Identification of shootin1 by double proteome screenings
Cultured hippocampal neurons are a well-established system to study spontaneous neuronal polarization (Dotti et al., 1988
; Craig and Banker, 1994
). They extend several minor processes during the first 12–24 h after plating (stages 1–2). One of these processes then begins to elongate continuously to become an axon (stage 3). The transition from stage 2 to 3 is the initial step of polarization ().
Figure 1. Identification, structure, expression, and intracellular localization of shootin1. (A) Differential 2DE analysis of proteins in stage 2 (cultured for 14 h) and stage 3 (cultured for 62 h) hippocampal neurons. The arrows indicate the protein spot of shootin1 (more ...)
To identify proteins involved in neuronal polarization, we performed two separate proteome analyses of cultured rat hippocampal neurons using a 93- × 103-cm large-gel 2DE (Inagaki and Katsuta, 2004
). One was to detect proteins up-regulated during neuronal polarization (): we screened ~6,200 protein spots on 2DE gels and detected 277 that were consistently up-regulated during the transition from stage 2 to 3 (n
≥ 3). The second analysis screened proteins enriched in axons (). Hippocampi dissected from embryonic day (E) 18 rat embryos were cut into ~1-mm blocks and cultured on plastic dishes, where they formed complicated networks of radial axons in 2 wk. The explants' somatodendritic parts were then separated from the axon networks, and both were compared by 2DE. By screening ~5,200 protein spots, we detected 200 spots enriched in the axon samples (n
A total of 23 spots were detected by both screenings. Tryptic digestion and mass spectrometry of one of them, located at a molecular mass of 60 kD and pI = 5.3 in gels (), identified 10 peptides whose sequences corresponded to the human cDNA sequence KIAA1598 encoding a 5′-truncated ORF of 446 amino acids. A BLAST search identified four human EST clones (BI598285, BG720033, BE568283, and BI457767) and suggested that 10 additional amino acids are present in the complete ORF. We then cloned the cDNAs for the rat and human ORFs and termed them shootin1.
Rat and human shootin1 encode proteins of 456 amino acids and predicted molecular masses of 52.4 and 52.6 kD, respectively (). Domain searching revealed that shootin1 contains three coiled-coil domains and a single proline-rich region (). It does not show significant homology to previously known polypeptides, however, suggesting that it belongs to a novel class of proteins. Database searches also identified a mouse orthologue of shootin1 () and partial ORFs in Macaca fascicularis, chick, zebrafish, and Fugu rubripes. Invertebrate homologues of shootin1 were not found in the databases. Thus, shootin1 is probably a late addition to the genome during the evolution of animals.
Shootin1 is brain specific and highly up-regulated during polarization
We raised an antibody against recombinant shootin1. It recognized a 60-kD band, corresponding to the apparent Mr of native and recombinant shootin1, in immunoblots of rat cultured hippocampal neurons (, arrowhead). Consistent with the 2DE data for the metabolically labeled protein (), the level of shootin1 expression increased remarkably during stage 2/3 transition (14.4-fold increase; n = 4; P < 0.005) and remained high until day in vitro (DIV) 14, thereafter returning to a low level by DIV28 when expression of the presynaptic protein synaptophysin increased (). Immunoblot analysis of various rat tissues detected shootin1 in postnatal day (P) 4 and adult brains but not in other tissues, suggesting that shootin1 is a brain-specific protein (). Expression of shootin1 was relatively low on E15, peaked around P4, and decreased to a low level in the adult brain (). Thus, the expression of shootin1 is up-regulated, both in hippocampal neurons and in brain, during the period of axon formation and elongation.
Shootin1 accumulates in axonal growth cones during the stage 2/3 transition
Next, we examined the localization of shootin1 in cultured hippocampal neurons. Immunocytochemical analysis showed a faint and diffuse staining of endogenous shootin1 in early stage 2 neurons (18–24 h in culture; unpublished data). In late stage 2, moderate amounts of shootin1 appeared in some growth cones of minor processes (). We used a volume marker, 5-chloromethylfluorescein diacetate (CMFDA), to measure the relative concentration of shootin1: it was calculated by using CMFDA as an internal standard (shootin1 immunoreactivity/CMFDA staining). The relative concentration of shootin1 accumulated in the growth cones of late stage 2 neurons was 2–4 times higher than that in the cell body (, arrowheads). In stage 3, shootin1 accumulated strongly in axonal growth cones (, arrows): 100% of axonal growth cones showed accumulation (n = 19). The relative concentration of shootin1 in the axonal growth cones of stage 3 neurons was ~10 times higher than that in the other regions. Notably, the accumulation seen at late stage 2 in minor processes mostly disappeared in stage 3 (, arrowheads), with only 12% of the processes showing accumulation (n = 68). Shootin1 concentration in the cell body remained low throughout stages 2 and 3 (, asterisks). The accumulation of shootin1 in axonal growth cones was observed until stage 5 (unpublished data).
