels of bacterial internalization when compared with
nontransfected HEp-2 cells (Table 1, N785I, P786A, and Y788E). In
addition, the deletion (residues 759-771) removing the cyto-1
region as well as the deletion (residues 771-790) removing the
cyto-2 region also abolished the ability of integrin-mediated bacterial
internalization (Table 1). In contrast, the single amino acid
substitutions F768A, F771L, and E784L as well as other mutations in the
region called cyto-3 (Fig. 1) (10) resulted in increased
efficiency of bacterial uptake relative to wild-type transfectants (Table 1). Several of the mutants that were more efficient at
promoting bacterial uptake than the parental integrin chain exhibited a reduction in focal contact association (Table 1)(10) .
These results indicate that residues in the membrane-proximal NPIY (cyto-2) region as well as the integrity of the cyto-1 region are critical for integrin-mediated bacterial internalization. The fact that single amino acid substitutions in the cyto-1 and cyto-3 regions depressing cytoskeletal association resulted in an increased uptake efficiency suggests that cytoskeletal association of integrins at focal contacts interferes with bacterial internalization.
To determine if the membrane-proximal NPIY
sequence could be substituted with another internalization sequence,
the cyto-2 NPIY sequence of the integrin chain was
replaced by PPGY, which determines rapid internalization of the
lysosomal acid phosphatase via coated pits(37, 38) .
As shown in Table 1, the integrin receptor containing the PPGY
sequence was competent to mediate bacterial internalization (Table 1, NPIY
PPGY) with an efficiency comparable to that
of other cytoplasmic mutants of the
chain that were
more proficient than the wild type. Binding studies performed on cells
expressing mutant integrins bearing the PPGY or N785I substitution
indicated that these receptors bound the CSAT mAb at a similar
efficiency (Fig. 2), ruling out the possibility that the
variations in bacterial uptake were due to a change in ligand-receptor
affinity(23) .
Figure 2:
Saturation curves of the CSAT mAb binding
to 3T3 transfectants expressing chicken/mouse integrins containing the
N785I or PPGY mutation. Two-fold serial dilutions of the 3T3
transfectants were plated in 24-well plates, and the cells were allowed
to adhere to the plastic surfaces. Monolayers were incubated with RPMI
1640 medium containing the CSAT mAb (2 µg/ml) for 3 h at 22 °C,
followed by incubation with rabbit anti-mouse antibody conjugated to
horseradish peroxidase for 2 h at 22 °C. Bound antibody was
quantitated using the 2,2`-azinobis(3-ethylbenzthiazoline-6-sulfonic
acid) substrate, reading the absorbance at 405 nm (see ``Materials
and Methods''). , 3T3 transfectant expressing the
chicken/mouse integrins with the N785I substitution;
, 3T3
transfectant expressing the chicken/mouse integrins with the PPGY
substitution;
, parental 3T3 cells.
To analyze the ability of this integrin
derivative to localize to focal contacts, immunofluorescence staining
of 3T3 transfectants using the anti-chicken -integrin
mAb W1B10 was performed on cells plated onto fibronectin-coated
surfaces as described(10) . As opposed to parental chicken
-integrins (Fig. 3C), the PPGY substitution
demonstrated a strong defect in localization to focal contacts with a
diffuse punctate staining (Fig. 3A). The defect in
focal contact localization appeared considerably more severe for the
PPGY substitution (Fig. 3A) than for the N785I
substitution (Fig. 3B), which had been previously
reported to show a defect in focal contact formation(10) . The
phenotype of the PPGY mutation was therefore a defect in cytoskeletal
association coupled with an enhancement of bacterial internalization.
Figure 3:
Substitution of the proximal NPIY sequence
with PPGY results in a defect in focal contact localization. 3T3
transfectants were plated on fibronectin-coated coverslips, and live
cells were stained with the anti-chicken -integrin mAb
W1B10 followed by an anti-mouse IgG antibody linked to fluorescein
isothiocyanate (see ``Materials and Methods''). Shown are 3T3
transfectants expressing
c/NPIY
PPGY (A),
c/N785I (B), and
c-wild type (C). The staining for the
c/NPIY
PPGY clone appears punctated.
c/N785I shows a slight defect in localization of
-integrin to focal contacts. The
c-wild type clone shows a typical focal contact
staining pattern. Bar = 10
µm.
