Biology Reference
In-Depth Information
21.2 The Mechanics of MTOC Repositioning
In an effort to visualize the cytoskeleton in general and in particular, the dynamics
of MTOC movement, we developed modulated polarization microscopy (MPM)
(Kuhn et al.
2001
). This microscope images cytoskeletal elements based on their
birefringence which allowed us to noninvasively image cells for extended lengths
of time without concerns of photobleaching. MPM imaging data showed that
MTOC movement was associated with development of tension in microtubules
that pulled the MTOC toward the contact site (Fig.
21.2
) (Kuhn and Poenie
2002
).
Once it reached the contact site, the MTOC oscillated laterally along the face of
the contact site. It turned out that the magnitude of these oscillations was signif-
icant, about three to four microns, which is about the same as the inner diameter of
the pSMAC. These oscillations were even more dramatic when two target cells
were bound to a single CTL. Here, the MTOC moved repeatedly from one contact
site to the other, a distance of approximately eight microns.
To explore this further, we examined numerous computerized 3D reconstruc-
tions of CTL-target pairs immunostained for tubulin and LFA-1. From this data, it
became clear that the MTOC could be positioned in various regions of the
cSMAC, but there were no examples where the MTOC crossed into the pSMAC
(Fig.
21.3
a and b). Thus, the inner margin of the pSMAC seemed to represent a
limit or barrier to oscillations of the MTOC under normal conditions. This raises
the question concerning what happens when the MTOC oscillates between two
target cells. Although we did not have specific examples of this in the immuno-
staining data, one often sees only partial rings of LFA-1. We might speculate that
in cases where two target cells are engaged, there could be a partial pSMAC at
each site which serves as endpoints for the oscillating MTOC.
The observation that tensioning of microtubules is associated with movement of
the MTOC toward the synapse suggests that there must be a motor protein,
anchored at the synapse, that reels in the microtubules. The most likely candidate
is the microtubule minus end-directed motor, dynein. Given that lateral oscillations
along the contact site can be in all directions (horizontal, vertical etc.), we sus-
pected that the motor must be distributed as a ring with dimensions similar to the
pSMAC. This would explain why the MTOC could only travel to the inner edge of
the pSMAC.
As the MTOC moves toward the synapse, the microtubules projecting from the
MTOC to the synapse take on the form roughly of a hollow cone, where the central
region of the cone is devoid of microtubules (Fig.
21.3
). The cone becomes progres-
sively wider as the MTOC comes closer to the membrane. Microtubules at the edge of
the cone appear to contact the cell surface or cortex in the region of the pSMAC and
then bend backwards toward the rear of the cell (Fig.
21.3
a-c) (Kuhn and Poenie
2002
). These bend points had the appearance of ''knuckles'' in the microtubules where
sharp bends were seen. This would make sense if motor proteins were anchored in the
pSMAC region of the synapse and suggested the possibility that microtubules interact
with a dynein ring that was in turn associated with sites where LFA-1 was clustered.
