nsistent with action at the level of the LET 23 receptor. This suggests that SEM 5 might regulate attenuation of LET 23 signalling through ubi quitination and subsequent endocytosis using two differ ent ubiquitin ligases, SLI 1 and CUL 4 DDB 1 CDT 2. Unfortunately, we have been order inhibitor as yet unable to directly assess LET 23 receptor localisation or endocyto sis during vulva development, immunostaining experi ments are inconsistent and current let 23,gfp transgenics are not fully functional. Tests of these mod els will require better reagents to investigate regulation of the LET 23 receptor. Ubiquitination and regulation of Notch signalling Receptor mediated endocytosis is important to termi nate or attenuate signalling, not only for EGFR but also for other signalling pathways, e. g. Notch.

During vulva development, LIN 12 signalling is required for establishment of the secondary cell fate and for the production of the anchor cell, which pro duces LIN 3. Interestingly, SEL 10, an F box and a WD40 containing protein that belongs to the CDC4 CUL 1 family of ubiquitin ligase, has been shown to play an important role in attenuation of LIN 12 signalling. SEL 10 was also shown to physically interact with LIN 12, implying that it regulates signal ling via ubiquitination of LIN 12. Herein we have not investigated the relationship between the CUL 4 DDB 1 CDT 2 ubiquitin ligase complex and LIN 12 signal ling. We did not observe any defects in anchor cell development, a process dependent on LIN 12, however, it has been previously shown for SEL 10 that a sensitised background is required to reveal its activity as an attenuator of LIN 12 signalling.

Therefore, we may not have detected a potential role for CDT 2 in attenuation of LIN 12 signalling. There is also an intimate link between LIN 12 and LET 23 signalling during vulva development. Indeed, high level of LET 23 signalling triggers expression of LIN 12 ligands in the primary P6. p cell. This activates LIN 12 signalling in the flanking secondary cells and ensures down regulation of LET 23 signalling in P5. p and P7. p cells. It is not impossible that the depletion of CDT 2 or CUL 4 impairs LIN 12 signalling and thereby prevents appropriate down regulation of LET 23 signalling in secondary cells, which would cause persistent expres sion of egl 17,cfp in secondary cells.

Localisation of CDT 2 The localisation of CDT 2 fused to GFP is predomi nantly nuclear in interphase and cytoplasmic during mitosis, which seems contrary with a function in endo cytosis. However, we Dacomitinib cannot exclude that a proportion of CDT 2,GFP below our limit of detection is cytoplas mic during interphase. Interestingly, early studies showed that human CDT 2 selleckbio can be detected in the cyto plasm, which would be consistent with a role in ubi quitination of cytoplasmic targets. Alternatively, the CUL 4 DDB 1 CDT 2 E3 ubiquitin ligase complex may be active in the cytoplasm only after nuclear breakdown. Further experiments will be required to establish w