Introduction
mTORC2, was known to modulate actin polymerization and cell morphology (Jacinto et al., 2004). In contrast to mTORC1, little is known about mTORC2 in the regard of regulation and signaling pathway. To date, the well defined downstream molecule of mTORC2 is Akt, while the other plausible kinase is belonged to the AGC kinase family with conserved turn motif. (Jacinto and Lorberg, 2008; Hoeffer and Klann, 2010).
mTORC2 phosphorylates PKC on Ser 657 is essential for facilitation at synapses. PKC Apl II of Aplysia as well as PKCε of vertebrates
perform regulating effects on neurotransmitter release via
phosphorylating SNAP-25 to modify the 5-HT induced reversal of
synaptic depression (Facchinetti et al., 2008; Jacinto and Lorberg, 2008).
In addition, rictor-null mice showed reduced dopamine content and
elevated norepinephrine expression. This could be influenced by lowering inhibition effect of Akt on the transcription of norepinephrine transporter This finding indicated that mTORC2 activated Akt modulated
monoamine homeostasis in brain (Siuta et al., 2010). Serum- and
glucocorticoid-inducible-kinase 1 (SGK1) is another target of mTORC2.
SGK1 transcription is regulated by cellular isotones and osmotic
(Rozansky et al., 2002). Hydration affects cellular volume to determine neuronal function. Dehydration would alter the SGK1 expression, which contributed to increase the activity of various ion channels and enzyme activity (Lang et al., 2006; Lang et al., 2010). It is also suggested that
SGK1 plays important role in memory formation though mediated memory facilitation and neuronal plasticity as well as maintained neuronal membrane potential (Lang et al., 2010). Recent studies also found that mTORC2 played a critical role in controlling dendritic spatial arrangement. In the PNS of Drosophila, the same types of class IV da dendrities would stop growing when their terminal encountered each other so that two neurons never overlapped or occupied the other’s receive field. This is called a tilling phenomenon which is controlled by the NDR family kinase Tricornered (Trc). The homozygous mutation of components of mTORC2, sin1, and rictor in da neurons could lead defects in dendritic tilling separately, whereas heterozygous mutation of the two genes caused no differences in tilling (Koike-Kumagai et al., 2009). However, as combining the mutation of Trc with rictor/sin1, significant defects of tiling would be shown. Similarly,
co-immunoprecipitation results demonstrated that human Trc homologue NDR1is associated with sin1/rictor, rather than raptor in mammalian Hela cells. This cooperation might function in fine-tuning actin cytoskeletal organization (Hergovich et al., 2006).
To date, except for abovementioned roles of mTORC2, rare
researches have provided forward insights in the effect of mTORC2 on neuronal cells (Koike-Kumagai et al., 2009). Hence, more detail
involving mTORC2 function and structure such as novel binding partner(s) is needed to be explored in order to move these researches further. Recently, it was found that mTORC2 could attach to ribosomes, which may regulate in apopotosis-related cascades (Zinzalla et al., 2011).
Currently, by utilizing rictor wild type vs. knockout mouse embryonic fibroblast (MEF) cells, and performing rictor immunoprecipitation, we discovered an associated protein via MS/SPEC analysis. It was
temporarily named RICAP (rictor associated protein). Bioinformatics analysis indicates that RICAP contains a RNA motif. As pathway regarding mTORC2 remains largely unclear, this finding makes us to address two issues-to investigate the relationship between RICAP and rictor/mTORC2 as well as to elucidate the effect of RICAP/mTORC2 on neuronal function. Hope this study will open an avenue to a novel field in signal transduction of mTORC 2 as well as in neuroscience.
Material and methods
Refer to Chapter 2 “General Materials and Methods”.
Results
To verify the association between RICAP and rictor
Recombinant FLAG tagged RICAP plasmids were co-transfected with HA-tagged rictor into HEK293T cells. As showed in Fig 13, FLAG-RICAP is capable to bind to HA-rictor. FLAG-empty and FLAG-P53 were included as two negative control while FLAG-mSin1 was used to be a positive control. This result further confirmed the association between rictor and RICAP in MS/SPEC analysis.