In stage 2, shootin1 shows fluctuating accumulation in multiple growth cones, concurrent with neurite elongation
To analyze the localization of shootin1 in living neurons, we monitored fluorescent images of EGFP-shootin1 expressed in hippocampal neurons under the cytomegalovirus promoter every 5 min. Although relatively high levels of EGFP-shootin1 appeared in the soma, indicating that the expression exceeds the endogenous levels, its distribution in neurites was virtually identical to that of endogenous shootin1 (see the following paragraph). Consistent with the immunocytochemical data, we observed accumulation of EGFP-shootin1 in the growth cones of minor processes in late stage 2 neurons (). As reported previously (Goslin and Banker, 1989
), minor processes showed competitive extension and retraction before polarization. Surprisingly, “hotspots” of EGFP-shootin1 accumulation repeatedly appeared and disappeared in the growth cones of individual neurites (n
= 11 cells; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1
). Most of the neurites elongated in conjunction with EGFP-shootin1 accumulation and, conversely, retracted as EGFP-shootin1 disappeared (). To measure relative concentration of EGFP-shootin1 in growth cones, we used the volume marker monomeric red fluorescent protein (mRFP): it was calculated by using mRFP as an internal standard (EGFP-shootin1/mRFP). By quantifying EGFP-shootin1 and mRFP in growth cones and neurite elongation speed, we found a clear dose dependency of neurite elongation rate on shootin1 concentration in the growth cones of stage 2 neurons ().
Figure 2. Dynamic accumulation of EGFP-shootin1 in growth cones of hippocampal neurons. (A) A stage 2 hippocampal neuron expressing EGFP-shootin1 was observed under a time-lapse fluorescence microscope every 5 min. The full video is presented in Video 1 (available (more ...)
Shootin1 accumulates asymmetrically in a single neurite before polarization
We continued observations until the neurons entered stage 3. Because long exposure to UV light damaged the cells, images were recorded every 30 min (n
= 3; ; and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1
). After stage 2, when EGFP-shootin1 accumulation fluctuated in individual neurites (), the neurons entered a phase in which one of the neurites was 10–15 μm longer than the others (; and Fig. S1). In most cases, this neurite would later become an axon (Goslin and Banker, 1989
). In the longest neurites, accumulation of EGFP-shootin1 stabilized in the growth cone ( and Fig. S1, neurite 1, arrows). Simultaneously, the level of EGFP-shootin1 in its sibling neurites decreased dramatically (neurites 2–5). In this period, the mean number of neurites that showed EGFP-shootin1 accumulation decreased to 1.13 (n
= 30). The longest neurites then underwent rapid elongation and the cells entered stage 3 (; and Fig. S1). Consistent with the immunocytochemical data (), EGFP-shootin1 remained highly concentrated in axonal growth cones during stage 3, whereas it disappeared from the growth cones of minor processes (; and Fig. S1). The dynamic shift of shootin1 accumulation into the nascent axon raises the possibility that it provides an intracellular asymmetric signal for neuronal polarization.
Excess levels of shootin1 disturb its asymmetric distribution and induce formation of surplus axons
To examine whether the asymmetric accumulation of shootin1 in a single neurite is important for neuronal polarization, we overexpressed EGFP-shootin1 or myc-tagged shootin1 (myc-shootin1) in hippocampal neurons under the stronger β-actin promoter. A high level of EGFP-shootin1 was detected in the soma, with its frequent transport from the soma to growth cones (, arrowheads; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1
). This in turn resulted in more continuous accumulation of EGFP-shootin1 in multiple growth cones (, arrows; compared with the dynamic fluctuation of a lower level of EGFP-shootin1 in , and Video 1) and ectopic accumulation of myc-shootin1 in minor process growth cones in stage 3 neurons (, arrowheads). These results suggest that the limited amount of shootin1 is essential for its asymmetric accumulation in a single neurite.
