Human Molecular Genetics, 2001, Vol. 10, No. 10 1061-1070
© 2001 Oxford University Press
DCX in PC12 cells: CREB-mediated transcription and neurite outgrowth
1Department of Molecular Genetics, The Weizmann Institute of Science, 76100 Rehovot, Israel and 2Institute of Evolution, University of Haifa, Haifa 31999, Israel
Received 11 January 2001 ; Revised and Accepted 13 March 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in doublecortin (DCX) result in X-linked lissencephaly in males. To explore the role of DCX in differentiation and signal transduction we overexpressed DCX in PC12 cells. Our results indicate that DCX stabilizes microtubules and inhibits neurite outgrowth in nerve growth factor-induced differentiation. However, neurite length is increased when differentiation is induced by epidermal growth factor and forskolin or by dibutyryl-cAMP. Furthermore, CREB-mediated transcription is downregulated, supporting the notion that cytoskeletal regulatory proteins can affect the transcriptional state of a cell. Using different constructs and mutations we reach the conclusion that microtubule stabilization is a key factor, but not the only one, in controlling neurite extension. Overexpression of a mutation found in a lissencephaly patient (S47R), completely blocks neurite outgrowth. We propose that these functions are important during normal and abnormal brain development.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The lissencephaly syndromes in humans involve abnormal cortical lamination and are medically categorized as neuronal migration defects (1,2). In type I, also known as ‘classical lissencephaly’ the cortex consists of four layers instead of the normal six, whereas in type II, also known as ‘cobblestone lissencephaly’ the cortex is unlayered (3). Two lissencephaly type I disease genes have been identified. Mutations in one allele of LIS1 (4,5) are sufficient to cause lissencephaly type I. The LIS1 protein regulates microtubule function and platelet-activating factor acetylhydrolase activity (reviewed in 6). A second lissencephaly type I gene mapped to the X chromosome (7,8), was cloned and termed doublecortin (DCX) (9,10). Mutations in the X-linked DCX gene result in lissencephaly in males or subcortical laminar heterotopia (SCLH), i.e. ‘double cortex’ in females (9,10).
DCX has been shown to be a microtubule-associated protein that stabilizes microtubules (11–13), the interaction with microtubules is via an evolutionarily conserved doublecortin (DC) domain (14,15). The domain is typically found in the N-terminal end of proteins and consists of two 80 residue repeats (later designated as pep1+2). Many missense mutations in DCX fall within the conserved regions. The expression and phosphorylation of DCX is regulated during brain development (11). In mice, DCX expression initiates at E12.5 and increases during development, but is absent in the adult brain (11). Developmental regulation was also observed in the human occipital cortex (12). In neuronal culture, DCX is only detected in young differentiating neurons concentrated in the distal regions of neurites (11). In light of these results, we have begun exploring the role of DCX in neuronal differentiation using overexpression in pheochromocytoma-derived cell line PC12 as a model system. In this line, cells grow neurite-like extensions in response to nerve growth factor (NGF) treatment. DCX localized along the neurites and stabilized microtubules in the presence of nocodazole. Interestingly, neurite extension following NGF induction in DCX-overexpressing cells was delayed. However, when differentiation was induced by epidermal growth factor (EGF) and forskolin or by dibutyryl-cAMP (dbcAMP), neurite length increased. Also, intracellular [Ca++] levels were significantly lower in DCX-expressing cells. CREB-mediated transcription and levels of phospho-CREB protein were reduced in DCX-transfected cells. These results suggest that overexpression of DCX can affect the transcriptional state of the cell. We postulate that these DCX-related functions are important during brain development.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DCX overexpression, cellular localization and microtubule stabilization