HEp-2 cell monolayers were incubated with E. coli MC4100/pRI203 (inv) for 2 h
at 22 °C to allow bacterial binding. After washing to remove
unbound bacteria, the cells were shifted at 37 °C for 10 min to
allow internalization (see ``Materials and Methods''), and
the samples were prepared using standard protocols(28) .
In
uninfected HEp-2 cells, coated pits appeared to be associated primarily
with the basolateral surface of the cell with a density of 11.2
pits/mm compared with 3.4 pits/mm for the apical surface (Table 2, column A). In HEp-2 cells challenged with bacteria, the
distribution of coated pits was dramatically different. In these cells,
most of the coated pits appeared to be located on the apical surface
with an incidence of 11.4 pits/mm of membrane, whereas the density of
basolaterally located pits decreased to 5.8 pits/mm of membrane (Table 2, column B). The vast majority of the pits (83%) that
were on the apical surface appeared to associate with nascent bacterial
phagosomes ( Table 2and Fig. 4C, curved arrows).
Also, large coated membrane lattices (>500 nm) were often visible at
the cell membrane region juxtaposing the engulfed bacteria (Fig. 4, A and B, arrows). In
uninfected cells, such structures were detected primarily at the basal
surface (Table 2). Consistent with these results, quantitation of
coated membranes indicated that in uninfected cells, clathrin
structures associated with
0.3% of the apical surface (Table 2), whereas 4.2% of the apical surface of phagocytizing
cells was covered by clathrin-coated membranes, with 90% of these
coated membranes associated with bacterial phagosomes (Table 2).
Figure 4:
Invasin-promoted uptake generates the
formation of large coated membrane lattices containing coated pit-like
structures. Monolayers of HEp-2 cells were infected with MC4100/pRI203 (inv) on ice for 2 h. Unbound bacteria were
washed away, and the cells were shifted to 37 °C for 15 min and
immediately fixed (see ``Materials and Methods''). The cells
were post-fixed and processed for transmission electron microscopy as
described(28) . In A and B, the arrows point to large coated membrane lattices underlying the bacteria
and containing coated pit-like structures. The arrowhead in B shows a similar structure at the ventral surface of the
cell. In C, the curved arrows point to coated pits
underlying bound bacteria, and the arrow indicates a coated
vesicle. In D, the cells were immunostained with the
anti-clathrin mAb X-22 followed by anti-mouse IgG linked to horseradish
peroxidase. Bound mAbs were revealed by staining with
3,3`-diaminobenzidine (see ``Materials and Methods''). The arrows indicate phagosomes associated with several coated
pit-like structures, and the arrowhead points to a bacterial
phagosome located closer to the cell nucleus devoid of such structures. Bars = 1.0 µm (A, B, and D) and 0.5 µm (C).
In these experiments, the thin sections showed an average of 3.85
cell-associated bacteria/cell section, with 25% of the cell-bound
bacteria associated with clathrin-coated membranes (Table 2).
This number increased to 50% for cell sections showing only 1 or 2
bound bacteria (data not shown). Some bacteria were internalized in
phagosomes showing several coated pit-like structures (Fig. 4D, arrows), whereas other phagosomes
were devoid of such structures (Fig. 4D, arrowhead).
Immunostaining with the anti-adaptor molecule AP2 antibody AP-6 also showed the formation of large adaptin lattices specifically associated with the plasma membrane juxtaposing the bacteria that were not detected in uninfected control cells (data not shown). These data indicate that clathrin tends to redistribute to the apical surface of HEp-2 cells that are internalizing invasin-expressing bacteria and that the majority of clathrin structures detected on the apical surface are associated with phagosomes surrounding bacteria being internalized.