We further cultured the neurons with overexpressed myc-shootin1 until DIV7. Remarkably, 47 ± 2.1% (n
= 3; 71 neurons examined; P < 0.0001, compared with myc-GST) of the neurons bore more than one (two to four) axons that were immunostained by the axon-specific markers anti–tau-1 () and anti-synaptophysin (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1
) antibodies but were immunonegative for the dendrite-specific marker anti-MAP2 antibody (Fig. S2 B). In contrast, only 2.5 ± 1.4% (n
= 3; 81 neurons examined) of control neurons with overexpressed myc-GST formed supernumerary axons. On DIV4, 32 ± 1.8% of neurons overexpressing shootin1 bore multiple axons (n
= 3; 209 neurons examined; P < 0.002, compared with GST) that were immunoreactive for tau-1 and anti-synaptophysin antibodies but were immunonegative for anti-MAP2 antibody. On the other hand, 10 ± 2.5% of control neurons overexpressing GST bore multiple axons (n
= 3; 191 neurons examined). At 50 h in culture, 21 ± 0.9% of neurons overexpressing shootin1 bore multiple axon-like neurites (n
= 3; 226 neurons examined; P < 0.001, compared with GST) that were immunoreactive for tau-1, whereas only 6 ± 1.2% of neurons overexpressing GST bore multiple axon-like neurites (n
= 3; 173 neurons examined). We also quantified the length of the neurites. Neurites labeled by axonal markers were markedly longer than dendrites (). Interestingly, the sum of the length of neurites in neurons overexpressing shootin1 was similar to that in control neurons on DIV7 () and DIV4 (not depicted). Hippocampal neurons elongate axons rapidly (43 μm/d) from stages 3 to 5 (DIV7; Dotti et al., 1988
). We consider that the limited amount of structural components produced in cell bodies similarly limits the total neurite elongation in shootin1-overexpressing and control neurons. A similar limitation of neurite growth in neurons with multiple axons was reported previously (Jiang et al., 2005
). Multiple axons were also induced by nontagged shootin1 cotransfected with EGFP (43 ± 2.6%; n
= 3; 67 neurons examined; P < 0.001, compared with EGFP), whereas a small population (1.6 ± 1.6%; n
= 3; 61 neurons examined) of control neurons expressing EGFP formed supernumerary axons, thereby ruling out the possibility that tagging myc to shootin1 influences the effects. These results suggest that the asymmetric accumulation of shootin1 is involved in neuronal polarization.
Repressing shootin1 expression inhibits neuronal polarization
We next suppressed shootin1 expression using a vector-based RNAi system that expresses microRNA (miRNA). To ensure a high level of expression of miRNA before polarization, hippocampal neurons prepared from E18 rat embryo and transfected with the expression vector of a miRNA designated against shootin1 or a control miRNA were plated on polystyrene plates without any coating. After 20 h for the induction of the miRNA expression, the cells were collected and cultured on coverslips coated with polylysine and laminin. The shootin1 miRNA reduced the level of neuronal shootin1 (, arrows), in comparison to control neurons (arrowheads) and neurons transfected with the control miRNA. Repression of shootin1 expression by the miRNA led to significant suppression of neuronal polarization at 50 and 70 h in culture, whereas the control miRNA had no such effect (). On the other hand, 100% of neurons transfected with the shootin1 miRNA (n = 25) became polarized on DIV7. As the 20-h delay in neuronal plating might affect time course of neuronal polarization after plating, we also performed similar experiments using E17 rat embryo. Essentially equivalent data were obtained with E17 rat embryo (). The significant suppression of neuronal polarization by shootin1 RNAi provides evidence that shootin1 is involved in neuronal polarization.
Shootin1 accumulation in growth cones stimulates neurite elongation during the stage 2/3 transition
As described, shootin1 showed fluctuating accumulation in growth cones concurrent with neurite elongation in stage 2 neurons, raising the possibility that shootin1 accumulation in growth cones stimulates neurite elongation. During the stage 2/3 transition, neurites of hippocampal neurons show dynamic elongation and retraction without a remarkable increase in total neurite length (Goslin and Banker, 1989
). In addition, the stage 2/3 transition is a critical period of neuronal polarization. Therefore, we examined the effect of shootin1 overexpression and RNAi during this period (24 and 48 h in culture). In contrast to the data of DIV7 () and DIV4, shootin1 overexpression induced a significant increase in total neurite length during this period (). Furthermore, repression of its level by RNAi resulted in a significant decrease in it (). Along with the time-lapse data, these results suggest that shootin1 accumulation in growth cones stimulates neurite elongation during the transition from stage 2 to 3.
