Spatial mapping of the neurite and proteomes reveals a functional Cdc42/Rac regulatory network

Olivier C. Pertz*†, Yingchun Wang*, Feng Yang‡, Wei Wang*, Laurie J. Gay*, Marina A. Gristenko‡, Therese R. Clauss‡, David J. Anderson‡, Tao Liu‡, Kenneth J. Auberry‡, David G. Camp II‡, Richard D. Smith‡, and Richard L. Klemke*§

*Department of Pathology and Moores Cancer Center, University of California at San Diego, La Jolla, CA 92093; and ‡Biological Sciences Division, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354

Edited by Masatoshi Takeichi, RIKEN, Kobe, Japan, and approved December 12, 2007 (received for review July 15, 2007) Neurite extension and navigation are guided by tion of the neurite and soma compartments for biochemical extracellular cues that control cytoskeletal rearrangements. How- analyses. Comparative analysis of the neurite and soma pro- ever, understanding the complex signaling mechanisms that me- teomes revealed the spatial relationship of thousands of diate neuritogenesis has been limited by the inability to biochem- proteins and specific signaling networks that operate in these ically separate the neurite and soma for spatial proteomic and distinct cellular compartments. In addition, using bioinformat- bioinformatic analyses. Here, we apply global proteome profiling ics and cell-based RNA interference approaches, we address a in combination with a neurite purification methodology for com- fundamental question pertinent to Rho family small GTPases, parative analysis of the soma and neurite proteomes of neuro- which couple extracellular cues to the during blastoma cells. The spatial relationship of 4,855 proteins were neuritogenesis (3). Although it is clear that Rac and Cdc42 play mapped, revealing networks of signaling proteins that control a fundamental role in regulating neurite extension, it is not integrins, the actin cytoskeleton, and axonal guidance in the understood why regulation of their activity relies on redundant extending neurite. Bioinformatics and functional analyses revealed upstream regulators such as guanine-nucleotide exchange a spatially compartmentalized Rac/Cdc42 signaling network that factors (GEFs) and GTPase activating proteins (GAPs) (5, 6). operates in conjunction with multiple guanine-nucleotide ex- These proteins are widely expressed and outnumber their change factors (GEFs) and GTPase-activating proteins (GAPs) to GTPase targets by a factor of three (5, 6). This prompted us control neurite formation. Interestingly, RNA interference experi- to investigate the spatial complexity of the Rac/Cdc42 signal- ments revealed that the different GEFs and GAPs regulate special- ing networks in the soma and neurite and determine the ized functions during neurite formation, including neurite growth functional relevance of multiple neurite-enriched GEFs and and retraction kinetics, cytoskeletal organization, and cell polarity. GAPs. Surprisingly, we find that the different Cdc42/Rac Our findings provide insight into the spatial organization of GEFs and GAPs control unique cellular functions, which signaling networks that enable neuritogenesis and provide a cooperate to fine tune neurite formation, rather than solely comprehensive system-wide profile of proteins that mediate this regulating neurite extension as proposed previously (3). process, including those that control Rac and Cdc42 signaling. Results GAPs ͉ GEFs ͉ neuritogenesis ͉ proteomics ͉ Rho GTPase Biochemical Purification of Neurites and Somata. To specifically isolate neurite and soma proteins, we used NIE-115 neuroblas- euritogenesis is a dynamic process involving the extension of toma cells as a model system for neuritogenesis. These cells can Nlong, thin protrusions called neurites that will subsequently be grown in large numbers for large-scale biochemical analysis differentiate into long or an elaborate dendritic arbor. This and have been well characterized for their -like proper- highly polarized process occurs through the segmentation from ties. Upon serum starvation in neuron differentiation medium, the soma periphery of a microtubule-rich shaft capped with a these cells express the neurofilament protein and readily extend growth cone, which is itself characterized by an actin-rich neurite-like processes that are morphologically similar to that of lamellipodium with numerous filopodial extensions and integrin- primary (7). Indeed, these neurites form actin-rich mediated adhesive contacts (1). Understanding this process is growth cones connected to a tubulin-rich shaft that can respond crucial, because it is necessary for proper wiring of the brain and to directional cues including immobilized extracellular matrix nerve regeneration, and has been linked to numerous neurode- proteins (ECM) and soluble extension and collapsing factors (8). ␮ generative diseases (2). When plated on the top of 3.0- m porous filters coated on the Although cultured neurons randomly form neurites in vitro, in lower surface with the ECM laminin, neurite extension occurs vivo this process is orchestrated by gradients of chemoattractants through the small pores exclusively to the lower surface of the and/or extracellular matrix proteins that precisely guide neurite filter [Fig. 1A and supporting information (SI) Appendix, Fig. initiation and advancement. This occurs in a polarized and highly 4A]. This response occurs within 24 h (Fig. 1 B and C), controlled manner and relies on spatially regulated mechanisms for exclusively on the laminin matrix (SI Appendix, Fig. 4B), is gradient sensing, membrane trafficking, integrin-mediated adhe- sion, and organization of the actin-microtubule (3). Author contributions: O.C.P., Y.W., D.G.C., R.D.S., and R.L.K. designed research; O.C.P., Although progress has been made in identifying such spatially Y.W., F.Y., W.W., L.J.G., and M.A.G. performed research; K.J.A. contributed new reagents/ regulated signals, this work has been limited primarily to single cell analytic tools; O.C.P., Y.W., F.Y., T.R.C., D.J.A., and T.L. analyzed data; and O.C.P. and R.L.K. analyses, using imaging-based techniques, precluding a large-scale wrote the paper. view of these signaling events during neuritogenesis. The authors declare no conflict of interest. Recently, we described a method for the purification of This article is a PNAS Direct Submission. pseudopodia from migrating cells, using a microporous filter †Present address: Institute for Biochemistry and Genetics, Department of Biomedicine, system (4). This model system, combined with contemporary University of Basel, CH-4003 Basel, Switzerland. large-scale protein mass spectrometry (LC-MS/MS), provided §To whom correspondence should be addressed. E-mail: [email protected]. global insight into the spatial organization of the signaling This article contains supporting information online at www.pnas.org/cgi/content/full/