To study the effects of DCX overexpression, we stably transfected PC12 cells with FLAG-tagged DCX pcDNA or with pcDNA-empty vector as control. The PC12 cell line is derived from a rat adrenal medullary pheochromocytoma tumor that can be differentiated into neuronal derivatives by NGF (16). Fifty clones of FLAG-tagged DCX pcDNA or pcDNA empty vector were grown and analyzed. All experiments were performed with two control lines (C1 and C2) and two lines overexpressing DCX (DCX 4 and DCX 7). DCX expression was examined by northern and western blot analysis (Fig. 1). In control cells (Fig. 1, lanes C1 and C2) there was no DCX expression (Fig. 1). However, doublecortin-like kinase (DCLK) is expressed as demonstrated by both northern (Fig. 1A, lower panel) and western blots (data not shown). The two DCX lines express DCX as demonstrated by northern (Fig. 1A) and western blot analysis (Fig. 1B and C). The size of the FLAG-tagged-DCX (45 kDa) is in agreement with the expected molecular mass and is 5 kDa larger than the endogenous protein (Fig. 1B). The two lines differ in the level of DCX expressed: DCX 4 expresses relatively low levels of protein, whereas DCX 7 expresses higher levels (data not shown). All experiments were performed with two control lines (C1 and C2) that do not express DCX and two lines overexpressing DCX (DCX 4 and DCX 7) as demonstrated by analyzing RNA and protein levels. Next, we examined the intracellular localization of exogenous DCX. Anti-DCX antibodies stained the neurites and growth cones of transfected cells but not those of control cells (Fig. 2A). Although DCX immunostaining extends into the growth cones, the distal tips are labeled only by phaloidin that binds to actin (Fig. 2A). Since DCX is known to be a microtubule-associated protein that stabilizes microtubules (11–13), we examined the effect of microtubule disruption in DCX-overexpressing cells. Following NGF treatment, PC12 cells extended neurites that were easily observed after 24 h. The cells were then treated overnight with nocodazole, a known microtubule-disrupting agent. Growth cones of the NGF-induced cells disappeared completely after nocodazole treatment (Fig. 2C) whereas the growth cones of DCX-overexpressing cells were retained (Fig. 2B). Microtubule stabilization was also seen in cells that were not treated with NGF (data not shown).
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DCX overexpression protects from nocodazole-induced apoptosis
Nocodazole and other microtubule-disrupting agents are known to cause a specific cell-cycle arrest at G2/M followed by apoptosis (17). Hence, we examined the change in cell-cycle and the appearance of apoptotic cells following nocodazole treatment in DCX-overexpressing and control PC12 cells that were not treated with NGF (Fig. 3). The sub-G1 area, appearing to the left of the G1 peak (2n) of the DNA distribution, represents the cell population undergoing apoptosis (Fig. 3A and B). It was obvious that the percentage of apoptotic cells is markedly decreased in the DCX-overexpressing cells (Fig. 3C). While 28% of the control cells were apoptotic, less than half this percentage (7.3 or 11.4%) were undergoing apoptosis in the DCX-expressing lines. The typical blockage of cell cycle in the G2/M phase (4n) was observed in both control and DCX-expressing cells. However, an area corresponding to the G2/M fraction was reduced in the control cells (43%) compared to DCX-expressing cells (average 66%), most likely due to the higher portion of apoptotic cells. No difference was observed in the G1 phase. This result extends the nocodazole protective effect previously seen by morphology.
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DCX, differentiation and [Ca++]
DCX is expressed in young differentiating neurons and their levels of expression increase when the neurons reached their final destination (11,12). We therefore postulated that DCX has a role in neural differentiation. To test this hypothesis, we treated stable control and DCX-expressing clones with NGF and examined the cells after 24, 48 and 72 h. Even though all clones extended neurites following NGF treatment, the neurites in DCX-expressing cells were much shorter compared with control cells (Fig. 4A). These differences were visible at all time-points tested. Similar results were obtained in transiently transfected PC12 cells (measurements in Fig. 4B). Next, we wanted to check whether this effect was specific to NGF-induced differentiation. It has been shown that the synergistic effects of EGF and cAMP result in PC12 differentiation in a way that is indistinguishable from NGF-induced differentiation (18). This was not the case when we applied EGF and forskolin, an activator of adenyl cyclase, to the transfected PC12 lines. Our results demonstrate that neurite length (Fig. 4C), and number of neurites per cell (data not shown) were substantially increased in the DCX-expressing cells. Comparable results were obtained when differentiation was induced by the long-acting non-hydrolyzable cAMP analog (dbcAMP) (Fig. 4D). NGF is known to affect several signal transduction pathways in PC12 cells (reviewed in 19–21). NGF induction affects more pathways than either EGF/forskolin or dbcAMP. Our results suggest that DCX is inhibiting one of the pathways that are induced only by NGF, pointing to an interesting role of DCX and cytoskeletal changes on growth-factor signaling.
