Investigation of Nucleoporin Antibodies Amanda DiGuilio 1, Yifei Bao 2, Tommy White 1, Adriana Compagnoni 2 and Joseph S. Glavy 1 1 Department of Chemistry, Chemical Biology and Biomedical Engineering, 2 Department of Computer Science Schaefer School of Engineering and Science, Stevens Institute of Technology, Hoboken, NJ Introduction The Nuclear Pore Complex (NPC) is an arrangement of proteins embedded in the nuclear envelope of eukaryotic cells that controls the transport of macromolecules between the cell cytoplasm and the nucleus (Figure 1). Each NPC has an eight-fold symmetric structure about its central pore, where transport occurs. There is an average Conclusions Solubility results for Nup 107, Nup 93 and those targeted by mAB414 have been determined under different conditions of cell lysis. These results have been applied to IP an individual NPC subcomplex and may be applied to the subcomplexes of a synchronized set of cells. IP with α-Nup 107 has shown at least two lysis conditions sufficient for solubilizing some amount of 5 members of the Nup subcomplex. The IP with α-Nup 107 was negative for Nup 37, Seh1 and Sec13. It is yet to be determined if their absence is due to insufficient antibody tagging, disruption of their interaction with the subcomplex or lack of solubility. IP with α-Nup 43 has shown that Nup 43 is not separated from the subcomplex by the conditions of cell lysis. Nup 160 was solubilized by conditions 3 and 5 and successfully extracted from the cell lysate by targeting Nup 107 and Nup 43. However, it was obtained in greater amounts by targeting Nup 43. An enrichment of Nup 160 by targeting Nup 43 may imply a stronger interaction between these two Nups. Technogenesis Research Scholars Summer 2009 Methods Mammalian Cell Culture and Lysis: HeLa cells were cultivated and harvested by centrifugation. The pellets that contain the cells were then frozen and thawed, which serves to crack the membranes, in the presence of lysis buffers. These lysis buffers consist of different detergents of various concentrations. Western Blotting: Cell lysate was electrophoresed and the proteins transferred to nitrocellulose membranes, then probed with newly developed antibodies. A developing reagent induced a chemical luminescent response from the antibody tagged Nups that can be recorded on film. Immunoprecipitation (IP): A protein, in our case a Nup, was targeted by its specific antibody. Then protein A sepharose beads were added which bind the antibodies and can be separated by centrifugation. This method is useful because it enables a protein and its subcomplex to be isolated. Isolated proteins can then be identified by western blotting. Affinity Purification of Antibodies: Serum was collected from rabbits that were inoculated with Nup segments to induce an immune response. The antibodies were purified from the serum by binding to their antigen on nitrocellulose membrane and collected by acid elution. Results Solubility Analysis: The solubility of three Nups has been analyzed upon lysis under six different conditions, shown in Table 1 according to their detergent concentrations. Conditions 3, 4 and 5 were followed by IP with specific antibody for Nup 107 (so named for a molecular weight (MW) of 107 kDa). Affinity Purification: Nup 43 was affinity purified. Figure 4 is a western blot probing for Nup 43 after IP with the affinity purified α-Nup 43. The antibody was used at different concentrations to probe two lanes of the same IP. The newly IP with Affinity Purified α-Nup 43: Specific antibody against Nup 43 was used to precipitate Nup 43 and its binding partners. Figure 5 shows that Nup 75 was isolated by targeting Nup 43. Similar results were obtained for Nup 107, Nup 133 and Nup 160. This implies that these Nups retained their interactions with the subcomplex under lysis conditions. The hypothesis that the lower MW members of the Nup subcomplex are lost during lysis is proved untrue. This work will lead to further research on the Nuclear Pore Complex. Mutations that occur in individual Nups result in altered pore structures and, therefore, abnormal transport. These mutations are known to be associated with several diseases including: leukemia, premature aging disorders, heart disease and cirrhosis of the