טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGruber Rita
SubjectImportin Beta Regulates the Seeding of Chromatin for Nuclear
Pore Assembly
DepartmentDepartment of Biology
Supervisor Professor Amnon Harel
Full Thesis textFull thesis text - English Version


Abstract

The nuclear envelope of higher eukaryotic cells re-forms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the “seeding” of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. In this study we use the in vitro nuclear reconstitution assay, which is based on Xenopus egg extracts. This nuclear reconstitution system allows us to manipulate its components in vitro, to dissect the assembly process, reveal the critical interactions between subunits and make progress towards a step-by-step mechanism. Combining this functional assay with classical biochemical methods for the purification of Nup107-160 complexe, provide us a tool to study the early stages of NPC biogenesis. Here, we show that the initial step of chromatin seeding is negatively regulated by importin b. Our chromatographic approach enabled us to separate two distinct sub-populations of the Nup107-160 complex from Xenopus egg cytosol. Only the sub-population which is pre-associated with ELYS is able to seed chromatin for NPC assembly. The inhibition by importin b occurs in cytosol, by the formation of a joint high molecular weight complex, containing importin b, ELYS and the Nup107-160 complex. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin b is only partially reversed by RanGTP. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin b and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.