Investigation of Nucleoporin Antibodies Amanda DiGuilio1, Yifei Bao2, Tommy White1, Adriana Compagnoni2 and Joseph S. Glavy1 1Department of Chemistry, Chemical Biology and Biomedical Engineering, 2Department of Computer Science Schaefer School of - PowerPoint PPT Presentation

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Investigation of Nucleoporin Antibodies Amanda DiGuilio1, Yifei Bao2, Tommy White1, Adriana Compagnoni2 and Joseph S. Glavy1 1Department of Chemistry, Chemical Biology and Biomedical Engineering, 2Department of Computer Science Schaefer School of

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Title: Investigation of Nucleoporin Antibodies Amanda DiGuilio1, Yifei Bao2, Tommy White1, Adriana Compagnoni2 and Joseph S. Glavy1 1Department of Chemistry, Chemical Biology and Biomedical Engineering, 2Department of Computer Science Schaefer School of


1
Investigation of Nucleoporin AntibodiesAmanda
DiGuilio1, Yifei Bao2, Tommy White1, Adriana
Compagnoni2 and Joseph S. Glavy11Department of
Chemistry, Chemical Biology and Biomedical
Engineering, 2Department of Computer
ScienceSchaefer School of Engineering and
Science, Stevens Institute of Technology,
Hoboken, NJ 07030
Technogenesis Research Scholars Summer 2009
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
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 a-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
a-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.
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
Affinity Purification Nup 43 was affinity
purified. Figure 4 is a western blot probing for
Nup 43 after IP with the affinity purified a-Nup
43. The antibody was used at different
concentrations to probe two lanes of the same
IP. The newly
Figure 4 Western blot probed for Nup 43. After
IP of the Nup 107-160 subcomplex with
affinity-purified a-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 a-Nup
43 (1100 and 1500 ratios of a-Nup 43 to 2
BSA, as shown).
Figure 1 The NPC embedded in the nuclear
envelope (courtesy of Daniel Stoffler Scripps
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.
purified antibody detected Nup 43 at a dilution
of 1100 but not at 1500. Nup 43 is visualized
at 45 kDa directly below the heavy chain in the
1100 lane.
IP with Affinity Purified a-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.
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 a-Nup 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 a-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 a-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 a-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.
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).
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.
Table 1 Data collected on Nup solubility by
Amanda DiGuilio and Yifei Bao (Ph.D candidate
from Computer Science
IP with a-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 a-Nup 43.
Nup 43 remains in cell extract, not in the IP.
The results for a-Nup 37, a-Sec13 and a-Seh1
were also negative while targeting Nup 107.
  • References
  • Glavy JS, Krutchinsky AN, Cristea IM, Berke IC,
    Boehmer T, Blobel G, Chait BT. Cell-cycle-dependen
    t phosphorylation of the nuclear pore Nup107-160
    subcomplex. Proc Natl Acad Sci U S A. 2007 Mar 6
    104(10)3811-6.
  • Schwartz, Thomas U. Modularity within the
    architecture of the Nuclear Pore Complex. Current
    Opinion in Structural Biology 2005, 15221-226.
  • 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.

Figure 2 Western blot probed for Nup 75 after IP
with a-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.
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