Characterization of interphase NPC assembly

• Applicant: Dr. Mohamed Ismail Hamed
• Subject Area: Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 508310822

Nuclear pore complexes (NPCs) are the gateways of the nuclear envelope that control the transport of molecules such as proteins, RNAs and ribonucleoprotein complexes between the nucleus and cytoplasm. In animal cells, the NPCs assemble and integrate into the nuclear envelope by two defined modes: at mitotic exit and during interphase. Mitotic assembly is a well-studied pathway that initiates at the decondensed chromatin. With the help of the DNA-binding nucleoporin, ELYS, other nucleoporins and NPC subcomplexes are directed to and assembled on the chromatin. The newly reforming NPCs and nuclear envelope establish the transport function between the nucleus and cytoplasm in a few minutes. On the contrary, interphase NPC assembly is less studied. It is initiated by a different set of nucleoporins that scaffold the inner nuclear membrane rather than binding to the chromatin and last over a period of several hours.Preliminary data from the host laboratory suggest that different nucleoporins have variable levels of contribution to interphase NPC assembly. Depletion of some nucleoporins inhibits the assembly process while depletion of other nucleoporins has no effect on interphase NPC assembly. Interestingly, some nucleoporins are dispensable for interphase but not mitotic NPC assembly and vice versa. I will expand on this by defining new nucleoporins necessary for interphase NPC assembly using NPC assembly assays in Xenopus egg extracts. Xenopus eggs provide a very reliable and specific platform for assembling interphase NPCs with a high level of distinction from mitotic NPCs. Furthermore, I will develop a robust imaging approach which implements photoswitchable fluorescent proteins to differentiate mitotic assembled NPCs from interphase assembled ones in tissue culture cells. Using CRISPR/Cas9 and RNAi mediated downregulation of specific nucleoporins, it will be possible to create a map of the nucleoporins crucial for interphase NPC assembly and to study their function.Through the following proposal I will decipher and differentiate interphase NPC assembly, at a molecular level, from mitotic NPC assembly. Specific disease conditions are provoked by the defects in NPC assembly resulting from mutations in one or more of the nucleoporins. This proposal can help answer fundamental biological questions as to which nucleoporins are contributing to specific human pathologies. Understanding the underlying mechanics of NPC assembly is a major step toward a clear distinction of the two assembly pathways and the role of individual nucleoporins in the assembly processes.

• DFG Programme: WBP Position