liver. Antibodies against these Nups can be envisioned as disease markers for diagnosis and prognosis of patients. Early detection of disease states increases the value of the antibodies we are presently developing. IP with α-Nup 107: Specific antibody against Nup 107 was used to precipitate Nup 107 and its binding partners. Figure 2 shows Nup 75 was precipitated by targeting Nup 107. Similar results were obtained for Nup 133 and Nup 160. Figure 3 is a western blot probed with α-Nup 43. Nup 43 remains in cell extract, not in the IP. The results for α-Nup 37, α-Sec13 and α-Seh1 were also negative while targeting Nup 107. References 1.Glavy JS, Krutchinsky AN, Cristea IM, Berke IC, Boehmer T, Blobel G, Chait BT. Cell-cycle-dependent phosphorylation of the nuclear pore Nup subcomplex. Proc Natl Acad Sci U S A Mar 6; 104(10): Schwartz, Thomas U. Modularity within the architecture of the Nuclear Pore Complex. Current Opinion in Structural Biology 2005, 15: Tran EJ, Wente SR. Dynamic nuclear pore complexes: life on the edge. Cell June 16; 125(6): Acknowledgements Dr. Joseph Glavy, Dr. Adriana Compagnoni, Tommy White, Yifei Bao, Stevens Institute of Technology and The Office of Academic Entrepreneurship for training, mentoring, support and funding. Figure 4: Western blot probed for Nup 43. After IP of the Nup subcomplex with affinity-purified α-Nup 43 proteins were separated on a 4-20% gradient gel and transferred to nitrocellulose. This was then probed with different dilutions of affinity- purified α-Nup 43 (1:100 and 1:500 ratios of α-Nup 43 to 2% BSA, as shown). Figure 5: Western blot probed for Nup 75. Proteins from the total (T), supernatant (S) and pellet (P) fractions of 3 different lysis conditions were separated on a 4-20% gradient gel. After IP of the Nup subcomplex with affinity-purified α- Nup 43 proteins were separated by electrophoresis and transferred to nitrocellulose for western blotting. of 2000 NPCs in the membrane of each cellular nucleus. The basic protein units that comprise the NPC are called nucleoporins (Nups). In each NPC these Nups are arranged into subcomplexes by protein-protein interactions. The Glavy Laboratory examines cells from the HeLa cell line, so named for cancer victim Henrietta Lacks from whom this immortal cell line was first cultured. These mammalian cells undergo open mitosis, which means the NPCs disassemble during cell division and are reassembled in the daughter cells. The NPC disassembles into subcomplexes whence they reassemble during telophase. Research in the Glavy Laboratory seeks to increase understanding of the assembly process by examining the relationship of individual Nups to their respective subcomplexes throughout mitosis. Because the protein-protein interactions responsible for the existence of subcomplexes and their solubilities are affected by the conditions of cell lysis, experiments were performed to optimize lysis conditions of the solubilization and isolation for different NPC components. purified antibody detected Nup 43 at a dilution of 1:100 but not at 1:500. Nup 43 is visualized at 45 kDa directly below the heavy chain in the 1:100 lane. Figure 2: Western blot probed for Nup 75 after IP with α-Nup 107 antibodies. Immunoprecipitated proteins of the Nup subcomplex were separated on a 4-20% gradient gel after cell lysis under conditions 3, 4 and 5shown in Table 1. Cell extract (C.E.) was probed for comparison. Figure 3: Western blot probed for Nup 43 with α-Nup 107 antibodies. Immunoprecipitated proteins of the Nup subcomplex were separated on a 4-20% gradient gel after cell lysis under conditions 3, 4 and 5shown in Table 1. Cell extract (C.E.) was probed for comparison. Nup 43 was not detected in the IP but does appear in the cell extract. Table 1: Data collected on Nup solubility by Amanda DiGuilio and Yifei Bao (Ph.D candidate from Computer Science Figure 1: The NPC embedded in the nuclear envelope (courtesy of Daniel Stoffler Scripps Research Institute). These are the lower MW members of the Nup subcomplex. Lysis conditions may disrupt these protein-protein interactions. Figure 3 shows the heavy and light chains of the α-Nup antibody which are known to compete with antibody tagged Nups. Lower MW binding partners may not be tagged sufficiently when competition exists.