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b
Fig. 21.4 Dynein forms a ring at the immunological synapse. a-f Jurkat T cells were paired with
Raji B cells coated with the superantigen Staphylococcus enterotoxin E (SEE). Cells were
immunostained and the fluorescence images were processed to obtain 3D images. In a, mouse
anti-ADAP formed a ring at the synapse with microtubules and the MTOC (green) in the center.
Cell pairs were also immunostained with rabbit anti-dynein intermediate chain (DIC) (b)or
mouse anti-DIC (c). Both antibodies stained a ring at the synapse. Jurkat cell pairs were stained
and a merged image of d, anti-ADAP, and e, mouse anti-DIC is shown in f. ADAP (red) and DIC
(green) colocalized in a ring at the synapse. Scale bars 5 microns. g, h Jurkat cells expressing
GFP-DIC were activated with SEE-coated Raji cells. The GFP fluorescence alone (g) and
immunostaining cell pairs for GFP (h) both show GFP-DIC accumulation at the synapse. Figures
4(a-f) are reprinted from Combs et al. (
2006
). Copyright (2006) National Academy of Sciences,
U.S.A
In studies of MTOC polarization in Jurkat cells, having found that LFA-1 was
not absolutely required, the question remained as to why there was a correlation
between microtubule anchor points and the pSMAC. One possibility was that
microtubule contact points and possibly dynein were more closely correlated with
ADAP, a molecule needed for LFA-1 clustering (Wang et al.
2009
; Kliche et al.
2012
). Immunostaining and computerized 3D reconstructions of ADAP and
microtubules showed that ADAP forms a ring at the synapse that is closely related
to where microtubules contact the pSMAC (Fig.
21.4
a). Furthermore, when ADAP
expression was reduced by introduction of antisense morpholino oligonucleotides,
MTOC translocation was blocked (Combs et al.
2006
). On the other hand, when
T cells were prepared from ADAP
-/-
mice, MTOC translocation was essentially
normal. The reason for this difference is not clear.
The Jurkat cell studies were extended to an examination of dynein. Immuno-
staining using two different antibodies against the dynein intermediate chain (70.1,
1467) showed that dynein was present as a ring at the synapse, closely related to
ADAP and other markers for the pSMAC Fig.
21.4
(b-f) (Combs et al.
2006
).
Furthermore, immunoprecipitation of dynein also pulled down ADAP suggesting
that the two molecules were linked in some way. Finally, when ADAP expression
was reduced using morpholino oligonucleotides, there was also a loss of dynein at
the synapse. These data suggested there was a link between ADAP, dynein, and
MTOC translocation in Jurkat T cells.
Although previous studies showed a link between dynein and MTOC translo-
cation they were not directly interfering with dynein. Recent studies by Martin-
Cofreces et al. showed that use of siRNA to reduce dynein expression led to a loss
of MTOC translocation (Martin-Cofreces et al.
2008
). They reported similar
effects due to overexpression of dynactin, which disrupts dynein complexes. More
recently, we have used molecular traps against the dynein intermediate chain
(Varma et al.
2010
) to show that when the trap is activated by dimerization,
MTOC translocation is reduced by more than 50 % (Christian and Poenie,
unpublished observations). Thus, several lines of evidence suggest that dynein is
important for MTOC translocation.
