10 A niversary of IIMCB

Fig. 3: mTOR inhibition impairs microtubule regrowth after depolimerization with nocodazole and increases CLIP170 binding to microtubules. mTOR
inhibition impairs microtubule regrowth after depolimerization with nocodazole and increases CLIP170 binding to microtubules a) Rat-2 cells were
treated with nocodazole or nocodazole together with rapamycin for 1 hour. After incubation cells were washed and put in a fresh medium or a medium
with rapamycin, rescectively. Cells were fixed immediately after incubation or 5 minutes after washing and stained for α-tubulin. b) HeLa cells were
treated with nocodazole and rapamycin as described above. Cells were fixed 5 minutes after washing out nocodazole and stained for α-tubulin and
CLIP-170. Arrows indicate CLIP-170 staining localized on microtubules tips (author: Łukasz Świech).
mTOR regulated genes and proteins potentially involved in
morphological changes occurring to cells of the nervous
system during TSC development. We identified 20 genes,
knockdown of which resulted in the cell soma size return
to control level, involved in such cellular processes as: gene
expression, translation, cytoskeleton dynamics, cell signaling
and cellular membranes turnover.
In 2008 our group started another collaborative project
within the 7FP EU focusing on understanding mechanisms
underlying GSK3 kinase functions in neuronal plasticity
in physiology and Alzheimer’s disease. The major task of
the Laboratory of Molecular and Cellular Neurobiology is
to understand links between mTOR and GSK3 in neurons
during various neuronal plasticity situations and check for
disturbances of this interaction in AD animal models.
Collaborative projects
We also closely collaborated with the group of Dr.
Hoogenraad (Erasmus MC, Rotterdam, Netherlands) in order
to study the role of microtubule dynamics in dendritic spine
development. Our research focused mostly on +TIP protein
EB3. Together, we showed that growing microtubule plusends
decorated by EB3 penetrate into dendritic spines and
can modulate spine morphology. Inhibition of microtubule
dynamics, as well as knockdown of either EB3 modulates
spine shape by exerting an effect on the actin cytoskeleton.
Further investigations showed that indeed EB3 exerts its
effect through binding partner, p140CAP, a known regulator of
actin. Additionally, cortactin was identified as binding partner
for p140CAP, important for actin regulation involved in spine
stabilization by invading dynamic microtubules. Importance
of spine shape and number regulation by EB3 is supported
by the fact that inhibition of microtubule dynamics by
application of low doses of nocodazole, resulted in inhibition
of long term synaptic plasticity (LTP). These observation has
been recently published in Neuron (Jaworski et al., 2009). In
the nearest future we hope for further close collaboration
regarding role of mTOR in microtubule dynamics in frame of
HEALTH_PROT Center of Excellence EU grant.
Finally, due to our group expertise in neuronal
physiology and siRNA technology, we are involved in
several collaborations at the IIMCB (Prof. J. Kuźnicki; Dr. M.
Miączyńska; Dr. U. Wojda, grant # N30110932/3854) and at the
Ochota Campus (Prof. L. Kaczmarek; Dr. G. Wilczyński, grant
NN301314733; Dr. W. Kłopocka, grant # N303017933).
Our research plans for 2009 include:
• further research on genes identified in shRNA screens in
context of dendritogenesis and TSC development
• further investigation of the role of phosphorylation of
mRNA binding proteins: Staufen1, Staufen2 and hnRNPA
for their biological functions
• conducting the kick off proteomic screens for mTOR
interacting partners in neurons under pathological
(epilepsy) conditions
• investigate reciprocal regulations loops between mTOR and
GSK3 under physiological and AD mimicking conditions.
Laboratory of Molecular and Cellular Neurobiology 65