Correspondence to Louis F. Reichardt: lfr{at}cgl.ucsf.edu; or Zhen Huang: zhenh{at}itsa.ucsf.edu
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This article contains online supplemental material.
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Introduction |
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The origin recognition complex (ORC; Bell and Stillman, 1992) is a hexameric protein complex key to initiating DNA replication during the cell cycle (Kelly and Brown, 2000; Bell, 2002; Bell and Dutta, 2002). It is part of the protein machinery responsible for one of the central processes of life, genome replication (Baker and Bell, 1998). Structurally the mammalian ORC is composed of four core (Orc25) and two peripheral subunits (Orc1 and Orc6; Dhar et al., 2001; Vashee et al., 2001), among which three (Orc1, Orc4, and Orc5) belong to the AAA+ family of ATPases (Neuwald et al., 1999). During early interphase when the Cdk activity level is low, the ORC initiates the assembly of a prereplication complex, which triggers DNA replication when the Cdk activity level rises as the cells undergo G1S transition. At the same time it prevents replication reinitiation through multiple mechanisms including an ATPase-dependent conformational change of the large subunit Orc1 and modification of several other subunits, which result in the inactivation of the complex (Lee and Bell, 2000; Nguyen et al., 2001; Li and DePamphilis, 2002; Mendez et al., 2002). Thus, the ORC acts as a Cdk-regulated ATPase-dependent molecular switch for initiating DNA replication during the cell cycle, ensuring that each wave of Cdk activation is translated into one and only one round of genome replication.
The extraordinary properties of the ORC and its associated signaling circuits make it an ideal protein machine for coupling Cdk activation and genome replication. Recent evidence suggests that the ORC may be reused later in the cell cycle to couple Cdk activation with cytokinesis, the process where the cytoplasm of a cell is divided into two parts so that each would inherit one copy of the genome replicated earlier in the cell cycle. Orc6 has been found to localize to the spindle midzone of mitotic cells and its loss of function leads to accumulation of multinucleate cells (Prasanth et al., 2002; Chesnokov et al., 2003). Orc2 has also been found to associate with centrosomes and centromeres, where it is required for proper segregation of replicated chromosomes (Prasanth et al., 2004). Thus, the ORC can orchestrate not only the nuclear event of DNA replication but also cellular morphogenetic processes such as cytokinesis where the exact division of one cell into two, once per cell cycle, is equally critical for safeguarding genome integrity. In this report, we describe a novel role for the ORC in initiating dendritic branch and spine formation in postmitotic neurons and discuss the implication of its unexpected function in the nervous system.
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Results |
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Association of ORC with neuronal membranes
The coexpression of the ORC core subunits in the brain and their colocalization in neuronal processes suggest that, as in DNA replication, these subunits (Orc25) may also exist and function as a complex in neurons. To determine if this is the case, we tried various immunoprecipitation approaches but without success. We then used the approach of brain homogenate fractionation. First we fractionated brains of early postnatal rat pups into cytoplasmic and membrane fractions. We found that, interestingly, all three subunits we examined, Orc35, were detected in the membrane fraction except for Orc3, which was also detected in the cytosol (Fig. 2 A). This suggests that the majority of the ORC subunits are associated with neuronal membrane while a fraction of Orc3 may exist in a second cytoplasmic pool. To determine the nature of neuronal membrane with which ORC is associated, we further separated it into microsome, synaptic vesicle, and lysed synaptosome fractions. We found that most of Orc35 proteins were associated with the microsome and synaptosome fractions and very little of them, particularly for Orc4 and Orc5, were detected in the synaptic vesicle fraction (Fig. 2 B). Similar patterns of subunit distribution were also observed in adult brain fractions (Fig. S2). In addition, consistent with the fact that none of the ORC subunits possess transmembrane motifs, we found that either high salt or chaotropic conditions would disrupt their association with the membrane. These results are thus consistent with the idea that the ORC subunits may exist primarily as a complex in the nervous system.