networks that control this process. Here, we extend this 0706545105/DC1. CELL BIOLOGY technique to neuroblastoma cells enabling the specific isola- © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706545105 PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 ͉ 1931–1936 Downloaded by guest on September 29, 2021 Fig. 1. Neurite purification assay, biochemical, and proteomic analyses. (A) Schematic of microporous filter system. (B) Neurite lysate protein amount on the filter bottom was determined for the indicated times from filters coated with laminin on the top, the bottom, or both sides. Standard deviations from three independent experiments are shown. (C) Fluorescence micrographs of ␣-tubulin (red) and F-actin (green, phalloidin) immunostained neurites extending to the lower filter surface for the indicated times. (Scale bar, 20 ␮m.) (D) 3D reconstruction and volume rendering of a confocal series of ␣-tubulin stained neurons on filter. (E) Equal amounts of neurite and soma lysates were separated by SDS/PAGE and either silver stained or Western blotted for phosphotyrosine (pY). (F) Rac and Cdc42 activity (GTP-Cdc42, GTP-Rac) was determined from equal amounts of neurite and soma protein fractions, using a GST-PBD pulldown assay and Western blot analysis. ERK served as a protein loading control. (G) Erk activity in neurite and soma fractions was determined by Western blot analysis with phosphospecific antibodies to the phosphorylated activated form of ERK. (H) Gene ontology analysis of the most significant canonical pathways present in the neurite (blue, 10 of 39 shown), soma (yellow), or equally distributed proteins (red). Green dotted line represents significance threshold as measured by Fishers’s test (P Ͻ 0.05).

mediated by ␤1-integrins (SI Appendix, Fig. 4C), and is robust However, as described for the pseudopodia purification system with Ͼ80% of somata extending neurites (SI Appendix, Fig. 4D) (4), this issue can be easily resolved by assuming equal protein with a mean of 1.8 Ϯ 0.8 neurites per cell (five fields of view, n ϭ density in the neurite and soma fractions, which allows for 85 cells, data not shown). Polarized neurite extension also works normalization based on protein concentration. As expected, on fibronectin (SI Appendix, Fig. 4E) and with pheochromocy- when equal amounts of neurite and soma lysate were analyzed toma PC-12 cells on laminin and fibronectin (SI Appendix, Fig. from cells expressing green fluorescent protein (GFP), which 4 F and G). The polarized neurites and their somata can then be acts as a soluble exogenous protein marker, GFP was equally stained and visualized by microscopy or manually separated abundant in the neurite and soma fractions (SI Appendix, Fig. from either side of the filter into the appropriate lysis buffer for 5A). This is also true for the cytosolic protein Erk2, which serves protein concentration determination and biochemical analyses as a loading control in these biochemical experiments (SI as described in Material and Methods. Confocal imaging through Appendix, Fig. 5A). However, the possibility remained that our the upper and lower filter surface illustrates the high level of protein profile reflected an enrichment of plasma membrane morphological polarity (Fig. 1D) achieved by these cells after versus cytosolic proteins, rather than real changes in protein stimulation with ECM proteins and reveals a typical neurite abundance in both subdomains. This was excluded by the finding morphology consisting of an F-actin rich growth cone with that an exogenously transfected GFP-fusion with the C-terminal numerous filopodia attached to a dense tubulin-rich shaft (Fig. CAAX domain of K-Ras4b, which localizes exclusively to the 1C). Biochemical and signaling polarity are illustrated by the plasma membrane, was equally abundant in the neurite and soma selective localization of nuclear histones in the soma and ele- fractions (SI Appendix, Fig. 5A). Together, these findings dem- vated phosphotyrosine (Fig. 1E), Cdc42, Rac (Fig. 1F), and ERK onstrate that microporous filters coated with ECM proteins activation (Fig. 1G) in the neurite compartment. It is important provide a simple system to selectively purify large numbers of to note that, because the neurite fraction contains much less neurites from their somata for biochemical analysis. protein than the soma fraction (typically 40 ␮g of neurite versus 800 ␮g of soma lysate for 1.2 ϫ 106 cells) and because multiple Characterization of the Neurite and Soma Proteomes. To quantify neurites often emanate from one neuron, direct comparison of relative changes in protein abundance of specific proteins in the neurite-soma equivalents on a per-filter basis is not possible. neurite and soma proteomes, we used two dimensional liquid

