David Fortuna

BIOL-1615
BIOL 1615 Research Paper Summary

The NPC (Nuclear Pore Complex) is responsible for transport between the inside
of a cells nucleus and the surrounding cytoplasm. It shows much variation between
different organisms, and, for many of them, remains largely unmapped. Understanding
this structure could help us understand how various species differentiated from one
another throughout history.
Over the past few years, scientists have identified the structure of several
proteins that typically compose the NPC (Nuclear Pore Complex). However, similar
proteins do not seem to directly relate to similar NPC structures. Finding the reasons
for the differences and mapping the NPCs of various species could help further the
scientific understanding of the divergence of life, which was the researchers purpose
for this study (The Introduction).
Obado, Brillantes, Uryu, Zhang, Ketaren, Chait, Field, and Rout set out to
identify key components of the NPC (Nuclear Pore Complex) of a single-celled
organism, called trypanosomes, map the structure and complexity of the NPC of
differing organisms, and, [...] propose a model of stepwise acquisition of
nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of
macromolecular complexes provides fuller understanding of evolutionary processes.
(pg. 1) In short, the researchers suggest that the NPC divergence can be explained by
the concept that, through small mutations over millions of generations, the NPCs
became ever more complicated, one step at a time, and that this would be evident in
closely-related organisms.

Obado and company gathered their information on trypanosomes (a singlecelled, parasitic organism) NPC (Nuclear Pore Complex), by marking NPC proteins with
nucleoporins and mapping them with a state-of-the-art computer program. They
experimented using T. Brucei (trypanosomes) cells grown in a culture of SDM-79 (The
Materials). Their procedures included In Situ Genomic Tagging, using pMOTag4G
vectors to tag some of the proteins of trypanosomes NPC, Affinity Isolation, where the
researchers freeze the cells in liquid nitrogen, pulverize them, clean them, tag them with
magnetic beads, extract the tagged particles from the pulverized remains, and further
clean them several more times, Mass Spectrometry, to analyze the tagged proteins, a
computer program that generates a 3-dimensional protein structure, another form of
tagging, called Immunogold Tagging, which includes staining and freezing the proteins,
and viewed the nuclear envelope sections with an electron microscope in a process
called Immunoelectron Microscopy. (The Methods)
After repeating the procedures many times, the researchers mapped the
complete NPC (Nuclear Pore Complex) of the trypanosomes (a single-celled parasitic
organism), and compared it to the relatively well-known NPCs of yeast and humans.
They noticed a common protein ring found in the NPC of most, if not all other Eukaryotic
cells. This discovery supports their claims of a relation between all Eukaryotic
organisms.
While the NPC of trypanosomes showed some similarity to that of humans and
yeast, some new, unique proteins were discovered, still showing a distinct difference.
Thus, while connections between TbNup65 [, a commonly known protein among most
NPCs] and the NPC appear largely conserved [, meaning they are largely shared
among most Eukaryotes], the mechanism anchoring the NPC to the pore membrane
appears to be distinct, and the moieties used to anchor the NPC to the pore membrane
(TM domains, ALPS motifs) are interchangeable. (pg. 9)

While most of the results were predicted by the researchers, it was not expected
that the NPC of trypanosomes would be nearly symmetrical, which seemed very odd.
The information gathered from their experiments supported their stepwise acquisition
model. The data reported here provide the first comprehensive survey of the
architecture of the NPC from a highly divergent organism, providing key insights into
evolutionary origins of function and mechanism at the nuclear envelope. (pg. 13) It
appears their model for the divergence of species seems to be accurate, given the level
of conservation most Eukaryotes share within their NPCs (The Results).
Though ground-breaking, their study was limited by 2 factors. First of all, further
research into the NPC composition of various species is needed to confirm the accuracy
of their model of divergence. Secondly, the researchers assumed that, because their
treatment and modifications of the cells did not affect trypanosomes survivability, their
treatment did not alter their natural NPC structure or composition in any way. This
experiment should be replicated, but feature different species, as well as different
treatments for the organisms (The Discussion).
.

Works Cited:

Samson O. Obado, Marc Brillantes, Kunihiro Uryu, Wenzhu Zhang, Natalia E. Ketaren,
Brian T. Chait, Mark C. Field, Michael P. Rout. Interactome Mapping Reveals the
Evolutionary History of the Nuclear Pore
Complex. 2016. 30 pages. Print.