Figure 5:
A, effect of potassium depletion on
integrin-mediated bacterial uptake. For potassium depletion, HEp-2 cell
monolayers were subjected to a hypotonic shock and incubated for 30 min
in K-free buffer (see ``Materials and
Methods''). After this period, MC4100/pRI203 (inv
) or anti-
-integrin
mAb-coated S. aureus resuspended in the same buffer was added
to the monolayers at a multiplicity of infection of 10 bacteria/cell,
and the incubation was allow to proceed at 37 °C for 30 min. The
percentage of bacterial internalization was determined as described
previously(25) .
,
monolayers challenged with S. aureus coated with the
anti-
mAb AIIBII;
,
monolayers challenged with S. aureus coated with the
anti-
mAb VD1; INV,
monolayers challenged with MC4100/pRI203 (inv
). Hatched bars, buffer
containing potassium; solid bars, buffer without potassium as
described under ``Materials and Methods.'' Each bar represents the mean ± S.D. of three determinations. B, effect of potassium depletion on
I-labeled
transferrin (TFN) uptake. HEp-2 cells were potassium-depleted
as described above, and the uptake of transferrin was determined as
described previously(30) . Cells were incubated with
I-labeled transferrin for 5 min at 37 °C. The cells
were washed three times with ice-cold PBS, and the monolayers were
treated with 0.3% Pronase for 1 h on ice. The cells were then
transferred to Eppendorf tubes and centrifuged for 2 min, and the
pellet and the supernatant were counted for
-radiations.
Nonspecific internalization was determined by incubation with
I-labeled transferrin for 5 min on ice. The results are
expressed as percentage of total cell-associated counts. Each bar represents the mean ± S.D. of three
determinations.
Cell loading
of antibodies against clathrin and AP2 adaptor molecules has been shown
to result in inhibition of endocytosis of transferrin (44) . To
demonstrate a role for clathrin in integrin-mediated bacterial uptake,
HEp-2 cells were loaded with the anti-clathrin mAb X-22 (45) or
with the anti-adaptor AP2 mAb AP-6 (46) using the syringe
loading technique(31) . After loading with mAb, HEp-2 cells
were allowed to adhere to plastic surfaces and infected with
MC4100/pRI203 (inv) (Fig. 6, solid
bars). As a control, HEp-2 cell samples were also infected with S. typhimurium strain SL1344, which is internalized via an
integrin-independent pathway (Fig. 6, hatched bars).
Cell loading with the anti-clathrin mAb resulted in a 60% inhibition of
invasin-mediated bacterial internalization (Fig. 6, solid
bar, X-22) when compared with loading with the irrelevant
mAb CSAT (Fig. 6, solid bar, CSAT), whereas
loading with the anti-adaptor AP2 mAb resulted in
50% inhibition (Fig. 6, solid bar, AP-6). In contrast,
internalization of S. typhimurium was not significantly
affected by cell loading with anti-clathrin or anti-adaptor mAb (Fig. 6, hatched bars, X-22 and AP-6), indicating that the inhibition seen was not due to a
general defect in bacterial internalization, but was specific for the
integrin-mediated uptake pathway. These results are consistent with a
functional role for clathrin and AP2 adaptor molecules during
integrin-mediated bacterial uptake.
Figure 6:
Effect of syringe loading of anti-clathrin
and anti-AP2 mAbs on integrin-mediated bacterial uptake. HEp-2 cells
were resuspended in RPMI 1640 medium containing 5% newborn calf serum
and loaded with mAbs using a 27-gauge needle. Cells were allowed to
adhere to plastic surfaces for 60 min at 37 °C, washed with
prewarmed medium, and infected with bacteria for 30 min at 37 °C.
The percentage of internalized bacteria was determined by the
gentamicin protection assay (25) after normalization to the
value obtained for the CSAT mAb control (see ``Materials and
Methods''). Each bar represents the mean ± S.D. of
three independent determinations. Hatched bars, cells infected
with S. typhimurium SL1344; solid bars, cells
infected with MC4100/pRI203 (inv). Cells were
loaded with anti-adaptor AP2 mAb AP-6, anti-clathrin mAb X-22, bovine
serum albumin (BSA), or the irrelevant anti-chicken integrin
chain mAb CSAT.
