* Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310
Oslo, Norway
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum
Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark
As described in our title, "Internalization of cholera toxin by
different endocytic mechanisms", we studied the endocytic pathways by
which cholera toxin (CT) can be taken in from the cell surface. We have
previously studied CT transport through the Golgi apparatus and to the ER, as
well as cAMP production (Sandvig et al.,
1996), and we were among the first to demonstrate this transport
step for CT. Similarly, as first shown for Shiga toxin
(Sandvig et al., 1992
), other
toxins are transported retrogradely before entry into the cytosol (for a
review, see Sandvig and van Deurs,
2002
).
However, studies of CT entry are of interest not only because this toxin
can increase the level of cAMP in some cells and cause diarrhoea, but also
because CT has been commonly used to label GM1 and to study endocytosis from
caveolae. In our study (Torgersen et al.,
2001), we used three different model systems to modulate uptake by
endocytic pathways: (1) Caco cells transfected with caveolin to create
caveolae at the cell surface; (2) Hela cells with inducible synthesis of
mutant dynamin, which have been reported to inhibit pinching-off of vesicles
from both clathrin-coated pits and caveolae; and (3) BHK cells with inducible
synthesis of antisense-clathrin heavy chain (cells in which clathrin-dependent
endocytosis can be shut off selectively). The data obtained from the three
systems reveal that both clathrin-dependent and clathrin- and
caveolae-independent mechanisms can lead to endocytosis of CT. This study does
not allow us to conclude whether caveolae can be responsible for endocytosis
of cholera. Although caveolae (with caveolin) are quite stable structures in
some cell types (Thomsen et al.,
2002
), the toxin itself might affect the stability. To date, no
publications have addressed this question. However, our study
(Torgersen et al., 2001
)
clearly shows that different endocytic pathways can be involved. This is in
agreement with recent data published by other laboratories
(Nichols et al., 2001
;
Shogomori and Futerman,
2001
,Shogomori and Futerman,
2001
). We did not make any attempt to answer whether toxin taken
in by the various endocytic mechanisms can elicit a biological response
(Torgersen et al., 2001
). This
is of course an important question but was not addressed in our study.
When one investigates the effect of a certain drug (which quite often has
more than one effect on cells) or, for instance, the importance of cholesterol
(either by adding drugs, removing cholesterol or adding cholesterol) on the
action of a toxin, a reduced or increased effect can be caused by an effect on
the endocytic uptake, or by an effect on a later step, such as endosome to
Golgi transport of the toxin. Along these lines, it has recently been
published that transport of CT (Shogomori
and Futerman, 2001,Shogomori
and Futerman, 2001
), ricin
(Grimmer et al., 2000
) and the
Shiga toxin B subunit (Falguieres et al.,
2001
) from endosomes to the Golgi apparatus are affected by
changes in cholesterol. Also, when comparing the effect of a drug on the
cytoplasmic action of various toxins, there may not necessarily be a
difference because of the different endocytic mechanisms used by the toxins,
but a different response could be caused by different intracellular pathways
(cholera/diphtheria toxin). Alternatively, the drug could have a direct effect
on the target molecule, for instance on the activity of a membrane-associated
target such as adenylyl cyclase, which in itself could be regulated by, for
instance, cholesterol or drugs affecting cholesterol. Studies with
proteoliposomes have even shown that coupling between Gs and
adenylyl cyclase can be dependent on the cholesterol:phospolipid ratio
(Bai and Youguo, 1998
). In
order to avoid such complications in our endocytosis studies, we concentrated
on the endocytic uptake by directly measuring the internalization from the
cell surface (Torgersen et al.,
2001
).
It should be noted that there are cell-specific differences (as discussed
in our article) when it comes to uptake from the cell surface (as well as to
intracellular routing of toxins). Thus, whether a toxin is associated with
lipid rafts (Falguieres et al.,
2001), and to what extent it is transported to the Golgi
apparatus, is clearly cell-type dependent and can be dependent on the type of
fatty acid in the toxin receptor
(Falguieres et al., 2001
;
Sandvig and van Deurs, 2002
;
Lingwood, 1999
). That
endocytosis of CT can occur independently of filipin addition is supported by
a previous study (Shogomori and Futerman,
2001
,Shogomori and Futerman,
2001
). In fact, when clathrin-dependent endocytosis is reduced by
antisense-clathrin induction, or when dominant-negative mutant dynamin is
induced to inhibit both clathrin- and caveolae-dependent endocytosis, there is
no further decrease in the uptake of CT by extraction of cholesterol with
mßCD (M. L. Torgersen, B.v.D. and K.S., unpublished). Thus, in the cells
we have studied, CT can be endocytosed even under such conditions.
In conclusion, it is clear that CT can be taken in by various endocytic mechanisms, and that more has to be done to characterize these mechanisms as well as the intracellular transport of CT. To fully characterize the transport of CT, investigation of each step will be necessary.
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