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Two major intracellular signals that regulate neuronal function are cAMP and [Ca++]. Therefore, we next examined intracellular [Ca++] involvement in the DCX-expressing cells. The levels of free intracellular [Ca++] were measured in the PC12 cells using Fura-2 [Ca++] indicators. As shown in Figure 5, DCX-expressing cells have substantially lower basal levels of intracellular [Ca++] than control cells (44.99 versus 94.23 nM). We did not examine cAMP levels. The differences in neurite length could be the result of multiple events, such as [Ca++]-mediated signal transduction pathway, microtubule assembly, rate of neurite elongation or and neurite stability (20,22–25). Furthermore, it has been demonstrated that changes in the microtubule-polymerization status also affect gene expression through different signaling pathways (26,27). Therefore, we decided to investigate whether there are differences in specific signal transduction pathways in the transfected cells.
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DCX and CREB-reporter-mediated transcription
One well studied target of both [Ca++] and cAMP is the transcription factor cAMP-responsive element binding protein (CREB) (28). Regulation of CREB has been shown to be crucial in many aspects of neuronal functioning (29,30), thus suggesting that it may be important to examine CREB function in our model system. A significant decrease in CREB-mediated luciferase activity was seen in DCX transient transfections (Fig. 6A). The luciferase activity was reduced to 22% of control values in cells treated with NGF, and to 35% without NGF treatment. A 3-fold decrease in luciferase activity was also observed in the stable DCX-expressing lines (Fig. 6B). The repression in CREB activity was overridden by co-transfection of DCX with protein kinase A (PKA) (Fig. 6C), that can directly phosphorylate CREB at Ser133 (31). This result is in agreement with the notion that CREB is typically activated by phosphorylation (32). Likewise, elevating cAMP by stimulating the PC12 cell lines with forskolin resulted in activation of CREB-mediated luciferase activity (Fig. 6D). Together, these results suggest that adenylate cyclase/PKA activation can function properly in the DCX-transfected cells.
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Endogenous CREB
To assess the endogenous phosphorylation status of CREB in the PC12 lines, we used an antibody that specifically recognizes the Ser133-phosphorylated form of CREB (31). Western blot analysis of nuclear-enriched preparations (Fig. 7A) showed reduced phospho-CREB immunoreactivity in the DCX-expressing cells (Fig. 7A), which remained at low levels following NGF treatment. In control cells the amount of phospho-CREB increased. The total amount of CREB was similar in the two cell lines (Fig. 7A). Phospho-CREB immunostaining emphasized this result (Fig. 7B and C); although phospho-CREB immunostaining was very strong in control cells (Fig. 7B), it was barely detectable in DCX-transfected cells (Fig. 7C). We tested the phosphorylation status and total levels of proteins that are known to mediate signaling pathways that affect CREB phosphorylation: ERK, AKT, MEK, p38 and JNK. To our disappointment we did not detect significant differences between control and DCX-overexpressing cells before addition of NGF and at different time points following NGF addition (15 min, 1, 3, 5 or 18 h).