Shootin1 is anterogradely transported to the growth cones with wave-like structures and diffuses back to the soma
We next asked how shootin1 accumulates asymmetrically in hippocampal neurons. As already noted (, arrowheads), the series of time-lapse imaging revealed active transport of shootin1 from the cell body to the growth cones in stages 2 and 3 neurons (). The shootin1 transport was observed along minor processes and axons. Ruthel and Banker (1998
) reported wave-like anterograde movement of growth cone–like structures along minor processes and axons of cultured hippocampal neurons. The transport rate of these “waves” was ~3 μm/min, similar to that of slow axonal transport component b, which transports actin (Lasek, et al., 1984
; Brown, 2003
). In addition, waves were enriched in F-actin and their movement was reversibly blocked by the actin-disrupting agent cytochalasin. Therefore, Ruthel and Banker (1998
) suggested that actin and other cytoskeletal components are transported as waves from the cell body to neurite tips via an actin-dependent mechanism. Shootin1 traveled as discrete boluses with growth cone–like structures at a mean rate of 1.0 ± 0.1 μm/min (n
= 12), which is similar to the speed of wave transport. We occasionally observed transient retrograde transport of GFP-shootin1. However, as in the case of the wave, retrograde transport was rare and short-lived, quickly reverting to anterograde movement. In addition, the boluses of shootin1 were enriched for F-actin () and the transport was arrested by the actin-disrupting agent cytochalasin D within 5 min (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1
), as reported for the waves. Blebbistatin, an inhibitor of myosin II (Straight et al., 2003
), also stopped shootin1 transport (). These results suggest that shootin1 is anterogradely transported with the wave-like structure by an actin- and myosin-dependent mechanism.
Figure 4. Shootin1 is anterogradely transported to the growth cones with wave-like structures and diffuses back to the soma. (A) Distal movements of EGFP-shootin1 within neurite shafts from the cell body to a growth cone. The arrows indicate boluses of EGFP-shootin1. (more ...)
Within 2 h of the cessation of the transport by blebbistatin or cytochalasin D, shootin1 accumulation in the axonal growth cones of stage 3 neurons disappeared ( and Fig. S3 B, arrows) and a relatively high level of shootin1 was observed in the soma, axonal shaft, and minor processes (arrowheads). To examine whether shootin1 returned back from the axonal growth cones to the cell bodies by diffusion or was locally degraded in the growth cones and newly synthesized in the cell body, we used the photoconvertible reporter Kaede (Ando et al., 2002
) to distinguish old shootin1 from newly synthesized shootin1. Kaede-shootin1 expressed in stage 3 hippocampal neurons was converted from green to red using UV light, and shootin1 transport was blocked by blebbistatin (). 1 h after the cessation of shootin1 transport, the accumulation of the red Kaede-shootin1 in the axonal growth cones decreased (, yellow arrows), whereas the red fluorescence of Kaede-shootin1 increased in the soma and shaft (yellow arrowheads). On the other hand, we could not detect new synthesis of green Kaede-shootin1 in the soma (, blue arrowhead). These data suggest that shootin1 passively diffuses back from the growth cones to the cell bodies.
Inhibition of shootin1 transport prevents its asymmetric accumulation in neurons
We next asked whether the anterograde transport of shootin1 is involved in its asymmetric accumulation in hippocampal neurons. As shown in and Fig. S3 C, cessation of shootin1 transport in stage 2 neurons by blebbistatin or cytochalasin D prevented accumulation of shootin1 in multiple growth cones. Stage 2 neurons were cultured for 36 h in the presence of blebbistatin or cytochalasin D. As described, in control neurons, shootin1 accumulates asymmetrically in growth cones of nascent axons during this period. On the other hand, shootin1 did not accumulate in single neurites in the presence of these drugs ( and Fig. S3 D). Cessation of shootin1 transport in already polarized stage 3 neurons also prevented accumulation of shootin1 in axonal growth cones, as described ( and Fig. S3 B). These data indicate that the actin- and myosin-dependent anterograde transport of shootin1 is necessary for its asymmetric accumulation in single growth cones.
