TY  - GEN
ID  - heidok9310
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/9310/
N2  - The nuclear envelope (NE) is a highly specialized membrane system that surrounds the interphase nucleus of eukaryotic cells. Nuclear pore complexes (NPCs) form gated channels through the NE and mediate nucleocytoplasmic transport. In metazoan cells, the NE breaks down and reforms during each cell cycle. These events are tightly coordinated in space and time with the formation of the mitotic spindle and the segregation of chromosomes to the two daughter cells. At the end of mitosis, new NPCs begin to assemble on chromatin and an intact NE reforms around the decondensing chromosomes. MEL-28/ELYS is a recently identified NE protein essential for nuclear integrity and function in many organisms. Genetic mutation or RNAi depletion of MEL-28 severely impair nuclear morphology and lead to loss of NPCs from the NE in a variety of cells and organisms. Our work and that of others shows that MEL-28 is critically involved in postmitotic NPC formation, but at the same time links between MEL-28 and other cellular processes are emerging. This thesis aims at thoroughly characterizing the role of MEL-28 in nuclear assembly. It addresses the function of MEL-28 in living cells and examines the contribution of MEL-28 to nuclear assembly in vitro. MEL-28 is an NPC/INM protein in interphase and partly localizes to kinetochores in mitosis. RNAi knockdown of MEL-28 in human cells results in loss of nucleoporins (nups) from the NE, but leaves the NE membranes intact, suggesting that it is specifically involved in NPC assembly. This phenotype is mirrored by employing MEL-28-immunodepleted Xenopus laevis egg extract for nuclear assembly in vitro, which gives rise to nuclei devoid of pores. MEL-28 acts in NPC formation by targeting nups to chromatin. It interacts with a subset of nups, the Nup107-160 complex, which is a central building block of the NPC. MEL-28 binds directly to chromatin through its AT hook and additional chromatin binding motifs in its C-terminus. My data show that MEL-28 anchors the forming NPC to chromatin. Addition of high concentrations of AT hook to a nuclear assembly reaction leads to inhibition of NPC assembly and recapitulates the MEL-28 depletion phenotype. Recombinant MEL-28 rescues the recruitment of the Nup107-160 complex to chromatin, indicating that the depletion phenotype in nuclear assembly can be specifically attributed to MEL-28. The function of MEL-28 is under control of the Ran GTPase. RanGTP enhances MEL-28 and nup binding to chromatin and thus triggers NPC formation. Moreover, MEL-28 chromatin binding is regulated during the cell cycle, possibly by phosphorylation. In conclusion, this study extends our current model of postmitotic NPC formation by demonstrating that targeting of nups to chromatin is mediated by and requires MEL-28. MEL-28 function is regulated spatially by the Ran GTPase and coordinated temporally with the cell cycle. The involvement of MEL-28 in NPC formation is its best characterized function to date, but it is likely that MEL-28 has additional roles in other cellular processes. In addition, this thesis contains an initial characterization of NET5, a conserved transmembrane protein of the INM. NET5 has a well defined domain topology and localizes to foci in the NE which are not identical to nuclear pores. RNAi knockdown of NET5 in human cells perturbs nuclear integrity and leads to distortion of the NE, suggesting that it has an essential role in nuclear organization. 
KW  - Kernpore 
KW  -  Kernporenkomplex 
KW  -  Kernhüllenuclear pore 
KW  -  nuclear pore complex 
KW  -  nuclear assembly 
KW  -  nuclear envelope
TI  - The role of MEL-28 in nuclear pore complex formation
Y1  - 2009///
A1  - Walczak, Rudolf
AV  - public
ER  -