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DCX structure and function
To map the region within DCX that is important for CREB down-regulation and for neurite extensions, we used the microtubule binding domain pep1+2 (amino acids 52–257) and two DCX mutations (D246X, S47R) (scheme and summary presented in Fig. 8C) (14). The S47R mutation affects a potential phosphorylation site and D246X is a truncation mutation at amino acid 246 that removes part of pep2 and the serine-rich C-terminal tail. We also tested the DCX homolog from the blind subterranean mole rat, Spalax judaei (2n = 60) (33) (GenBank accession no. AJ298280) which differs from the human protein in only four amino acids: substitutions Thr14
Ala, Thr289Ala, Thr293Ser and missing Val347. The Spalax homolog was used since this rodent underwent dramatic evolutionary changes in its brain cortex. Among the residues changes, two are potentially changes in phosphorylation. As mentioned earlier, a marked decrease in CREB-mediated luciferase activity was seen in transient transfections of DCX (Fig. 8A). Mutations found in patients S47R, D246X or naturally occurring substitutions in Spalax and pep1+2 did not result in a significant reduction of CREB-mediated luciferase activity (Fig. 8A). Since none of the DCX variants used affected CREB-mediated transcription, no obvious correlations between this activity and neurite length can be drawn.
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We then questioned whether we could map activity leading to the reduced length of NGF-induced neurite extensions. PC12 cells were co-transfected with the same constructs, induced to differentiate by NGF, and neurite length was measured after 72 h. As shown in Figure 8B, DCX overexpression inhibited neurite extensions. Spalax and D246X did not influence the length of the neurites. Pep1+2 further inhibited neurite extensions (Fig. 8A–C). The effect of introducing DCX harboring the S47R mutation was very striking as it completely blocked neurite formation. Neurite number per cell was appreciably reduced when expressing pep1+2, and completely abolished in the case of the S47R point mutation (data not shown). Cell radius was also reduced when either the pep1+2 or the D246X mutation was expressed (not shown).
To test whether endogenous CREB phosphorylation correlated with the luciferase reporter assay we co-transfected PC12 cells with either S47R + GFP (Fig. 8D and E) or Spalax + GFP (Fig. 8F and G) and stained with anti-phospho-CREB antibodies (Fig. 8D and F). Transfected cells (marked with GFP in Fig. 8E and G or by white arrows in Fig. 8D and F) did not exhibit reduced staining of phospho-CREB in comparison to untransfected cells (marked by yellow arrows in Fig. 8D and F), suggesting that CREB phosphorylation was not affected by these mutations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The characteristic laminar organization of the cerebral cortex is disrupted in lissencephaly patients. The process of attaining the final structure of the cerebral cortex involves multiple orchestrated events. These events include: specification of cortical tissue, neuronal proliferation and fate determination, migration of neurons from the proliferative zones to their final positions, elaboration of neuronal extensions and establishment and refinement of connections (reviewed in 34). DCX apparently has a role in migrating neurons in the developing brain (11,12,35). However, considering that DCX is mainly expressed in the cortical plate (11,12,36), it is plausible that it is involved in neuronal differentiation. Furthermore, it has been suggested that DCX is involved in signaling (9,10,14), though no specific pathways have been demonstrated. Our results indicate that overexpression of DCX affects growth factor-mediated signaling in PC12 cells. DCX reduced neurite outgrowth in NGF-treated cells but increased their growth in EGF/forskolin- and dbcAMP-treated cells. These findings point to interesting variability in the impact of DCX and cytoskeletal changes on growth factor signaling. Also, DCX overexpression dramatically reduced the basal level of free [Ca++] and CREB-mediated transcription, suggesting that cytoskeletal regulatory proteins can alter transcription.
DCX as a stabilizer of microtubules
Previous work demonstrated that DCX stabilizes microtubules (11–13). Here, we extended this work using neuronal cells expressing DCX. When treated with both NGF and nocodazole the DCX cells retained their growth cone in contrast to non-expressing cells. Microtubule-interfering agents are known to induce cell cycle arrest at the G2/M phase, thus initiating apoptosis (37–39). Indeed, treatment of the DCX-expressing and non-expressing PC12 cells with nocodazole resulted in a typical G2/M arrest in cell cycle. However, overexpression of DCX resulted in a significantly smaller portion of apoptotic cells, most likely due to increased microtubule stability.