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To determine other potential effects, we next examined expression of the dendritic marker MAP2. We found that, despite their simplified dendritic trees, orc3 knockdown neurons showed relatively normal MAP2 staining (Fig. 3, GG'' and HH''), suggesting that the identity of these processes was not affected by loss of Orc3. Consistent with the selective dendritic localization of Orc3, we also observed relatively normal growth and morphology of axons and staining of the axonal marker Tau1 (unpublished data) as well as normal size of neuronal somas (Fig. S4 available at http://www.jcb.org/cgi/content/full/jcb.200505075/DC1). In addition, we also observed relatively normal expression of the neuron-specific tubulin isoform TubßIII (unpublished data). Thus, orc3 seems to be quite specifically involved in regulating dendritic growth and branching of hippocampal neurons at this stage of differentiation. However, we did frequently observe an additional effect in siRNA1- or 4-transfected neurons in that their dendrites seemed to be somewhat thicker and they appeared to have a looser organization of microtubules (Fig. S4). Because we did not observe this with an orc5 siRNA construct (see Fig. 4), it suggests that orc3 may have an additional function separate from the core complex in neurons. This is consistent with our finding that, unlike other subunits, Orc3 was also found in the cytoplasmic fraction of brain homogenates (Fig. 2).
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ORC is required for initiating dendritic spine formation
Dendritic arbor development of hippocampal neurons is followed by formation of dendritic spines, tiny protrusions on dendrites where the majority of excitatory synapses in the brain are located (Yuste and Bonhoeffer, 2004). To determine if the ORC is involved in spine development, we transfected 14 DIV neurons with siRNA1 or 4 and examined them on 17 DIV. We found that orc3 knockdown severely impeded the development of dendritic spines (Fig. 5, AC). Normally dendrites of 17 DIV neurons are covered by a large number of spines that are spaced more or less evenly along the dendrites; in the orc3 knockdown neurons, however, very few spines were found along the dendrites and they appeared irregularly. For control neurons, the density of spines along dendrites is 4.6 per 10 µm. For siRNA1-treated neurons, however, the density dropped to
1.1, a 76% decrease; for siRNA4-treated neurons, it dropped to
1.0, a 78% decrease (Fig. 5 F). These results suggest that orc3 is required for the development of dendritic spines on the dendrites of hippocampal neurons. Moreover, consistent with the results on dendritic development, siRNA11, which targets orc5, also caused a 76% decrease in spine density from
4.6 to 1.1 per 10 µm in transfected neurons (Fig. 5 F and Fig. S5). Thus, the ORC is required for spine development along neuronal dendrites.
Dendritic spines normally undergo a gradual process of maturation from thin, long filopodia to mushroom-shaped protrusions (Hering and Sheng, 2001; Yuste and Bonhoeffer, 2004). In control neurons, the majority of dendritic spines on 17 DIV assume a shape with enlarged heads (Fig. 5 A), consistent with a relatively high degree of maturation. In siRNA1-, 4-, or 11-transfected neurons, we noticed that the few spines that developed also had a similar morphology (Fig. 5, B and C, and Fig. S6 B available at http://www.jcb.org/cgi/content/full/jcb.200505075/DC1), suggesting that they might have matured properly. To test this, we examined the expression of a number of postsynaptic markers in these spines. We found that, as in spines of control neurons on 17 DIV (Fig. 5 D), nearly all the spines of siRNA1- or 4-transfected neurons exhibited strong signals for the major postsynaptic scaffold protein PSD95, as revealed by a PSD95EGFP reporter (Fig. 5 E). This pattern was further confirmed by immunostaining for endogenous PSD95 protein with an antibody. Similarly, the vast majority of these spines were also found to be positive for the AMPA (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluR1 by immunostaining (unpublished data). These results indicate that, despite the dramatic reduction in the number of developing spines, the absence of ORC function does not seem to affect their maturation, suggesting that the ORC might be required in an early step of spine development.
Recent in vivo imaging data indicate that dendritic branching seems to depend on the formation of synapses on newly extended filopodia, which subsequently stabilize a subset of the filopodia and allow them to mature into branches (Niell et al., 2004). It has been suggested that dendritic spine development may also take place in a similar synaptotropic fashion. To determine if the ORC is required for filopodial stabilization, we next examined neurons of earlier stages when most of the dendritic protrusions are still in the form of filopodia. We transfected siRNA constructs into 11 DIV neurons and examined them on 14 DIV. We found that on 14 DIV, orc3 knockdown resulted in a severe loss of dendritic filopodia on transfected neurons. We normally observed large numbers of filopodia on the dendrites of control neurons (Fig. 5 G). However, very few such protrusions were found on siRNA1-treated neurons (Fig. 5 H). These results thus suggest that the ORC may be required in the very early step of spine development, the initiation of dendritic filopodia, although we cannot exclude the possibility that it may also have a role in their stabilization. To determine how the ORC may regulate filopodial formation, we then probed the dendritic cytoskeleton using a number of EGFP reporters. We found that in control neurons, EGFPMena, a marker that labels filopodia before their emergence (Svitkina et al., 2003; Mejillano et al., 2004), displayed periodic clusters along dendrites and decorated tips of protruding filopodia (Fig. 5 I). By contrast, very few such clusters were observed along the dendrites of siRNA1-treated neurons (Fig. 5 J). Similar results were observed using an EGFPVASP reporter (unpublished data). As Orc6 has been found to interact with the septin proteins, we also used a mCDC10 reporter to determine its localization but found relatively normal clusters of EGFPmCDC10 along dendrites in orc3 knockdown neurons (Fig. 5, K and L). These results thus indicate that the ORC might regulate the organization of the actin cytoskeleton in emerging dendritic filopodia. As its role in controlling dendritic branching also seems to depend on filopodial formation (Dailey and Smith, 1996; Niell et al., 2004), our data thus indicate that the ORC may regulate both dendrite and spine development through controlling the key common step of dendritic filopodial initiation. Depending on the stage of neuronal differentiation, the failure in filopodial initiation may either dramatically reduce dendritic branching or severely impair spine development.
