Investigation of Nucleoporin Antibodies
Amanda
1
1
DiGuilio ,
Yifei
2
Bao ,
1
White ,
Tommy
Adriana
1
Joseph S. Glavy
2
Compagnoni
and
Department of Chemistry, Chemical Biology and Biomedical Engineering, 2Department of Computer Science
Schaefer School of Engineering and Science, Stevens Institute of Technology, Hoboken, NJ 07030
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
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 Figure 1: The NPC embedded in the nuclear envelope (courtesy of Daniel Stoffler Scripps
cell line was first Research Institute).
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.
Technogenesis Research Scholars
Summer 2009
These are the lower MW members of the Nup107-160 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.
Figure 3: Western blot probed for Nup 43 with α-Nup
107 antibodies. Immunoprecipitated proteins of the
Nup 107-160 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.
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 Figure 4: Western blot probed for Nup 43. After IP of the Nup 107-160 subcomplex with
concentrations
to 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 affinityprobe two lanes of the purified α-Nup 43 (1:100 and 1:500 ratios of α-Nup 43 to 2% BSA, as shown).
same IP. The newly
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.
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 107-160 subcomplex
are lost during lysis is proved untrue.
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).
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 107-160 subcomplex with affinity-purified αNup 43 proteins were separated by electrophoresis and transferred to nitrocellulose for western blotting.
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 107-160 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.
Table 1: Data collected on Nup solubility by Amanda DiGuilio and Yifei Bao (Ph.D candidate from Computer Science
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.
Figure 2: Western blot probed for Nup 75 after IP with α-Nup
107 antibodies. Immunoprecipitated proteins of the Nup 107160 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.
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.
References
1. Glavy JS, Krutchinsky AN, Cristea IM, Berke IC, Boehmer T, Blobel G, Chait BT. Cell-cycle-dependent
phosphorylation of the nuclear pore Nup107-160 subcomplex. Proc Natl Acad Sci U S A. 2007 Mar 6;
104(10):3811-6.
2. Schwartz, Thomas U. Modularity within the architecture of the Nuclear Pore Complex. Current Opinion in
Structural Biology 2005, 15:221-226.
3. Tran EJ, Wente SR. Dynamic nuclear pore complexes: life on the edge. Cell. 2006 June 16; 125(6):1041-53.
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.