DCX localization, neurite extension, [Ca++] and CREB
Our results demonstrate that the exogenous DCX in NGF-differentiated PC12 cells is localized to the neurites and growth cones. Neuritogenesis is a complex process that requires coordinated Trk A-dependent signaling cascades, rapid activation of cytoplasmic kinases and induction of neuronal specific genes (40–42). In addition, NGF influences growing axons at the growth cone level by differential regulation of actin and microtubule assembly (43,44). This process is mediated by actin-binding proteins and microtubule-associated proteins (45,46). We therefore hypothesized that DCX may be important in respect to neurite growth. Indeed, DCX affected neurite outgrowth even though the effect was dependent upon the differentiation-inducing agents used. In the case of NGF-induced differentiation, DCX overexpression inhibited neurite outgrowth. In contrast, when dbcAMP or EGF/forskolin were used, the presence of DCX augmented neurite extensions. The differences may be due to the fact that NGF activates multiple pathways, whereas dbcAMP or EGF/forskolin activates only a subset of pathways. NGF, when bound to its receptor(s), induces an increase in most identified secondary messengers such as cAMP, cGMP, [Ca++], phosphoinositides, arachidonic acid and glycosylphosphatidylinositol metabolites (reviewed in 47). DCX activity is likely to be inhibiting at least one of these NGF-induced pathways. Intracellular [Ca++] was significantly reduced in DCX-expressing cells. The transcription factor CREB is a target of both [Ca++] and cAMP (28). This transcription factor has been shown to be involved in many aspects of neuronal functioning (reviewed in 30), thus, we examined CREB function in our cell lines. CREB binds as a dimer to a conserved cAMP response element (CRE) found in the promoters of numerous eukaryotic genes (48). A critical event in CREB activation is phosphorylation of Ser133 (32), which induces an increase in CREB transactivation potential by allowing its recruitment to co-activators such as the CREB-binding protein (CBP) (49). CREB is phosphorylated by many different kinases: PKA, members of the [Ca++]/calmodulin-dependent kinase family and the extracellular signal-regulated kinase (ERK)-stimulated RSK and MSK kinases (reviewed in 28). These kinase families were reported to mediate the actions of both [Ca++] and growth factors on CREB phosphorylation. Our results revealed less CREB-mediated transcription using the luciferase reporter system. The addition of PKA or increased levels of cAMP alleviated this inhibition. We also showed that DCX-transfected cells exhibit lower levels of phospho-CREB but not of total CREB. [Ca++]-dependent CREB response has been suggested to play a central role in mediating neurotrophin actions in neurons (50–52). Interestingly, DCX and CREB expression are partially overlapping in the developing brain. CREB was found to be expressed in all layers of the developing neocortex from E10 to E14, and decreased thereafter (53), whereas expression of DCX initiates at E11.5 and increases later on (11,14). Therefore, we suggest that the involvement of DCX in reducing CREB-mediated transcription may be relevant to the processes of brain development.
Structure and function
Down-regulation of CREB-mediated transcription requires the presence of a full length DCX. Neurite outgrowth is more complicated, extends over a longer time, and is probably influenced by microtubule stabilization and other signaling events. DCX reduced neurite length while both the Spalax homolog and the D246X mutation showed no effect. These results imply that the Spalax homolog has a different activity to human DCX. Spalax is the blind subterranean mole rat, S.judaei (2n = 60) (33), belonging to the Spalax ehrenbergi superspecies in Israel. The differences in activity may be due to evolutionary adaptations of Spalax to life underground (reviewed in 54). Alternatively, since the Spalax homolog was cloned from an adult, it may have different activity from an embryonic form. Since it is not possible to breed this animal in captivity, we cannot test this hypothesis directly. The different activity of the Spalax homolog underscores the importance of the four different amino acids, and we postulate that the possible changes in phosphorylation may be of importance. D246X is missing both the serine-rich tail (again, a region that is phosphorylated) and part of pep2 that is required for microtubule binding. Pep1+2, which is sufficient to bind microtubules and stabilize them, reduced neurite outgrowth to a further extent than the normal DCX. This result suggests that merely affecting microtubule stabilization is enough to repress neurite extension, and that domains missing pep1+2 may modulate this activity. In our previous study (14), we noted the possibility that DCX mutated in amino acid 47 may result in increased stabilization of microtubules. Here we show that this mutation (S47R) completely blocks neurite outgrowth. All together, these results support the notion that dynamic instability of microtubules is very important in the process of growth cone exploration before extending the stable microtubules in the neurites (44,45,55).