Orc4 ATPase motif mutants and dendritic branching
One of the critical properties of the ORC in regulating DNA replication is its ability to act as a molecular switch that not only precisely couples origin firing with Cdk activation but also triggers complex inactivation once replication is initiated. Among the mechanisms that contribute to ORC inactivation is the intrinsic ATPase activity of the ORC that induces a conformational change upon ATP hydrolysis (Lee and Bell, 2000). During DNA replication, Orc1 seems to be the main subunit responsible for this function and mutations in its ATPase motifs have been found to interfere with DNA replication (Chesnokov et al., 2001; Klemm and Bell, 2001). Among the core subunits expressed in the nervous system, Orc4 and Orc5 also belong to the AAA+ family of ATPases, which typically contain two well-conserved motifs termed Walker A and B (Neuwald et al., 1999). However, only Orc4 has both of the ATPase motifs conserved (Chesnokov et al., 2001), raising the possibility that these motifs may play a role in regulating ORC activity in neurons.
To test this, we generated a point mutation in the Walker B motif of Orc4 where the glutamate residue (Glu 157) known to be essential for ATP binding/hydrolysis in other ATPase family members is replaced by glutamine (O4EQ). We first tried to determine the effects of overexpressing this construct on dendritic spine development. However, possibly due to the relatively low level of O4EQ protein expression and/or its inability to incorporate itself into the ORC complex in well-differentiated neurons, we were unable to observe consistent and convincing effects. We next turned to examine its effects on dendrite development of earlier stage neurons. We found that, interestingly, when transfected into neurons at the time of plating, O4EQ significantly increased the elaboration of dendritic branches of hippocampal neurons. In 7 DIV neurons transfected with wild-type orc4 (O4WT), the dendritic branches were mostly confined near the cell body and were mostly of low branchpoint orders (Fig. 6, A and A'). In neurons overexpressing O4EQ, however, dendrites of many cells frequently extended away from the cell body and had higher order branches (Fig. 6, B and B'). Quantification revealed that the total branchpoint number increased more than 60%, from 24 for O4WT cells to over 39 for O4EQ cells (Fig. 6, D and E). Moreover, the branchpoint density showed an average increase of 32% from
2.1 for O4WT cells to over 2.7 for O4EQ cells (Fig. 6 F). Thus, O4EQ, behaving as an apparent gain of function mutant, seemed to affect dendritic development mainly through promoting branchpoint formation, suggesting that the Walker B motif of Orc4 may be normally involved in down-regulating ORC activity.
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Discussion |
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The ORC, acting as a switch in initiating DNA replication, plays a key role in maintaining genomic integrity that ensures exactly one round of genome replication per cell cycle. As it is one of the key components of the DNA replication machinery, this raises the question how it may regulate the process of neuronal morphogenesis. Recent findings of ORC function in the later events of the cell cycle including cytokinesis and mitosis have provided some clues. Here the Orc6 and Orc2 subunits are associated with the spindle midzone, cleavage furrow, and centrosomes and are required to coordinate these processes of major cytoskeletal reorganization for the proper division of the cytoplasm and the accurate transmission of the genome to daughter cells (Prasanth et al., 2002, 2004; Chesnokov et al., 2003). Although the mechanistic details of ORC function in cytokinesis and mitosis are still unclear, these findings nonetheless demonstrate that the ORC is not limited in its capacity to regulating DNA replication but can also interact with components of the actinmicrotubule cytoskeleton and the cell membrane and participate in processes of cellular morphogenesis outside the nucleus.
