The integrity of the genome is the most important biomedical consideration. It is therefore essential to identify and to characterize how this structure is formed, protected, and regulated. Each human cell contains a genomic fortress, the nucleus, perforated with complex entry portals, the nuclear pores. At every cell division, after th genome is duplicated, the cell must take down the fortress and assemble a large mitotic spindle for genome separation. Following, the cell must once again build the nucleus, enclosing the genomes in nuclear membranes with nuclear pores. Mitosis is thus characterized by the construction of three massive structures: the spindle, the nuclear membranes, and the nuclear pores. Interference with any step can be lethal. Thus, understanding these mechanisms is essential.
In AIM l, we address the mechanism of assembly of the nuclear pore, building on our extensive expertise and tools. Because the pore is massive (120 megadaltons), consists of 30 nucleoporins, and requires the unique creation of a 1200 A structure within a long-lived membrane channel, our task is not trivial. It has and continues to involve devising innovative assays, novel drugs, and new intermediates.
In AIM l, we ask how the pore- initiating protein ELYS binds post-mitotic chromatin, how this sets in motion creation of an early intermediate on the inner nuclear membrane, how this then initiates an outer membrane counterpart, and potential fusases and/or mediators, such as Torsin A, mutated in human Torsin Dystonia, for creating the membrane channel.
In AIMs ll and lll, we turn to karyopherins, the known nuclear import and export receptors, and address their expanded role in global cellular regulation. It is now clear from our work and others that Importin Beta and its distant karyopherin relative, Transportin, have been co-opted by the cell at mitosis to act as global regulators of the three major mitotic assembly events: spindle assembly, nuclear membrane assembly, and nuclear pore assembly, all of which occur around chromatin at different points in mitosis. In this mechanism, Transportin and Importin beta mask factors required for assembly in areas of low RanGTP, but free these factors near mitotic chromatin to promote assembly. The spatial """"""""GPS""""""""-like aspect of the regulation derives from their """"""""dueling"""""""" counterpart, RanGTP, produced in active form only near chromatin. We will probe whether, as we expect, this control logically extends to representatives of the other 17 disparate karyopherins, both importins (AIM ll) and exportins (AIM III). If so, as we fully expect, the potential targets could be so numerous that much of the human cell would be under karyopherin regulation at mitosis. Expansion of this exciting regulatory paradigm would open new avenues of control for many well-known gene regulatory, cancer, and signaling proteins, adding a spatial aspect to their control previously unsuspected.
The structure and integrity of the genome are of immense biological and biomedical importance. In fact, the human genome of each of us is contained within a fortress, the nucleus, and can be accessed only through intricate gateways or nuclear pores. No signals can reach our genome, nor messages be sent from it except through these gateways. It is therefore essential to learn how this structure is formed, protected, and regulated. The receptors that carry cargo in and out through the gateways are also of great significance having, in essence, a high security clearance. We will focus on the gateways and receptor control in normal and dividing cells. Our work points toward new avenues of control for many cell signaling and cancer proteins, even adding a spatial aspect to their control that was previously unsuspected.
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