Neuronal differentiation and epithelial integrity: the role of Drosophila Short stop
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Abstract
The nervous system is the most complex organ in animals and the ordered
interconnection of neurons is an essential prerequisite for normal behaviour. Neuronal
connectivity requires controlled neuronal growth and differentiation. Neuronal growth
essentially depends on the actin and microtubule cytoskeleton,
and it has become increasingly
clear, that crosslinking of these cytoskeletal fractions is a crucial regulatory process. The
Drosophila Spectraplakin family member Short stop (Shot) is such a crosslinker and is crucial
for several aspects of neuronal growth. Shot comprises various domains: An actin binding
domain, a plakin-like domain, a rod domain, calcium responsive EF-hand motifs, a
microtubule binding Gas2 domain, a GSR motif and a C-terminal EB1aff domain. Amongst
other phenotypes, shot mutant animals exhibit severely reduced dendrites and neuromuscular
junctions, the subcellular compartmentalisation of the transmembrane protein Fasciclin2 is
affected, but it is also crucially required in other tissues, for example for the integrity of
tendon cells, specialised epidermal cells which anchor muscles to the body wall. Despite these
striking phenotypes, Shot function is little understood, and especially we do not understand
how it can carry out functions as diverse as those
described above.
To bridge this gap, I capitalised on the genetic possibilities of the model system
Drosophila melanogaster and carried out a structure-function analysis in different
neurodevelopmental contexts and in tendon cells. To this end, I used targeted gene expression
of existing and newly generated Shot deletion constructs in Drosophila embryos and larvae,
analyses of different shot mutant alleles, and transfection of Shot constructs into S2 cells or
cultured fibroblasts. My analyses reveal that a part of the Shot C-terminus is not essential in
the nervous system but in tendon cells where it stabilises microtubules. The precise molecular
mechanism underlying this activity is not yet elucidated but, based on the findings presented
here, I have developed three alternative testable hypothesis. Thus, either binding of the
microtubule plus-end tracking molecule EB1 through an EB1aff domain, microtubulebundling
through a GSR rich motif or a combination of both may explain
a context-specific
requirement of the Shot C-terminus for tendon cell integrity. Furthermore, I find that the
calcium binding EF-hand motif in Shot is exclusively required for a subset of neuronal
functions of Shot but not in the epidermal tendon cells. These findings pave the way for
complementary studies studying the impact of [Ca2+] on Shot function.
Besides these differential requirements of Shot domains I find, that most Shot domains
are required in the nervous system and tendon cells alike. Thus the microtubule Gas2 domain
shows no context specific requirements and is equally essential in all analysed cellular
contexts. Furthermore, I could demonstrate a partial requirement of the large spectrin-repeat
rod domain of Shot in neuronal and epidermal contexts. I demonstrate that this domain is
partially required in processes involving growth and/or tissue stability but dispensable for
cellular processes where no mechanical stress resistance is required. In addition, I
ndemonstrate that the CH1 domain a part of the N-terminal actin binding domain of Shot is
only partially required for all analysed contexts.
Thus, I conclude that Shot domains are functioning different in various cellular
environments. In addition my study lays the base for future projects, such as the elucidation of
Shot function in growth cones. Given the high degree of conservation between Shot and its
mammalian orthologues MACF1/ACF7 and BPAG1, I believe that the findings presented in
this study will contribute to the general understanding of spectraplakins across species
borders.