RICAP knockdown on primary cortex neuronal cells caused neurites disrupted and soma lysed gradually
Before clarifying the relationship of RICAP and rictor on neuronal cells, it is essential to estimate the respective effects of RICAP or rictor on neurons at first. So far, we have assessed the preliminary responses of neuronal cells under long-term infection of RICAP. Neuronal cells were isolated from the cortex of p2 mice and infected with pLKO.1- puro- luciferase/RICAP on day 5. As seen in Fig. 14 A, C, RICAP deficiency could cause cell death gradually but not immediately. Cellular phenotypes
with RICAP knockdown were normal at the beginning 3 to 5 days but soma/neurites destroyed became obviously with time after infected for 6 to 12 days. Correspondently, the RICAP amounts were declined and tend to be eliminated from day 4 to day 10 (Fig.14 B). This phenomenon indicated that RICAP should be correlated with the maintenances of neuronal soma/neurites and its importance might be crucial in a crucial timing which relied on more experiments to be clarified.
Discussion
The association between recombinant RICAP and recombinant rictor further supports the result shown in the mass-spec data done in MEF.
Since less is known in the regards of regulators of mTORC2, the finding also can provide as a platform to uncover the factor which can alter the association between these two. In addition, cortical neurons with
deficency of RICAP would result in death denoting the importance of RICAP in neuronal growth.
By mediating LTP (long-term potentiation) and LTM (long-term memory) to establish the process of memory consolidation, actin polymerization is invloved in and proposed to be a critical point
(Wullschleger et al., 2006; Guertin and Sabatini, 2007). As mentioned in the introduction section, mTORC2 was known to control actin
organization. This highlights its importance in determing synaptic mophology via re-organizing local neurotransmitter receptors, such as AMPA receptors. Rictor also playes critical roles from embryogenesis to adult, with highly expression in the brain (Guertin et al., 2006; Shiota et al., 2006). Rictor deficency might cause smaller brains, affect
morphology and function of Purkinje cells, as well as increase
monoamine neurotransmitter to lead schizophrenia-like behavior (Siuta et al., 2010; Carson et al., 2013; Thomanetz et al., 2013). Recent reaserches indicated that activating mTORC2 by A-443654 enabled to convert E-LTP (early-form LTP) to L-LTP (late-lasting LTP) through altering synaptic strength by promoting actin polymerization (Huang et al., 2013).
Although evidences demonstrated that mTORC2/Rictor contributed in
structural maintainance in CNS, the morphological alteration in terms of molecular mechanisms in Rictor knockdown was not clear, especially in our stem cell-derived neurons. It is plausible that some functions of mTORC2 in different region of brain or some neuronal types. Therefore, we will take more emphasis on exploring the influence of mTORC2 on primary cortical neurons or mESC-derived glutamatergic neurons in a long-term manner to observe whether eliminating endogenous mTORC2 components will interfere cytoskeletal organization and affect neuronal function.
Furthermore, different types of actin are also important in trafficking modulation of ribosomes, RNA binding proteins, and relative RNA to destination for local expression (Olson and Nordheim, 2010). Hence, to clarify whether Rictor engages in dynamic export of
ribosomes/RNP/RNA via binding or interplaying with RICAP remains to be further investigated.
Fig. 13.
Fig 13. RICAP exhibited as an associated partner of rictor obviously in overexpressed model. Recombinant FLAG tagged empty, RICAP, P53, or mSin1was co-transfected with HA-rictor into HEK293T cells.
Cell lysates were performed FLAG IP, run onto SDS-PAGE, and subjected to Western blot analysis. The PVDF membrane was blotted with anti-HA antibody on the top panel and anti-FLAG antibody at the bottom panel respectively. Lane 1~4 were from total lysate while lane 6~9 were from IP. The blot is representative of one experiment repeated four times.
A
B
C
Fig. 14.
Fig. 14. Neurites gradually loss in primary cortex neuronal cells infected with RICAP RNAi. A. After viral infection of
Luciferase-/RICAP-RNAi for 3 days or 12 days, images were taken to compare the morphological change. Arrowheads indicated the spot-like disrupted neurites. Scale bar, 40 μm. B. Cell lysates after infection for 4 days and 10 days were collected to examine the knockdown efficiency.
The lysates were loaded onto SDS-PAGE and followed by Western blot analysis using anti-RICAP as first antibody. Actin was used as a loading control. C. Neuronal cells infected with either luciferase or RICAP RNAi were cultivated for 12 days. Cells were fixed and performed ICC to
further compare the morphological change. The upper pannel was stained with βIII-tubulin (green). Nucleus were stained with DAPI (blue). Scale bar, 40 μm. The blot is representative of one experiment repeated two times.