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February 16, 2016
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Dynamic properties of APC-decorated microtubules in living cells
Journal Name, Vol.: page no, date
Introduction:
The adenomatous polyposis coli(APC) protein is a 312kDa proten that is the product of the
tumor suppressor gene APC. This gene is mutated in familial polyposis, a condition that
predispose the carriers to colorectal cancer.
The APC protein is also found in wide variety of tissues and in great amounts in brain
where it is is concentrated in growth cones of developing neurons. Most studies of this protein
explored its role in the Wnt signalling pathway where it controls beta catenin phosphorylation (in
activation) and export of beta catenin from the nucleus. Thus through its combined effect on
degradation and nuclear export, APC plays a major role in the control of nuclear accumulation of
β-catentin and, hence in the modulation of β-catentin mediated gene transcription.
Another function of APC is to bind to microtubules, promote their assembly and induce
bundling in vitro and in transfected cells. The APC binding site on microtubules is at the Cterminus. Mutation of APC alters both the microtubule binding domain and the beta catentin
binding domain. The β-catentin binding domain appears to be sufficient for the tumor suppressing
ability of APC. However, there are some pathogenic mutants retaining β-catentin binding sites but
lack the microtubule binding domain.
In epithelial cells APC is concentrated at the tip of the cells, however it is found in
uniformly significant lengths of microtubules that have been suggested to be important in active
cell migration. APC can form complexes with another MAP, the microtubule end binding protein
EB1. There is a considerable analogy between EB1-APC protein complexes and the complexes
formed by the microtubule binding proteins CLIP-170/115 and CLASPs. The important point
regarding the significance of APC binding to microtubules is to establish how the dynamics of
microtubules is influenced by the binding of APC.
Materials and methods:
Using a cDNA encoding full length human APC in the mammalian expression vector
,pMCVneo was used for the expression of a fusion protein consisting of N-terminally truncated
APC tagged at the N-terminus with enhanced green fluorescent protein(EGFP). They cultured the
COS-7 and PtK2 cells and then for microtubule depolymerization cells were plated on collagencoated coverslips and treated with nocodazole for fixation. APC was probed with the polyclonal
antibody against the C-terminus total α-tubulin and acetylated α-tubulin were probed with
monoclonal antibody. Then they performed western blotting on cells and studied them with
immunofluorescence microscopy.
Results:
The authors of this paper used a cDNA encoding full length human APC in the
mammalian expression vector pCMV neo. They labelled the N-terminus of the protein with
enhanced green fluorescent protein (EGFP). Also APC was probed with the polyclonal antibody
APC-C 20.
The authors of this paper found out that COS cells were transiently transfected with a
vector coding for full-length human APC, and expression of transgene was confirmed by western
blotting. Cells transfected with APC gene presented the protein in the expected areas. In many
APC expressing cells , APC localized to distinctively curled thick filamentous structures
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February 16, 2016
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throughout the cytoplasm. The sinuous appearance of microtubules in APC transfected cells is
quiet distinct from the typical organization of microtubules in untransfected cells in which they
radiate from the centromere to the periphery of the cell. In addition, cytoplasmic extensions
sometimes forming very thin processes were frequently observed in cells expressing APC.
They also saw that unlike their proximal part, the distal regions of microtubules
terminating in cytoplasmic extensions of cells expressing EGFP-APC were uniformly decorated
by APC and extensively interwoven.
In cells expressing EGFP-APC in high levels, microtubules were decorated by APC during
their entire length. And the authors observed that APC stabilize microtubules in vitro and to
protect microtubules against depolymerization by nocodazole in transfected PtK2 cells.
The distal ends of microtubules terminating in cytoplasmic extensions of transfected cells
were uniformly decorated by APC and formed tightly interwoven three dimensional arrays. In
addition , when expressed at high levels, APC induces the reorganization of the microtubule
network into sinuous filamentous structures that are likely to consist of bundled microtubules, as
suggested by earlier electron microscopy studies.
Reorganization of the microtubule network into sinuous bundles is not a property unique
to APC as it can be obtained after over expression of a number of other MAPs.
In particular formation of sinuous microtubule bundles seems to be a characteristic feature
of MAPs with an affinity to plus ends of microtubules, such as CLIP-170 or CLIP-115. Another
microtubule associated protein that induces the formation of whorls of microtubules is the
dynactin component P150. Interestingly EB1, a binding partner of APC, associates with P150.
Therefore all the proteins mentioned above may be part of functionally similar structures.
Discussion and critique:
The authors find out that human APC moved as points or clusters along microtubules in
COS cells with a translocation velocity of approximately 5micrometer/min. At the end they
conclude that this is indicative of microtubule longevity. Several mechanisms can be suggested to
explain the behaviour of APC decorated microtubules. The most likely explanation is that the
transient bending of microtubules is the consequence of compression forces acting on
microtubules reaching the edge of the cell while elongating. In other cases APC decorated
microtubules indicate in a way that they undergo length variation and thus that they retain the
known dynamic instability of microtubules.
I think that although this paper explains some aspects of APC gene and its effect on
microtubules it can not establish a relationship between these characteristics and the disease
conditions in brain and colon. This was what I expected while reading this article because all the
authors are from neuroscience and psychiatry department.