Introduction stages of cancer. State of the art

Introduction

 

A
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. 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.   

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State
of the art

 

Previous
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 this process 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.

 

Recent
findings

 

The
polarity interaction network of C.
elegans (CePIN) consitst out 296
proteins in which 439 interactions have been identified. These results were
collected by using baitclones which screened two Gal4 AD libraries and a
mixed-stage C. elegans AD-cDNA library
which re tested all the identified interactions. 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
positive. 

 

The
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. Defects in early embryonic
polarity were examined. By expressing mCherry::PAR-6, the anterior was examined, the
excretory canal was examined by expressing VHA-5::GFP. Furthermore, the process
of yolk protein uptake by oocytes, which is induced by par-3, par-6,
pkc-3 and cdc-42 genes, and strain expressing the yolk-protein
fusion by VIT-2::GFP was investigated. 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. It turned out that when
only protein pairs that interact were analysed, the pairs already had increased
enriched high GO similarity scores and that no further enrichment was caused by
the overlap in phenotype. However, a significant reduction of low GO similarity
pairs was noticed. So, a phenotypical overlap with low GO simularity correlates
negatively and positive with a high GO similarity. This suggests that it is
possible to capture important interactions to cell polarity via phenotypic
profiling and may lead to understanding the individual proteins that are not
know to act in the common process. 

 

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.

Discussion

 

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.

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