Cell loading with higher concentrations of the anti-clathrin and anti-adaptin mAbs resulted in similar levels of partial inhibition of integrin-mediated bacterial internalization (data not shown). These partial levels of inhibition could reflect the limitations of the technique used(31, 44) , but they could also indicate the existence of multiple bacterial internalization mechanisms, some of which may be independent of clathrin and the AP2 adaptor.
The results presented here clearly indicate that
integrin-mediated bacterial internalization and focal contact
localization have different requirements. Previous studies demonstrated
that the cytoplasmic domain of the subunit allows
association of integrins with the cytoskeleton as well as localization
of integrins to focal contacts (9, 32) . Three
clusters of amino acids distributed along the cytoplasmic domain of the
subunit, denoted cyto-1, -2 and -3, contribute to
this process (Fig. 4)(10) . Substitution mutations in
each of these clusters have weak but distinct effects on focal adhesion
formation. In contrast, substitution mutations depressing bacterial
internalization are centered in cyto-2. Such mutations have very strong
effects on bacterial internalization (Table 1) and weak effects
on focal contact formation(10) .
These results were
unexpected because it had been thought that association of integrins
with the cytoskeleton was required for bacterial uptake. This
hypothesis was based on the fact that invasin-mediated uptake by
cultured epithelial cells is accompanied by a reorganization of F-actin
around the internalized bacteria (21) and is inhibited by
cytochalasins(22) . Also, the role of actin in phagocytosis has
been well established as actin filaments as well as actin-binding
proteins associate with nascent phagosomes in macrophages (47) . Electron micrographs of HEp-2 cells, however, show that
during the early phases of bacterial uptake, actin filaments,
visualized by decoration with myosin S-1 pieces, associate mainly with
cell protrusions at the tip of the nascent phagosome, with only a few
nucleation sites associated with the host cell membrane. ()Clearly, actin is involved in bacterial uptake, but
whether it directly binds to integrin receptors during uptake is
questionable based on the results of this work.
In this study, we
found that deletions removing the cyto-1 and cyto-2 regions as well as
amino acid substitutions of residues 785-788 (NPIY) in cyto-2
abolished the ability of the integrin receptor to mediate bacterial
uptake. Physical studies using short peptides similar to NPIY indicate
that tyrosine-containing motifs have a high propensity to promote
tight-turn configurations(36, 48) . When these
sequences are proximal to an -helical segment, they have been
shown to allow receptor localization to clathrin-coated
pits(33) , perhaps by interacting with adaptor
molecules(34) . Although the NPIY sequence corresponding to
cyto-2 and the predicted
-helical cyto-1 appear to follow this
paradigm closely, there has been little previous evidence that these
determinants allow integrin association with clathrin-coated pits. For
example, it has been reported that mutations in the NPXY
sequences do not prevent internalization of the
-integrin in Chinese hamster ovary
cells(49) . On the other hand, immunofluorescence and
immunocolloidal gold experiments have shown that a portion of the
fibronectin receptor population localizes within clathrin-coated
pits(50, 51, 52) . It is possible that
-integrins are internalized via multiple pathways, one
of which requires the
chain cytoplasmic sequence NPIY
and clathrin-coated pits.
In this study, large lattices of clathrin and AP2 adaptor complexes are formed beneath bound bacteria in the early stages of the internalization process. These structures were not detected in uninfected cells and appeared to be induced during the bacterial internalization process. Consistent with this observation, a redistribution of the clathrin coat from the basolateral to the apical cell surface occurred in cells that were in the process of internalizing bacteria. The recruitment of coated pits from the basolateral to the apical surface of the cell, in specific association with bacterial phagosomes, suggests that the accumulation of clathrin is a specific event triggered by the bacterial internalization process.
