Dissertation committee:
Καθηγητής Σταύρος Ι. Χαμόδρακας Τμήμα Βιολογίας Πανεπιστήμιο Αθηνών (επιβλέπων), Ερευνητής Α Κώστας Ιατρού Ινστιτούτο Βιολογίας Ε.Κ.Ε.Φ.Ε. Δημόκριτος, Αν. Καθηγητής Σπύρος Ευθυμιόπουλος Τμήμα Βιολογίας Πανεπιστήμιο Αθηνών
Summary:
Members of the adaptor protein family CAV(CAP, ArgBP2, Vinecin), which is
characterized by thepresence of three SH3 domains at the C-terminal and a SoHo
domain at the N-terminal of its members are known to participate in various
signaling pathways that lead to cytoskeletal reorganization or growth hormone
responses. In this study, we present the identification of BmSH3, a new member
of the CAV family found in the domesticated silkmothBombyx mori. BmSH3 was
initially identified as a protein interacting with the orphan nuclear receptor
BmE75C, a known transcription factor found to be expressed in the epithelium of
the silkmoth ovarian follicles and known to repress the expression of the gene
encoding the egg specific protein (ESP) during the transition from
vitellogenesis to choriogenesis.
Three putative transcripts originating from a single bmsh3 gene have been
identified, BmSH3-A1, BmSH3-A2 and BmSH3-B. We studied the transcription
pattern of BmSH3 and deduced the presence of BmSH3 mRNA in all studied tissues.
In particular, for the three identified transcripts we found that BmSH3-A1 is
expressed in all studied tissues, BmSH3-A2 is expressed in the ovary and the
head, whereas transcripts of BmSH3-B were identified only in the ovary. The
expression of BmSH3-A1 and BmSH3-B transcripts occurred in all stages of
oogenesis.
To study BmSH3 at the protein level, we generated two antibodies, anti-BmSH3.1
that recognizes the linker area between the SoHo and the first SH3 domain and
BmSH3.2 that recognizes the SH3 domains. Using Western immunobloting, multiple
possible protein isoforms were detected in various silkworm tissues. For the
specific identification of BmSH3, we used mainly the anti-BmSH3.1 antibody,
based on its specificity for the follicles. The expression of BmSH3 was
verified also by Western immunoblotting of follicular tissue, where two
distinct isoforms were detected of approximately 75 and 85 kDa,which we propose
to be specific proteolytic cleavage products of the original BmSH3-A1 isoform,
that was identified intact in the sheath.
The subcellular distribution BmSH3 was investigated by transient expression of
EYFP-BmSH3-A1 chimeric proteins in silkmoth cultured cells and examination of
the subcellular distribution of BmSH3 in developing follicles.
Following the establishment of the cytoplasmic distribution of BmSH3-A1 by
immunofluorescence in transiently transfected cell lines, we examined the
localization in cell lines co-transfected with BmSH3-A1 and expression vectors
for BmE75A or BmE75C. Although the subcellular localization of BmSH3-A1
remained cytoplasmic, a clear change was established in the distribution of
BmE75C, which in addition to the nucleus, was also found in the cytoplasm and
more specifically in filopodia and lamellopodia formations.
The colocalization of BmSH3-A1 with BmE75C was verified by immunoprecipitation
assays. We confirmed the interaction of BmSH3-A1with BmE75C in insect cell
limes co-transfected with respective expression constructs. In the same assays,
after the co-transfection of the same insect cell lines with vectors expressing
BmSH3-A1 and BmE75A, we could observe the interaction of these two proteins as
well.
This observation is significant because it implies new functions for BmE75C and
BmE75A. Specifically, for the BmE75C isoform we speculate, based on its
subcellular distribution, that is also involved in cell movement via the
BmSH3-A1 interaction. Hence, BmSH3 could modify the known role of BmE75C and
BmE75A as transcription factors to other functions.
A cytoplasmic distribution of BmSH3 as represented by BmSH3-A1, was also found
to exist in developing follicles, both during vitellogenesis and choriogenesis.
Of particular interest was the investigation of the spatial distribution of
BmSH3-A1 during choriogenesis, which revealed a dynamic change in subcellular
distribution that progressed from a diffuse cytoplasmic occurance in the middle
sections of the cells of the follicular epithelium during early choriogenesis,
to a polarized distribution at the apical and basal sites of the follicular
epithelium during late choriogenesis. At the apical site of the follicular
cells of late choriogenic follicles, BmSH3 was observed at specific foci that
may reflect the emerging structure of the developing chorion. Because of this
distribution, we propose that BmSH3 is also involved in the mechanism of
chorion protein secretion during eggshell formation.
Apart from the cytoplasmic distribution of BmSH3 at the middle sections of the
follicular cells during early choriogenesis, BmSH3 was also faintly observed at
the apical site of newly formed cell-cell adhesions, which appear as thick
polygonal shapes during late choriogenesis. Based on such observations, we also
propose that BmSH3 plays a role in the loss of patency during the transition
from vitellogenesis to choriogenesis.
Keywords:
Silkworm, Oogenesis, Protein interactions, Cytoskeleton, Bombyx mori