In summary, our findings imply an exciting role for DCX-elevated expression in the cortical plate as a participant in the regulation of CREB-mediated transcription and in neurite extension progression. Based on our results with DCX mutations, it is likely that these normal functions are disrupted in lissencephaly patients, and are thus important in the formation of brain structure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture
PC12 cells were grown at 37°C with 7% CO2 and 95% air in DMEM nutrient medium (Gibco-BRL) supplemented with 10% fetal calf serum (FCS) and 10% horse serum, 4 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. For inducing differentiation, plates were coated with 0.5 mg/ml collagen type I from rat (Sigma), the serum was reduced to 1% FCS and 1% horse serum and 50 ng/ml NGF (Sigma) was added. Transfections were done using lipofectamine (Gibco-BRL) according to the manufacturer’s instructions.
Cloning of Spalax DCX and other plasmids
Total RNA and cDNA were prepared from brain tissue of two S.judaei (2n = 60). The animals used were adults captured in the field and kept for at least 3 months before use in the animal facility of the Institute of Evolution, University of Haifa. The oligonucleotides used for the RT–PCR cloning were designed according to the published sequence of human and mouse DCX (GenBank accession nos AF040254 and AB011678). 5'-sense oligo: 5'-ggttccaccaaaatATGg (ATG initiator) and a 3'-antisense oligo 5'-TCAcatggaatc(g/a)ccaag (TGA terminator codon). The PCR product was subcloned into pGEM-T easy vector (Promega) and two positive colonies for each individual were sequenced on both strands. All inserts (DCX, pep1+2, D246X and S47R) were cloned first into pECE (56) with FLAG epitope at the N-terminus and then were transferred into pcDNA3 vector (Invitrogen). Spalax was cloned directly into pcDNA3 vector.
Antibodies
Anti-
-tubulin (monoclonal, clone DMIA), anti-FLAG (monoclonal, clone M2) were purchased from Sigma. Anti-CREB and anti phospho-CREB on Ser133 (polyclonal) were purchased from New England BioLabs Anti-doublecortin antibody mouse polyclonal and rabbit polyclonal were produced against GST–DCX (57). Peroxidase-conjugated affinipure goat-anti mouse IgG (H+L); lissamine/rhodamine-conjugated affinipure goat anti-mouse IgG (H+L); Cy3-conjugated affinipure goat anti-rabbit IgG (H+L); FITC-conjugated affinipure goat anti-mouse or anti-rabbit IgG (H+L). Goat anti-rabbit alkaline-phosphatase (Santa-Cruz Biotechnology). Fluorescein-phalloidin at a final concentration of 50 µg/ml (Sigma).
Immunostaining and live-cell fluorescent microscopy
Cells were permeabilized and stained as described (58). For microtubule disruption, cells were treated with 5 µM nocodazole (Sigma) overnight or pretreated with 50 ng/ml NGF for one overnight and 5 µM nocodazole was added for the second overnight. For live-cell microscopy, PC12 cells were plated on coverslips and cotransfected with DCX-expressing constructs and GFP expression vector (Clontech) in a 1:1 ratio. Cells were visualized using a confocal microscope (BioRad). PC12 cells that were stably transfected with DCX-pcDNA or pcDNA empty vector were plated on glass coverslips coated with 0.5 mg/ml collagen type I from rat, and were subjected to different treatments: NGF 50 ng/ml, dbcAMP 0.1 mM, forskolin 25 µM, EGF 100 ng/ml (Sigma). Cells were visualized in PBS using an Olympus phase microscope (IX50 model).
