Nuclear positioning, which includes both migration and anchorage of the nucleus, is an important process in C. elegans development. The outer nuclear membrane (ONM) KASH proteins UNC-83 and ANC-1 are required for nuclear migration and anchorage, respectively. The inner nuclear membrane (INM) SUN protein UNC-84 is required for both nuclear migration and anchorage. The UNC-83 and ANC-1 KASH domains are thought to interact with the UNC-84 SUN domain in the perinuclear space. This allows force to be transferred from the cytoskeleton to the nucleoskeleton during nuclear positioning. KASH protein localization is dependent on SUN proteins, but SUN protein localization is not dependent on KASH proteins. Using a membrane-bound yeast two-hybrid system (Igor Stagljar, Dualsystems Biotech), we have shown that the KASH domain directly interacts with two separable domains of UNC-84, the SUN and linker domains. This interaction is required for nuclear migration and anchorage. Using a molecular genetics approach, we found that the UNC-84 SUN domain is required for KASH protein localization and function in vivo. We also demonstrated that point mutations in the KASH domain disrupt the function but not the localization of UNC-83. This suggests a stronger interaction, presumably to allow a large transfer of force during nuclear positioning, is required to move or anchor the nucleus than is required to simply localize the KASH proteins to the ONM (McGee, et al. Mol Biol Cell. 2006:1790-801). This KASH and SUN interaction appears to be conserved across eukaryotes. To identify residues in the linker domain that are required for interaction with the UNC-83 KASH domain, we are performing a mutational analysis of the UNC-84 linker domain in the membrane-bound two-hybrid system. We are also using this two-hybrid system in a screen to test whether there are unidentified binding partners with the SUN domain of UNC-84. In order to test the conservation of function between human and C. elegans KASH and SUN domains, we are testing whether the human domains in the C. elegans KASH and SUN proteins localize and are functional. We have found that a human KASH domain in UNC-83 is able to partially rescue UNC-83 function in migrating
hyp-7 nuclei and localize to the nuclear envelope. We have also found that this
hyp-7 rescue is dependent on the UNC-84 SUN domain. This suggests that the human KASH domain is able to interact with the UNC-84 SUN domain in vivo. Further experiments are testing the functional rescue of human SUN domains in UNC-84.