Figure 5. Inhibition of shootin1 transport prevents its asymmetric accumulation in hippocampal neurons. (A) Stage 2 hippocampal neurons treated with 50 μM blebbistatin for 1 h were double stained with anti-shootin1 antibody (red) and a volume marker CMFDA (more ...)
Shootin1 regulates the localization of PI 3-kinase activity in hippocampal neurons
Recent studies indicate that PI 3-kinase is located at a critical upstream position in signaling pathways for neuronal polarization (Arimura and Kaibuchi, 2005
; Wiggin et al., 2005
). We finally examined whether shootin1 interacts with the PI 3-kinase pathway. The physiological association of shootin1 and PI 3-kinase was examined by coimmunoprecipitation assay. When shootin1 was immunoprecipitated from P5 rat brain lysates, coimmunoprecipitation of the p85 subunit of PI 3-kinase was detected (). Shootin1 was also reciprocally coimmunoprecipitated with p85, indicating that it associates with p85 in vivo. PI 3-kinase activity, indirectly visualized by the phosphorylation of Akt at Ser473 (P-Akt), was enriched in the axonal growth cones of stage 3 neurons (, arrows) as reported (Shi et al., 2003
) and preferentially colocalized there with shootin1 (, insets). We exogenously coexpressed shootin1 and p85 in HEK293T cells but could not detect coimmunoprecipitation between shootin1 and p85 (not depicted). Thus, shootin1 may interact with PI 3-kinase through unidentified neuronal proteins. As shown recently (Yoshimura et al., 2006
), overexpression of constitutively active PI 3-kinase (Myr-PI 3-K p110) induced formation of multiple axons ( and Fig. S4 B, available at http://www.jcb.org/cgi/content/full/jcb.200604160/DC1
), as in the case of shootin1 overexpression. Jiang et al. (2005)
also reported that overexpression of constitutively active Akt (Myr-Akt), a downstream kinase of PI 3-K, induced formation of multiple axons. On the other hand, inhibition of PI 3-kinase activity by 20 μM LY294002, a specific inhibitor of PI 3-kinase, delayed neuronal polarization (), as reported previously (Menager et al., 2004
) and as in the case of shootin1 RNAi. These results suggest that shootin1 interacts with PI 3-kinase and is involved in a similar pathway mediating neuronal polarity.
Figure 6. Shootin1 regulates the localization of PI 3-kinase activity in hippocampal neurons. (A) Brain lysates from P5 rat brain were incubated with anti-shootin1 antibody, anti-p85 antibody, or control IgG. The immunoprecipitates were analyzed by immunoblotting (more ...)
Next, we examined whether shootin1 functions upstream of PI 3-kinase or vice versa. Shootin1 RNAi decreased its level in axonal growth cones, which in turn inhibited accumulation of PI 3-kinase activity there (, arrows), suggesting that shootin1 in axonal growth cones is required for accumulation of PI 3-kinase activity there. Conversely, myc-shootin1 overexpression induced its ectopic accumulation in the growth cones of minor processes, which in turn resulted in ectopic accumulation there of P-Akt (, arrowheads), thereby suggesting that accumulation of shootin1 can recruit PI 3-kinase activity. On the other hand, inhibition of PI 3-kinase activity by LY294002 did not affect the accumulation of shootin1 in axonal growth cones (, arrows). Shootin1 overexpression or RNAi did not change the activity of PI 3-kinase in hippocampal neurons (), ruling out the possibility that the expression level of shootin1 changes the total activity of PI 3-kinase in neurons. These results suggest that shootin1 regulates subcellular localization of PI 3-kinase activity in hippocampal neurons.
We further examined the functions of shootin1 and PI 3-kinase within the cell polarity pathways. Inhibition of PI 3-kinase activity by LY294002 led to a reduction in the percentage of neurons with multiple axons induced by shootin1 overexpression ( and Fig. S4 A). On the other hand, multiple axon formation by overexpression of constitutively active PI 3-kinase was not inhibited by shootin1 RNAi ( and Fig. S4 B). Collectively, these results provide evidence that shootin1 functions upstream of PI 3-kinase and is required for spatially localized PI 3-kinase activity, which is essential for neuronal polarization (Shi et al., 2003