10 A niversary of IIMCB
10 A niversary of IIMCB
10 A niversary of IIMCB
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Fig. 3: mTOR inhibition impairs microtubule regrowth after depolimerization with nocodazole and increases CLIP170 binding to microtubules. mTOR<br />
inhibition impairs microtubule regrowth after depolimerization with nocodazole and increases CLIP170 binding to microtubules a) Rat-2 cells were<br />
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<br />
with rapamycin, rescectively. Cells were fixed immediately after incubation or 5 minutes after washing and stained for α-tubulin. b) HeLa cells were<br />
treated with nocodazole and rapamycin as described above. Cells were fixed 5 minutes after washing out nocodazole and stained for α-tubulin and<br />
CLIP-170. Arrows indicate CLIP-170 staining localized on microtubules tips (author: Łukasz Świech).<br />
mTOR regulated genes and proteins potentially involved in<br />
morphological changes occurring to cells <strong>of</strong> the nervous<br />
system during TSC development. We identified 20 genes,<br />
knockdown <strong>of</strong> which resulted in the cell soma size return<br />
to control level, involved in such cellular processes as: gene<br />
expression, translation, cytoskeleton dynamics, cell signaling<br />
and cellular membranes turnover.<br />
In 2008 our group started another collaborative project<br />
within the 7FP EU focusing on understanding mechanisms<br />
underlying GSK3 kinase functions in neuronal plasticity<br />
in physiology and Alzheimer’s disease. The major task <strong>of</strong><br />
the Laboratory <strong>of</strong> Molecular and Cellular Neurobiology is<br />
to understand links between mTOR and GSK3 in neurons<br />
during various neuronal plasticity situations and check for<br />
disturbances <strong>of</strong> this interaction in AD animal models.<br />
Collaborative projects<br />
We also closely collaborated with the group <strong>of</strong> Dr.<br />
Hoogenraad (Erasmus MC, Rotterdam, Netherlands) in order<br />
to study the role <strong>of</strong> microtubule dynamics in dendritic spine<br />
development. Our research focused mostly on +TIP protein<br />
EB3. Together, we showed that growing microtubule plusends<br />
decorated by EB3 penetrate into dendritic spines and<br />
can modulate spine morphology. Inhibition <strong>of</strong> microtubule<br />
dynamics, as well as knockdown <strong>of</strong> either EB3 modulates<br />
spine shape by exerting an effect on the actin cytoskeleton.<br />
Further investigations showed that indeed EB3 exerts its<br />
effect through binding partner, p140CAP, a known regulator <strong>of</strong><br />
actin. Additionally, cortactin was identified as binding partner<br />
for p140CAP, important for actin regulation involved in spine<br />
stabilization by invading dynamic microtubules. Importance<br />
<strong>of</strong> spine shape and number regulation by EB3 is supported<br />
by the fact that inhibition <strong>of</strong> microtubule dynamics by<br />
application <strong>of</strong> low doses <strong>of</strong> nocodazole, resulted in inhibition<br />
<strong>of</strong> long term synaptic plasticity (LTP). These observation has<br />
been recently published in Neuron (Jaworski et al., 2009). In<br />
the nearest future we hope for further close collaboration<br />
regarding role <strong>of</strong> mTOR in microtubule dynamics in frame <strong>of</strong><br />
HEALTH_PROT Center <strong>of</strong> Excellence EU grant.<br />
Finally, due to our group expertise in neuronal<br />
physiology and siRNA technology, we are involved in<br />
several collaborations at the <strong>IIMCB</strong> (Pr<strong>of</strong>. J. Kuźnicki; Dr. M.<br />
Miączyńska; Dr. U. Wojda, grant # N301<strong>10</strong>932/3854) and at the<br />
Ochota Campus (Pr<strong>of</strong>. L. Kaczmarek; Dr. G. Wilczyński, grant<br />
NN301314733; Dr. W. Kłopocka, grant # N303017933).<br />
Our research plans for 2009 include:<br />
• further research on genes identified in shRNA screens in<br />
context <strong>of</strong> dendritogenesis and TSC development<br />
• further investigation <strong>of</strong> the role <strong>of</strong> phosphorylation <strong>of</strong><br />
mRNA binding proteins: Staufen1, Staufen2 and hnRNPA<br />
for their biological functions<br />
• conducting the kick <strong>of</strong>f proteomic screens for mTOR<br />
interacting partners in neurons under pathological<br />
(epilepsy) conditions<br />
• investigate reciprocal regulations loops between mTOR and<br />
GSK3 under physiological and AD mimicking conditions.<br />
Laboratory <strong>of</strong> Molecular and Cellular Neurobiology 65