CENTROSOME ATTACHMENT TO THE NUCLEUS REQUIRES THE SUN-1 MEDIATED LOCALIZATION OF ZYG-12 TO THE OUTER MEMBRANE OF THE NUCLEAR ENVELOPE IN C. ELEGANS EMBRYOS
Open Access
- Author:
- Minn, Il
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 23, 2008
- Committee Members:
- Wendy Hanna Rose, Committee Chair/Co-Chair
Christian Lorenzo Malone, Committee Member
Avery August, Committee Member
Melissa Rolls, Committee Member
Richard Cyr, Committee Member - Keywords:
- Centrosome
SUN-1
ZYG-12
Attachment - Abstract:
- SUN-1 and ZYG-12 are nuclear envelope proteins required for the centrosomal attachment to the nucleus in C. elegans embryo. We have shown that two isoforms of the ZYG-12, which contain KASH and transmembrane domains, depend on SUN-1 (also called MTF-1), to localize to the nuclear envelope. To map the domains of ZYG-12 necessary for SUN-1 mediated localization, we expressed C-terminal fragments of ZYG-12, which contain KASH and transmembrane domains. These C-terminal fragments are sufficient for nuclear envelope localization in a SUN-1 dependent manner. We have investigated the membrane topologies of ZYG-12 and SUN-1. Both epitope accessibility and fluorescence protease protection assays using in vivo nuclei in combination with proteinase K protection assays using microsomes revealed that ZYG-12 and SUN-1 are type II membrane proteins of the outer and inner membrane of the nuclear envelope, respectively. ZYG-12 directly interacts with SUN-1. The interaction occurs between the KASH domain of ZYG-12 and the internal domain of SUN-1, distinct from SUN domain. FRAP analyses using in vivo nuclei and expression of ZYG-12 and SUN-1 in HeLa cells revealed that SUN-1 confines and is sufficient for the localization of ZYG-12 to ONM which is contiguous with ER. Putative ATPases ASNA-1 and ASNA-2 are required for the cytokinesis of early C. elegans embryo. Mammalian ASNA-1 is known to be responsible for ER-localization of tail-anchored (TA) proteins. Double RNAi of C. elegans asna-1 and asna-2 did not affect the localizaion of TA protein ZYG-12. Interestingly the RNAi pheno-copied syn-4 mutants and syn-4 RNAi. SYN-4 is one of the syntaxin proteins of C. elegans and is a TA protein and required for the cytokinesis of early embryo. SYN-4 localizes to cell membrane. Whether ASNA-1 and ASNA-2 is responsible for SYN-1 membrane localization is to be examined.