Dendrite branching and spine formation, like cytokinesis, are also very different from the process of DNA replication. On the other hand, in vivo imaging studies have found that synaptic growth at the Drosophila NMJ, at least morphologically, is surprisingly similar to yeast budding, the unique cytokinetic process of budding yeast (Zito et al., 1999). This suggests that there may be commonalities underlying neuronal morphogenesis and cytokinesis. Indeed, the Orc3 protein has been found to localize to the Drosophila NMJ and orc3 mutants display impaired NMJ development and function (Rohrbough et al., 1999). Orc6 function during cytokinesis has also been found to depend on its interaction with a septin protein (Chesnokov et al., 2003), a component well known for its role in organizing the actin cytoskeleton at yeast budding sites. Our findings that the ORC may regulate dendrite and spine development through controlling the organization of the actin cytoskeleton are therefore consistent with these observations.
The ORC plays a key role not only in the interphase of the cell cycle by regulating genome replication but also in the later events of the cell cycle where it is involved in coordinating the processes of cytoplasmic division and chromosome segregation. As it is one of the cornerstones of the cell cycle machinery, our observation on ORC function in neuronal dendrite development suggests that there might be wider commonality between the cell cycle and neuronal morphogenesis than previously recognized. Indeed, many key components of the cell cycle machinery have been recently implicated in the differentiation of postmitotic neurons. For example, the kinesin-like protein mKLP-1, which plays an essential role in cytokinesis (Glotzer, 2001), has been found to localize to the dendrites and is required for dendritic differentiation (Yu et al., 1997, 2000). Family members of the cytoplasmic polyadenylation element binding (CPEB) protein, which coordinates mitotic progression through controlling local protein translation around the mitotic spindle (Groisman et al., 2002), also promote protein translation in neuronal dendrites (Huang et al., 2003) and regulate synapse-specific long-term facilitation (Si et al., 2003). The Polo family protein kinase SNK, whose family member Plk1 plays prominent roles in the cell cycle (Barr et al., 2004), has also been found to regulate activity-dependent spine synapse remodeling (Pak and Sheng, 2003). The anaphase promoting complex, well known for its role in cell cycle progression (Harper et al., 2002), plays key roles in the development of the Drosophila NMJ (van Roessel et al., 2004) and Caenorhabditis elegans synapses (Juo and Kaplan, 2004). The parallel functions of these key cell cycle components therefore suggest that the function of the ORC in dendrite development is not an exception, but may reflect an emerging underlying commonality between these two biological processes. They suggest that a large part of the cell cycle apparatus may have been coopted and tinkered with during evolution to fulfill new functions in postmitotic neurons.
The function of the ORC during the cell cycle is to faithfully translate each wave of Cdk activation into exactly another copy of the genome and, in most cases, another copy of the cell. Viewed from another angle, it may also be described as to translate the waves of Cdk activation a cell experiences into the copy number of the genome or the number of cells. From this perspective, it is interesting to note that the formation of new dendritic branches and spines in neurons also seems to involve an all or none decision that depends on ORC function. It is tempting to speculate that there might also be waves of Cdk-like activity in neurons that may be responsible for triggering the activity of the ORC and regulating dendritic branch and spine formation. Most interestingly, recent data have shown that dendritic spines continue to appear and disappear in the adult cortex and experience-driven plasticity is accompanied by increased spine synapse turnover (Grutzendler et al., 2002; Trachtenberg et al., 2002). As the ORC is continuously expressed in many areas of the adult nervous system, this raises the possibility that it may be a key part of the molecular machinery that translates patterns of neural activity into patterns of neuronal connectivity and that is believed to underlie long-term memory of sensory experience.
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Materials and methods |
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Neuronal culture and transfection
Primary hippocampal neurons were prepared from E18 rat embryos and cultured in high density on poly-L-lysinecoated coverslips in Neurobasal medium supplemented with B27 (Invitrogen). Animal use was in accordance with institutional guidelines. Neurons were transfected either with Effectene (Qiagen), Lipofectamine 2000 (Invitrogen) reagents, or using the calcium phosphate method. A ratio of 2.5:1 or higher between siRNA or cDNA expression constructs and reporter plasmids was maintained to ensure that labeled cells were transfected with the intended constructs. For siRNA constructs, neurons were transfected on 7, 11, or 14 DIV as specified in the text. For orc4 expression constructs, neurons were transfected on the day of plating and examined on 7 DIV.
