Biology Reference
In-Depth Information
Trafficking of adhesion components in complexes
It is becoming evident that signalling molecules may also tra
c to and from
functional regions of the cell via large, cytoplasmic complexes. GIT1, which is
an adaptor protein that contains an ARF-GAP domain, cycles in large
cytoplasmic complexes between the cytosol, adhesions and the leading edge
(Manabe et al., 2002). The GIT1 complexes are not vesicular, unlike what we
observed with a5 integrin, since they do not co-localize with a number of
different endocytic or Golgi markers. Instead they appear to represent large,
motile, multi-molecular signalling complexes that contain molecules such as
paxillin, PAK and PIX. By contrast, at least some of these adhesion
components may also reside in vesicular structures (Di Cesare et al., 2000;
Paris et al., 2002). These adhesion components can localize to distinct
subcellular compartments, although their function is presently not known.
Our studies indicate that the GIT1 complexes can target constitutively
activated PAK to adhesions and the leading edge suggesting that these
structures can regulate the intracellular distribution of signalling molecules.
Migration in vivo
Most studies examining mechanisms of cell migration have utilized cells
growing in various culture systems. However, the ECM and growth factor
environment in vivo are not generally known and differ from that in vitro. For
example, cells migrating in vivo, unlike cells in culture, are exposed to a three-
dimensional matrix of various ECM molecules. In vivo, cells encounter
gradients of many different growth factors from the surrounding tissue while
cells in culture are usually exposed to a homogeneous bolus of a single or
small set of growth factors. In this context, it is unclear whether cells in vitro
and in vivo use the same mechanisms to migrate. We have begun to address
this issue by developing systems for studying cell migration in situ. The goal is
to extend the measurements of migration, molecular localization and
dynamics that are usually made in culture systems to cells migrating in situ.
Our approach is to image migrating cells in 200-300 mm slices using two
different model systems. These include migration of muscle precursors from
somites to the limb buds during development and migration of neuronal
precursors from the subventricular zone to the olfactory bulb via the rostral
migratory stream (RMS). We use time-lapse microscopy to observe the
migration of individual cells that are either labelled with the fluorescent
marker, DiI, or by expressing GFP-tagged fusion proteins in the slices. These
studies have already provided unexpected differences in migration between the
slice cultures and dissociated cell cultures in vitro. For example, the long,
highly polarized, persistent protrusions observed in the slice cultures are not
usually seen in migrating fibroblasts in vitro.
