As a student I always marvelled at the sight of single cells in culture moving over
artificial surfaces and exhibiting membrane ruffles and protrusions. However, while I found
cultured cells fascinating I always wondered how cells are able to move and regulate their
shape in the context of a whole organism where so many space constraints exist and where
all cellular processes have to be tightly regulated. Some answers to my questions began to
emerge in a paper written by Baum and Perrimon (2001), in which the authors showed the
expression and regulation of the actin cytoskeleton and of actin binding proteins in a real
epithelium.
The cytoskeleton is a meshwork of protein polymers extending throughout the cytoplasm.
It not only provides structural support for the cell but also plays a central role in a
range of dynamic processes from signalling to endocytosis and intracellular trafficking. A
particularly clear example of this is the use of actin cytoskeleton as a “wool” for
knitting multiple dynamic structures such as lamellae, filopodia, and stress fibres. These
structures determine cell shape and also produce the driving force accompanying many types
of cellular movements including muscle contraction and cell division. We know many details
about some of the proteins that modulate the dynamics of actin in these structures.
However, most of them have been found biochemically and their function has been elucidated
primarily using in vitro and cell culture assays of actin assembly. What about these
proteins in the context of a developing organism? How do cells generate a spatially and
temporally ordered network of actin filaments represented at the tissue level? To answer
these questions, we need to move to experimentally accessible multicellular organisms, such
as
Drosophila , which offers virtually unlimited possibilities as a
model system for the genetic and molecular analysis of biological processes.
Baum and Perrimon (2001) analyzed the function of a number of proteins involved in actin
dynamics within the context of a developing epithelium—the follicle cells that surround the
germ line cyst during
Drosophila oogenesis. These cells have a simple polarised
arrangement of actin filaments, which provides a useful system to study the spatial
organisation of the actin cytoskeleton.
Taking advantage of the ability to generate clones of cells lacking specific proteins,
the authors identified new functional roles for actin regulators such as CAP (a
Drosophila homologue of adenylyl cyclase-associated proteins),
Enabled (Ena) and Abelson (Abl). These proteins had been well characterized in cell culture
and in vitro studies, but little was known about their function in a developing organism.
Clones of cells lacking CAP (Figure 1), a protein known to inhibit actin polymerisation,
maintained their epithelial polarity but had higher levels of actin and defects in the
apical actin organisation. This result indicates that the inhibitory activity of CAP is
restricted to one side of the cells, thus demonstrating that actin dynamics can be
independently modified at opposite poles of an epithelium. Ena, a member of the Ena/VASP
family proteins that catalyse filament formation, and Abl, a protein kinase that binds CAP
in mammalian cells, were found to work with CAP in this process. The authors proposed that
CAP, Ena, and Abl regulate the level and spatial organization of actin in the follicle
cells.
In contrast to the spatially restricted functions of CAP, Ena, and Abl, profilin and
cofilin were shown to regulate actin filament formation throughout the cell cortex, a more
global function that matches the results obtained in cell culture experiments. In summary,
this study showed how proteins can organise actin in space and began to highlight some of
the differences and similarities between cells in culture and in vivo. The functions
revealed in the follicular epithelium were consistent with the roles previously shown in
mammalian systems, but the experiments on intact tissue began to reveal a spatial and
temporal functional dimension that could not have been observed in cell culture.
These experiments could be expanded to large-scale screens (St Johnston 2002), but this
would be time consuming and could encounter the problem that some genes will be cell
lethal, preventing the analysis of their function in actin dynamics. However, two more
recent reports (Kiger et al. 2003; Rogers et al. 2003) describe a complementary and
exhaustive search for regulators of cytoskeletal dynamics by taking advantage of genomic
resources and the powerful RNA interference (RNAi) technique (Hutvágner and Zamore 2002).
RNAi allows individual genes to be knocked out in a simple and controlled fashion.
Kiger et al. (2003) used RNAi in two different cell lines of
Drosophila to screen a number of genes involved in signalling
and cytoskeletal dynamics. They targeted 994 genes, of which 160 produced phenotypes in the
experiment. The range of phenotypes varied from specific defects in the actin and tubulin
cytoskeleton to others affecting cell cycle progression, cytokinesis, and cell shape. They
also showed that only about 40% of the genes had similar loss-of-function phenotypes in
both cell lines. This alone indicates an important limitation of many tissue culture
experiments, since the same protein can have different effects depending on the cell type.
Another valuable element of this work is that clustering of genes with similar phenotypes
leads to the identification of pathways and networks of genes that are involved in
cytoskeletal function.
Rogers et al. (2003), using only one
Drosophila cell line, studied the effects of proteins involved
in the formation of lamellae. The authors looked at the effects of loss of function in 90
genes known to be involved in actin dynamics and the formation and activity of the lamella.
As well as confirming the function of many proteins already known to play a role in this
process, this analysis allowed them to find interactions between genes and to build genetic
pathways.
Together these two studies reveal that RNAi screens in tissue culture can be a powerful
tool for finding new functions of known and uncharacterized genes, and new relationships
between genes. However, this is only the beginning, and the genes identified in this manner
will have to be tested in vivo, in systems like that of Baum and Perrimon, where specific
functions can be assessed in time and space within the confines of real organisms. The
focus must be to understand how all these molecular events and regulation cascades operate
in individual cells to contribute to the generation of changes in a whole individual.
Increasingly, the attention of developmental biologists is being drawn from genes and their
products towards cells (Kaltschmidt and Martinez Arias 2002). The future, it seems to me,
lies in the combination of in vitro systems, cell culture, and in vivo studies. I hope to
apply this view in my analysis of the process of dorsal closure in
Drosophila embryos, as an example of how signalling pathways
coordinate and regulate the activity of the cytoskeleton in the generation of shape and
morphogenetic movements (Jacinto et al. 2002).