Protein gel electrophoresis and immunoblots
Proteins were extracted in PBS–TDS buffer supplemented with protease and phosphatase inhibitors (PBS: 140 mM NaCl, 130 mM disodium hydrogen phosphate, 10 mM sodium dihydrogen phosphate; TDS: 1% Triton X100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate; inhibitors: 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM PMSF, 10 mM tetrasodium diphosphate decahydrate, 2 mM sodium orthorthovanadate and 50 mM NaF). For nuclear extracts, cells were washed and incubated for 15 min in cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT and 0.5 mM PMSF) and NP-40 was added to 0.5%. The extract was centrifuged for 30 s at 10 060 g. at 4°C. Pellets were resuspended in buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA and 1 mM PMSF). The dissolved pellets were centrifuged for 5 min at 9760 g. at 4°C. The supernatant was transferred to fresh tubes and stored at –70°C. Proteins were separated on 10% SDS–PAGE gels (59) and electroblotted. Detection was done with alkaline phosphatase anti-rabbit antibodies. The reaction was developed using nitro blue tetrazolium (NBT) 330 µg/ml and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) 165 µg/ml (Sigma) in alkaline phosphatase buffer (100 mM Tris–HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2).
Fluorescence cell sorter (FACS)
Cells were washed twice with PBS, centrifuged, and resuspended in 100% ethanol while vortexing. Permeabilization and fixation were performed by incubation in ethanol for 30 min at room temperature. Cells were precipitated, resuspended in PBS with 500 U/ml RNase A (Sigma) and incubated at 37°C for 15 min. Before analyzing in the FACScan, propidium iodide was added to a final concentration of 25 µg/ml.
Luciferase reporter assay
Cells were divided 1 day prior to the transfection to 50% confluency in 6-well plates and transfected on the following day. After 24 h cells were collected and analyzed using the Dual-Luciferase reporter system according to the manufacturer’s instruction (Promega). Negative control (supplied with the Pathdetect kit, Stratagene) was the DNA-binding domain of the GAL4 protein.
Intracellular [Ca++] measurements
Cells were loaded with 3 µM Fura-2 AM, prepared from a stock DMSO solution of 1 mM (Molecular Probes). Dye loading was in a HEPES-buffered (10 mM) recording medium containing Ca++ (2 mM), Mg++ (1 mM), NaCl (130 mM), KCl (4 mM) and glucose (10 mM). The medium had a pH of 7.4, and the osmolarity was adjusted to 320 mOsm with sucrose. The cultures were exposed to the dye for 60 min, and then used for imaging. The culture coverslips were glued to the bottom of a small, 0.5 ml flow chamber placed in an inverted Nikon microscope. The cells were imaged with a cooled CCD camera (Photometrics) linked to a Macintosh computer. Images were taken at 5 s intervals (60). The ratio of fluorescence emitted from the cells to excitation wavelengths of 340 and 380 nm were calculated. The intensity of excitation with the two wavelengths was equated with neutral density filters to yield a ratio of 
1 at resting [Ca++]i. Groups of cells were compared using ANOVA.
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ACKNOWLEDGEMENTS |
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The authors thank Prof. Menachem Segal for help in [Ca++] measurements, Prof. Rony Seger for useful discussions and Michal Caspi for critical reading of the manuscript. This work was supported in part by the Israeli Science Foundation (grant no. 19/00), Minerva Foundation, Fritz Thyssen Stiftung and HFSP (grant no. RG283199). O.R. is an Incumbent of Aser Rothstein Career Development Chair in Genetic Diseases.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +972 8 934 2319; Fax: +972 8 934 4108; Email: orly.reiner@weizmann.ac.il
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REFERENCES |
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