These observations are similar to previous reports that demonstrated that particles internalized by phagocytic cells were encompassed by clathrin-coated phagosomes(39) , but the functional significance of these previous findings was unclear. In the results reported here, inhibition of uptake following cell loading with anti-clathrin or anti-adaptin mAb indicated that coat-associated proteins are required for integrin-mediated bacterial uptake. In addition, potassium depletion (Fig. 5), high concentrations of sucrose, and cytosol acidification (data not shown), treatments that have been shown to prevent coated pit-mediated endocytosis, resulted in a strong inhibition of integrin-mediated bacterial uptake. These results suggest that clathrin plays a role in integrin-mediated bacterial internalization, although it is not clear if processes different from those seen in clathrin-mediated endocytosis are also involved.
Whether the sole role of the proximal NPIY sequence of the
chain cytoplasmic domain during bacterial
internalization is to allow association of integrins with AP2 complexes
remains to be determined. Mutations of these residues lead to total
elimination of bacterial uptake, whereas introduction of anti-clathrin
antibody in the cell cytosol only inhibits internalization by
60%.
Perhaps these residues are critical for multiple bacterial
internalization pathways, some of which do not require clathrin.
Alternatively, clathrin association with integrins may be required for
bacterial internalization, and the partial inhibition observed in the
antibody loading experiment may be due to inherent technical
limitations.
Several mutations in the cytoplasmic domain of the
chain caused impaired integrin localization to focal
contacts, yet resulted in a bacterial internalization efficiency that
was higher than that promoted by the parental
-integrin. Similarly, substitution of the proximal
NPIY sequence by a structurally related sequence, PPGY (cyto-2) (Fig. 2), resulted in an integrin receptor highly efficient in
promoting bacterial uptake, yet defective in focal plaque localization.
One explanation for this observation is that the proximal NPIY sequence
is involved in integrin recycling(53) , but this process is
inefficient unless the receptor breaks its association with the
cytoskeleton. Presumably, the PPGY mutation shifts the balance from
integrin association with focal contacts to a recycling receptor
readily available for bacterial internalization. The role of the more
distal NPIY region is less clear. Although the mutations in the more
distal NPXY sequence (NPKY) stimulate uptake, we cannot
eliminate a possible role for this region in the uptake process. It is
possible that the presence of the amino acid changes used in this work
may not disrupt signaling functions in cyto-3 that are important for
uptake.
Previous studies on Fc receptors in the macrophage have
suggested that phagocytosis and endocytosis of the receptor involve
different processes(54) . Mutational analysis of the
cytoplasmic domain of the Fc receptor for IgE in macrophages indicates
that residues involved in endocytosis of the receptor are different
from residues involved in phagocytosis of IgE-coated
erythrocytes(54) . On the other hand, as pointed out above,
coated pits have been shown to associate with early phagosomes during
Fc receptor-mediated phagocytosis in macrophages (55) . Perhaps
with some receptors, clathrin associates with phagosomes mainly to
ensure receptor recycling, rather than playing an active role during
phagocytosis, as indicated by our results with integrin-mediated
bacterial internalization. To illustrate that phagocytic
internalization of different receptors may occur via different
pathways, phagocytosis via the receptors for C3 derivatives is not
accompanied by a strong release of HO
, whereas
Fc receptor-mediated uptake results in such a burst(56) . It is
possible that phagocytosis via Fc receptors in macrophages involves
different processes than integrin-mediated bacterial internalization in
normally non-phagocytic HEp-2 cells, and the results described here may
not apply to all classes of receptors.
Phagocytosis via either Fc
receptors or integrins seems to require tyrosine phosphorylation (57, 58) and formation of
F-actin(21, 22) . Although the integrin chain presents a potential tyrosine phosphorylation site at
Tyr
(59) , its substitution with an alanine
residue did not influence bacterial uptake. It is likely, however, that
tyrosine phosphokinases play an active role in transmitting signals
necessary for bacterial internalization steps, and a number of studies
indicate that a variety of cytoplasmic components are phosphorylated in
response to adhesion of integrins to
substrates(4, 13, 60) . Further studies aimed
at identifying these signals should allow a better understanding of the
internalization process.