A Also, a lack of cell polarity

Acomplex organism arises by cell division in which a cell or a group of cellshave different purposes.

The spatial process that induces the divergence inshape, structure and function of these cells is called cell polarity. Theestablishment of cell polarity is motivated by cortically localized proteinswhich determine functionally distinct domains. In most cellular processesproteins play an important role and the polarization of cells is no exception.Earlier studies have shown that several protein complexes and proteins caninduce cell polarization. For example, apical domain identity is induced by theprotein complex PAR-3-PAR-6-aPKC and Crumbs-SDT-PATJ, whereas the proteincomplex LGL-SCRIB-DLG stimulates the basolateral identity. It is important togain knowledge on cell polarity and understanding this process because forinstance a loss in cell polarity could lead to a disturbance of shape,structure or organization of cellular components. Also, a lack of cell polarityis associated with diseases like cystic firbosis, renal cystic diseases and iscorrelated with early stages of cancer.

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    Stateof the art Previousstudies have indicated that cortical polarity regulators create polarity viamutual exclusion. As little is know about how cortical polarity is involved incytoskeletal rearrangement and the functional specialization of membranedomains it is still unclear how cortical polarity layers are devided inspecific domains. To better understand cell polarity it is necessary to gainknowledge of the involved proteins and the molecular interactions between them.Via Y2H and phenotypic profiling. 100 protein pairs were found of which thepolarity process resulted in the same phenotype which are likely to have auseful in vivo interaction. Furthermore,in this study the proteins PAR-6 and PAC-1 ARHGAP21 are studied in more detail andit is testified how the synergy between those proteins are essential for radialpolarization of the C.

elegans embryo. Recentfindings Thepolarity interaction network of C.elegans (CePIN) consitst out 296proteins in which 439 interactions have been identified. 54 of the 439interactiones were identified previously of which 19 have been studied in moredetail.

Most interactions were found by small(er) fragments, indicating anincreased detectability. Theinteractions and proteins were collected by using baitclones which checked twoGal4 AD libraries and a mixed-stage C.elegans AD-cDNA library.

For each interaction a minimal region ofinteraction (MRI) was defined. The average length of the MRI’s was 60% of thetotal length of the protein, averaging 408 amino acids. The accuracy of theMRI’s was proven by comparing to previous studies (93% overlap) and a coaffinity purifcation analysis in which 10 out of 19 MRI synergies scoredpositive.   Thequality of the CePIN is determined byinvestegating whether the interacting proteins share other aspects whichexpress a functional association.

Via phenotypic profiling by RNAi in ninedifferent strains it was examined if fluorescently tagged proteins wereinvolved in several polarity related processes. The results suggestthat the CePIN was significantlyenriched for an overlap in phenotype when compared to all non interactingprotein pairs. The protein pairs that overlap in phenotype are enriched for ahigh Gene Ontology (GO) and are weakened of a low GO similarity score and forpresence in WormNet.

Furthermore, several interactions were certified withco-affinity purifications from embryonic-kidney-293 cells of humans and werecompared with multiple disclosed C.elegans mammalian protein-protein interaction trap (MAPPIT).  The outcomeswere examined in different ways.

It was investigated whether coorperatingproteins with complementary phenotypes were more likely to be earlier examinedin literature. 9% of the interactions indicated to have overlay in appearanceand were described in literature already, compared to 2% of the remaininginteractions. This suggests that it is possible to detect importantinteractions via phenotypic profiling. At last, the individualproteins that were studied in more detail are PAR-6 and PAC-1. It turned outthat an interaction between PAR-6 and PAC-1 is necessary for radialpolarization. To demonstrate the validity of the used approach to discoverfunctional important interactions, the interaction between the proteins PAC-1and PAR-6 are studied in more detail. It seems that PAR-6 and PAC-1 do notlocalize together in polarized cells, they are found at cell contacts whenpolarity is already established. Additinally, the interaction between PAR-6 andPAC-1 were tested via Y2H.

It turned out that both proteins needed used a aminoacid domain around 100 basepairs to bind to eachother. This interaction wasconfirmed by co-affinity purification from mammalian HEK293 cells. Furthermore,when PAC-1 and PAR-6 were both expressed they co-localized which is distinctfrom the localization parts when expressed seperately. These observationssuggest that there indeed is a physical interaction between PAR-6 and PAC-1. Whenfunctional relevance of the interaction is studied, it was checked whetherPAC-1 can functionally substitue for a wild-type protein in the absence ofPAR-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 theproteins and there is no radical polarization. Discussion The results of the study indicatethat CePIN consits out of 296proteins and 439 interactions. When 93 randomly chosen protein pairs wereretested on reliability it turned out that different tests wereindistinguishable but were significantly higher than the control tests. Thesevalidations emphasise the quality of the network. For each of the interactions ofthe network a MRI was determined. The results indicate that small fragments ofbait constructs increase the detectability of interactions when compared tofull length proteins (359/439 interactions).

This might be caused by smallerMRI’s (60% of total amino acids) which are able to bind more easily than thecomplete protein. When a random sample of interactions was retested only 48% ofthe interactions was succesfully reproduced. This was due to false negativesbut still this is comparable to previously validation rates and own previousfindings. These interactions are not always well studied and might show promissingopportunities. 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 futurestudies which are trying to gain knowledge on the mechanism of cell polarity.


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