Immunochemistry
Hippocampal neurons were fixed in a 1:1 mixture of culture medium and 8% paraformaldehyde/8% sucrose/PBS for 10 min at room temperature, except for PSD95 staining, when it was followed by a 5-min fixation in 20°C methanol. Primary antibodies used for immunofluorescent staining included: affinity-purified rabbit anti-Xorc3 (0.6 µg/ml; gift of P. Carpenter, University of Texas, Houston, TX), custom rabbit anti-mOrc3 serum (1:1,000), mouse monoclonal anti-MAP2 clone AP-20 (1:250; Sigma-Aldrich), monoclonal antiTau-1 (1:400; Chemicon), monoclonal antiß-tubulin isotype III (1:100; Sigma-Aldrich), monoclonal anti-PSD95 (1:200; Affinity Bioreagents), rabbit anti-GluR1 (1:200; Upstate Biotechnology), and Alexa488-conjugated rabbit anti-GFP (1:500; Molecular Probes). Secondary antibodies used for immunostaining included: Cy3-conjugated goat antimouse IgG (1:500; Jackson ImmunoResearch Laboratories) and Cy3-conjugated goat antirabbit IgG (1:500; Jackson ImmunoResearch Laboratories). Rabbit anti-mOrc3 and chicken anti-mOrc5 antibodies were custom raised against bacterially expressed mOrc3 and mOrc5 protein inclusion bodies at Covance Research Products, Inc. and affinity purified with recombinant proteins bound to nitrocellulose filters. Goat anti-ORC4L antibody for Western blot analysis was purchased from Abcam, Inc.
Brain fractionation
Rat brain homogenate was fractionated after standard protocols (De Camilli et al., 1983; Cho et al., 1992; Schilling et al., 1999; Lee et al., 2001; Ehlers, 2003). In brief, P10 rat or adult mouse brain homogenate was centrifuged at 1,000 g to remove nuclei and large debris (P1), and the postnuclear supernatant (S1) was centrifuged at 100,000 g to obtain crude cytosol (C) and membrane (M) fractions. To subfractionate the membrane fraction, supernatant S1 was centrifuged at 10,000 g to obtain a crude synaptosomal pellet fraction (CS). The supernatant (S2) was further centrifuged at 140,000 g to obtain cytosol (C) and microsomal (Mi) fractions. In parallel, the crude synaptosomal fraction (CS) was lysed by hypoosmotic shock and centrifuged at 25,000 g to obtain a synaptic vesicleenriched fraction (SV) and a lysed synaptosomal fraction (LS). The membrane fractions were solubilized in nuclear lysis buffer (50 mM Tris, pH 7.5, 0.5 M NaCl, 1% NP40, 1% deoxycholate, 0.1% SDS, 2 mM EDTA plus protease inhibitors) and all fractions quantified using the Bio-Rad Dc protein assay. Samples were separated on SDS-PAGE gradient gels (Bio-Rad) and transferred to nitrocellulose membrane for Western analysis.
Microscopy and quantification
Glass coverslips with cultured neurons were mounted with ProLong antifade medium (Molecular Probes) after the appropriate immunochemical procedure. Digital images of neurons were captured using a LSM 5 PASCAL confocal microscope (Carl Zeiss MicroImaging, Inc.) using a Plan-Neofluar 40x (NA = 1.30; Carl Zeiss MicroImaging, Inc.) oil objective at room temperature. Neuronal dendritic trees were traced manually using NIH Image software (version 1.62). Data were collected from at least three duplicate experiments for each construct. For analysis of dendritic morphology, branches shorter than 3.3 µm (15 pixels) were ignored in the tracing process. For spine density analysis, all protrusions on dendrites were counted whether or not they had enlarged heads. Statistical analysis was done using the Student's t test and P values smaller than 0.01 (P < 0.01) were considered significant.
Online supplemental material
Fig. S1 shows that the DNA replication components downstream of ORC are not expressed but Orc3 and Orc5 proteins are detected in the adult mouse brain. Fig. S2 shows that the ORC subunits colocalize with endogenous Orc3 as EGFP fusion proteins and cofractionate in adult mouse brain homogenate. Fig. S3 shows that overexpression of a truncated form of Orc3 impaired dendritic growth and branching. Fig. S4 shows that soma size is unaffected but dendritic microtubule organization is altered in orc3 knockdown neurons. Fig. S5 shows that orc5 is required for dendritic spine formation. Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200505075/DC1.
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Acknowledgments |
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Z. Huang was supported by a Helen Hay Whitney fellowship. L.F. Reichardt is an investigator of the Howard Hughes Medical Institute.
Submitted: 12 May 2005
Accepted: 29 June 2005
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References |
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