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As yet, only interaction between Nup358 and
microtubules via dynein/BicD2 at G2 phase has been characterised. The
underlying mechanism is poorly understood. More importantly, attachment and
interaction microtubules to nuclear pores complexes is still not yet known. In
this study, we have obtained an insight into attachment mechanism since it is
observed that long microtubules with protein-like structure lie above nuclear
pores. We have suggested them to be motor protein i.e dynein. They should be
labelled with BicD2 antibody if further work will be done. Also, although size
of filaments shown on images matches the expected size, but we are not
certainly sure if they are microtubules. Bio-maker like tau should be used to
immune-stain filament since tau is microtubules associated protein in nucleus
and confirm its identify (Lu, Li, He, Bartlett &
Götz, 2014). Fortunately, we have identified some
possible methods to investigate association between nuclear pore complexes and
microtubules such as using Taxol and chemical fixer like paraformaldehyde to
stabilise microtubules at G2 phase of xenopus oocytes. A high concertation of
Taxol can result in stabilising microtubules by binding to Taxol binding site,
paclitaxel and can be observed under microscope (Amos & Löwe, 1999).

Thus, an increase in concentration of Taxol above 3.0×10^-3M to see whether
there is a larger network of microtubules. While paraformaldehyde fixation
before nuclei isolation is also a workable method, however, it disrupts the
morphology of nuclear pores at some degrees. Alternatively, glyoxal can be used
to chemically fix oocytes before dissection because it can cross-link with
protein with a high effectiveness and nuclear pores morphology can be preserved
better for immunostaining (Richter et al., 2017). From
the above results provided, there is no interactions and attachment are found
between microtubules and nuclear pore complexes via BicD2 and Nup358.

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Microtubules are observed to lie over nuclear pores and suggested scanning
electronic microscope is not working well to obtain images beyond images.  So, direct linkage could be seen with
transmission electron microscope couple with microinjection paramecium axonemal
tubulin (Geuens, 1989).


Interestingly, Clathrins present at the
entrance of nuclear pores on outer nuclear membrane with Taxol treatment.

Cathrin is a protein forms lattice-like coated vesicles and is recognised  in endocytosis to recycle receptors engaged
in signal transduction and nutrient uptake etc. It also operates some membrane
trafficking during interphase (Kirchhausen, Owen &
Harrison, 2014). While Taxol is anticancer drug that
stabilise microtubules by binding to them directly at a molecular level. This
leads to interphase microtubules bundling and suppression of microtubules
activity, hence, cell is mitotic arrested and under cell death by apoptosis (Wang et al., 2013). We believe clarthrin have a separate
function other than membrane trafficking. Cathrin is moonlighting protein like
Nup358 which has various functions (Kirchhausen, Owen &
Harrison, 2014). It is suggested clathrin plays a role in decrease
permeability of nuclear pores and block the binding domain of nucleoporins when taxol is added. Firstly, according
Figure 2x, there is gold-labelling spot found on clathrin-bineded pore. So,
clathrin might block the binding domain of nucleoporins. Secondly, since taxol can causes apoptosis
by blocking cells in G2 phase of the cell cycles (Wang et al., 2013). Cathrin
blocks the nuclear pores thereby information such as genetic material cannot
move into nuclear and no mitosis can be done. With stabilisation of
microtubules and decreasing permeability of nuclear pores, Taxol can induce
cell apoptosis. The underlying mechanism of how clathrin disrupt nuclear pore
complex permeability is barely studied yet. It is suggested
Phenylalanine-Glycine rich proteins and clathrin inhibitor Pitstop-2 contribute
to membrane permeability (Liashkovich et al., 2015). When
clathrin binds to nuclear pores, this activates clathrin inhibitor Pitstop-2 to
decrease permeability of nuclear pores. This prediction could be examined by
looking at expression of Pistop-2. Other anti-mitotic drugs should be put under
investigate to see whether clathrin is attached to nuclear pores. Attachment of
clathrin gives us an insight into anti-cancer drug development.


Previous studies showed that dynein and
Nup358 plays an important role in linking both microtubules to nuclear pores,
however, no mechanism have been suggested yet. Possibly kinesin-1 involves in
attachment by acting as antagonist of dynein. Kinesin-1 and dynein are both
motor proteins but they transport microtubules towards different terminal (Ellefson & McNally, 2009). They can work on its own or act together. We
suggested that if dynein really involves in attachment of microtubule and
nuclear pores complexes, gene of kinesin-1 will be silent or turned off and
express gene dynein motor protein only. Alternatively, they can compete with
each other for binding site. Studies show kinesin-1 and dynein both bind Nup358
on the same binding same when they interact with Nup358 directly (Cai, Singh, Aslanukov, Zhao & Ferreira, 2001).


Speaking of mechanism of how this attachment
activates and how to recruit suitable motor proteins, it is obvious that there
are other molecules other than Nup358 and BicD2 involve in this nuclear
pores-microtubules attachment. There are other nucleoporins in nuclear pore
complexes and dynein accessory factors like LIS1 and NudE which they can
enhance and dynein-microtubule interaction during dynein force generation (Reddy et al., 2016). Some cell cycle-dependent regulator like
CDKs or PLK1 are likely to involve in motor protein activation by regulating
phosphorylation (Mochida & Hunt, 2012).  Involvement of mentioned regulators could be
tested by gene knockdown and western blotted once attachment of microtubules
and nuclear pores is confirmed.     


On the other hand, KASH domain protein plays
an important role in anchorage and movement on outer nuclear membrane with
cytoskeleton systems (Wilhelmsen, Ketema, Truong
& Sonnenberg, 2006). Nesprins, one of the KASH domain protein
which is reported to affect position of nuclear and centrosomes in G2 phase by
removing nesprins, yet the impact of depleting dynein on microtubules and
nuclear membrane is much more extreme (Tanenbaum, Akhmanova &
Medema, 2011). Nesprins form complexes with SUN protein
when binding to nuclear pores. Results from immunefluorescence and
immune-electron microscopy revealed either depletion of Sun1 by RNA
interference or overexpression of dominant-negative sun1 demonstrate clusters
of nuclear pore complexes (Liu et al., 2007). This
suggests SUN1 is one of the important factors in determining nuclear pore
complexes distribution on outer membrane surface (Liu et al., 2007). In
addition, this implicates nesprins may also recruit microtubules motor protein
to nucleoporins on nuclear pores like how Nup358 recruits BicD2 and causes
attachment to oocur. Hence, KASH domain protein like nesprins contributes to
attachment of nuclear pores complexes and microtubules under certain situation
such G2 phase.


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