complex organism arises by cell division in which cells have different
functions. The spatial process that induces the differences in shape, structure
and function of these cells is called cell polarity. The establishment of cell
polarity is driven by cortically localized proteins which determine
functionally distinct domains. In most cellular processes proteins play an
important role and the polarization of cells is no exception. Earlier studies
have shown that several protein complexes and proteins can induce cell
polarization. For example, apical domain identity is induced by the protein
complex PAR-3-PAR-6-aPKC and Crumbs-SDT-PATJ, whereas the protein complex
LGL-SCRIB-DLG stimulates the basolateral identity. It is important to gain
knowledge on cell polarity and understanding this process because for instance
a loss in cell polarity could lead to a disturbance of shape, structure or
organization of cellular components. Furthermore, a lack of cell polarity is
associated with diseases like cystic firbosis, renal cystic diseases and is
correlated with early stages of cancer.
of the art
studies have indicated that cortical polarity regulators create polarity via
mutual exclusion. As little is know about how cortical polarity is involved in
cytoskeletal rearrangement and the functional specialization of membrane
domains it is still unclear how cortical polarity layers are devided in
specific domains. To better understand cell polarity it is necessary to gain
knowledge of the involved proteins and the molecular interactions between them.
Via Y2H and phenotypic profiling. 100 protein pairs were found of which the
polarity process resulted in the same phenotype which are likely to have a
useful in vivo interaction. Furthermore,
in this study the proteins PAR-6 and PAC-1 ARHGAP21 are studied in more detail and
it is demonstrated how the synergy between those proteins are essential for
radial polarization of the C. elegans embryo.
polarity interaction network of C.
elegans (CePIN) consitst out 296
proteins in which 439 interactions have been identified. 54 of the 439
interactiones were identified previously of which 19 have been studied in more
detail. Most interactions were detected by small(er) fragments, indicating an
interactions and proteins were collected by using baitclones which screened two
Gal4 AD libraries and a mixed-stage C.
elegans AD-cDNA library. For each interaction a minimal region of
interaction (MRI) was defined. The average length of the MRI’s was 60% of the
total length of the protein, averaging 408 amino acids. The accuracy of the
MRI’s was proven by comparing to previous studies (93% overlap) and a co
affinity purifcation experiment in which 10 out of 19 MRI interactions scored
quality of the CePIN is determined by
investegating whether the interacting proteins share other aspects which
express a functional association. Via phenotypic profiling by RNAi in nine
different strains it was examined if fluorescently tagged proteins were
involved in several polarity related processes. The results suggest
that the CePIN was significantly
enriched for an overlap in phenotype when compared to all non interacting
protein pairs. The protein pairs that overlap in phenotype are enriched for a
high Gene Ontology (GO) and are depleted of a low GO similarity score and for
presence in WormNet. Furthermore, several interactions were validated with
co-affinity purifications from embryonic-kidney-293 cells of humans and were
compared with multiple published C.
elegans mammalian protein-protein interaction trap (MAPPIT).
results were examined in different ways. It was investigated whether
interacting proteins with simular phenotypes were more likely to be earlier
examined in literature. 9% of the interactions showed to have overlap in phenotype
and were described in literature already, compared to 2% of the remaining
interactions. This suggests that it is possible to detect important
interactions via phenotypic profiling.
Finally, the individual
proteins that were studied in more detail are PAR-6 and PAC-1. It turned out
that an interaction between PAR-6 and PAC-1 is necessary for radial
polarization. To demonstrate the validity of the used approach to discover
functional important interactions, the interaction between the proteins PAC-1
and PAR-6 are studied in more detail. It seems that PAR-6 and PAC-1 do not
localize together in polarized cells, they are found at cell contacts when
polarity is already established. Additinally, the interaction between PAR-6 and
PAC-1 were tested via Y2H. It turned out that both proteins needed used a amino
acid domain around 100 basepairs to bind to eachother. This interaction was
confirmed by co-affinity purification from mammalian HEK293 cells. Furthermore,
when PAC-1 and PAR-6 were both expressed they co-localized which is distinct
from the localization parts when expressed seperately. These observations
suggest that there indeed is a physical interaction between PAR-6 and PAC-1. When
functional relevance of the interaction is studied, it was checked whether
PAC-1 can functionally substitue for a wild-type protein in the absence of
PAR-6. It turned out that other proteins aren’t able to replace PAR-6. Thus,
without the presence of one another there is no interaction between the
proteins and there is no radical polarization.
The results of the study indicate that CePIN consits out of 296 proteins and
439 interactions. When 93 randomly chosen protein pairs were retested on
reliability it turned out that different tests were indistinguishable but were
significantly higher than the control tests. These validations emphasise the
quality of the network. For each of the interactions of the network a MRI was
determined. The results indicate that small fragments of bait constructs
increase the detectability of interactions when compared to full length
proteins (359/439 interactions). This might be caused by smaller MRI’s (60% of
total amino acids) which are able to bind more easily than the complete
protein. When a random sample of interactions was retested only 48% of the
interactions was succesfully reproduced. This was due to false negatives but still
this is comparable to previously validation rates and own previous findings.
These interactions are not always well studied and might show promissing opportunities.
For example interactions that function in the endocytic pathway, neuronal
development and defects in yolk endocytosis. This indicates that CePIN is a good resource for future
studies which are trying to gain knowledge on the mechanism of cell polarity.