PhD thesis
Functional analysis of the Drosophila gene smallish (CG43427)

Publication Details
Beati, S.
Publication year:


The establishment and maintenance of cell polarity is crucial for
the function of many cell types in multicellular organisms. Especially
in epithelial tissues, cell polarity is connected to the regulation of
cell adhesion and regulated by a complex hierarchy of highly conserved
proteins. These can be subdivided into three groups of genes, the
bazooka and crumbs groups, which encode apically localizing proteins,
and the discs large group that encodes laterally localizing tumor
suppressor proteins. Among these classes of proteins, Bazooka (Baz), the
Drosophila homolog of vertebrate Par-3, plays a predominant role as
shown by genetic epistasis experiments.
In a yeast two-hybrid screen we identified the protein encoded by the
annotated gene CG43427 which we named smallish (smash), as a new
interaction partner of Baz. The gene product of smash possesses a
C-terminal PDZ binding motif and a LIM domain close to the C-terminus.
Endogenous Smash colocalizes with Baz apically in epithelial cells, a
region harboring the adherens junctions (AJs). Co-immunoprecipitation of
Baz and an N-terminally tagged version of Smash-PI (an isoform encoding
for the last 889 amino acids of Smash) has confirmed that these
proteins interact in vivo in embryos.
To analyze the function of smash during the development of Drosophila,
we generated two different knockout alleles by transdeletion, one
representing a null allele and the other a C-terminal truncation
affecting the part of the protein carrying the LIM domain and the PDZ
binding motif. We found that smash is not an essential gene, as
homozygous mutants for both alleles are viable and fertile. The
subcellular localization of polarity markers such as Baz were not
affected upon smash knockout. On the other hand, overexpression of Smash
using the UAS/Gal4 system and transgenes encoding for N-terminally
GFP-tagged versions of Smash caused lethality in embryonic and larval
stages. Rare eclosing escaper flies were decreased in body size.
Overexpression of Smash in epithelial cells resulted in reduction of the
apical surface area, indicating that Smash may function in apical
constriction, a process important for morphogenetic rearrangements in
epithelia. Overexpression of Smash during eye development caused a rough
eye phenotype and reduction of eye size. Upon ubiquitous overexpression
of Smash in embryos, many embryonic cuticles exhibited anterior and
dorsal holes.
Following up on these findings, we showed that the non-receptor tyrosine
kinase Src42A binds N-terminally GFP-tagged versions of Smash-PI in
vitro in S2 cells and is furthermore able to phosphorylate GFP-Smash-PI.
Endogenous Smash protein was found to be tyrosine phosphorylated in
vivo in embryos as well. Domain deletion versions of Src42A still showed
binding to Smash, indicating different binding mechanisims provided by
the fact that tyrosine phosphorylation of Smash was only abolished upon
deletion of the kinase domain.
A double mutant for Src64B, the second Src kinase encoded by the
Drosophila genome, and smash is lethal. However, embryonic cuticles did
not show defects and epithelial integrity appeared intact.

Last updated on 2019-25-07 at 18:51