1932 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706545105 Pertz et al. Downloaded by guest on September 29, 2021 chromatography-coupled tandem MS (LC-MS/MS) to analyze equal amounts of soma and neurite lysate. By calculating the ratio of peptide spectrum counts (neurite/soma) assigned to all of the peptides for each protein in each fraction, it is possible to quantify the relative abundance for a given protein in the two samples. A detailed description of this system is provided in Material and Methods and in our previous work on the pseudo- pod proteome (4). In total, 4855 proteins were identified from at least two peptides. A protein was considered to be increased/ decreased in a particular fraction if its neurite/soma ratio changed by 2-fold or greater or if it was detected in only one fraction (unique). All other proteins were considered to be equally abundant in the two fractions. Using these criteria, 1,229 proteins were enriched in the neurite, 1,676 proteins were enriched in the soma, and 1,950 proteins were equally distributed (SI Appendix, Fig. 5B). A complete list of these proteins is found in SI Table 1. To validate our proteomics data, we used quantitative Western blotting and densitometry to confirm the relative changes in protein abundance measured by LC-MS/MS (SI Appendix, Fig. 5C). There was good correlation of protein abundance ratios detected by the two different methods for several protein classes including cytoskeletal proteins (actin), cytoplasmic signaling proteins (p130Cas), and internal mem- brane proteins (␤-COP) (SI Appendix, Fig. 5C). However, in a few cases (Cdc42, RhoA, and Dock4) a correlation was not apparent. This likely reflects differences in the protein solubility Fig. 2. Potential Rac and Cdc42 interactomes. Interactome was generated as properties of the mass spectrometry compatible urea lysis buffer described in Material and Methods. Proteins are named by their official gene compared with the highly efficient 1% SDS buffer used for symbol according to Entrez Gene. Proteins with decreasing enrichment values Western blot analysis. Also, using the gene ontology resource are ordered from left to right. Subcellular localization is color-coded: blue, Babelomics (9), we performed a proteome-wide analysis of neurite enriched proteins; red, equally distributed proteins; yellow, soma protein subcellular localization (SI Appendix, Fig. 5D). Results enriched proteins. Solid lines represent interactions with Rac, whereas dotted from these studies revealed the expected subcellular distribution lines represent interactions with Cdc42. Green arrows point to proteins func- of neurite and soma proteins, which provides additional valida- tionally analyzed by siRNA in this study. A detailed description of each protein can be found in SI Appendix, Table 4. tion of our biochemical approach. Finally, several reported neurite marker proteins MAP1b (2.5ϫ), MAP2 (5ϫ), Tau ϫ ϫ ϫ (7.7 ), kinesin-1 (4.7 ), and kinesin-3 (3.5 ) (1) were enriched that apoptosis, JNK, and PDGF signaling pathways are highly in the neurite fraction. Together, these findings confirm our represented in the neurite relative to the soma (Fig. 1H and SI previous observations that LC-MS/MS can be used to determine Appendix, Fig. 5E). These pathways have not been previously quantitative differences in protein abundance between two linked to neurite formation. It is notable that such an analysis subproteomes obtained from fractionated cells. does not imply that all of the components of a defined signaling network are solely restricted to one subcellular domain. For Spatial Organization of Signaling Networks in Neurites and Somata. example, there are 6 proteins with the ‘‘axonal guidance signal- To find the functional interrelationship of the proteins enriched ing’’ descriptor enriched in the soma versus 64 in the neurite. in the neurite and soma proteomes and determine whether they Conversely, there are 15 proteins with the ‘‘estrogen receptor compartmentalize into distinct signaling networks, we used the signaling’’ descriptor enriched in the soma versus 8 in the neurite. Ingenuity Pathways Analysis (IPA) program and the Ingenuity Further description of the signaling networks present in the Pathways Knowledge Base (IPKB), which is a system wide database of biological pathways created from multiple relation- neurite and in the soma are provided in SI Tables 2 and 3 and ships of proteins, genes, and diseases (10). The IPA program can SI Appendix, Figs. 6 and 7 and SI Text. analyze large genomic or proteomic datasets to find the most statistically significant canonical pathways and protein networks Analysis of Rac and Cdc42 Signaling in Neuritogenesis. Previous work relevant to the dataset based on the calculated probability score has shown that Rac and Cdc42 regulate neuritogenesis by by searching the IPKB. The neurite proteome contained 39 coupling extracellular guidance cues to organization of the actin canonical pathways and the soma proteome contained 7 path- cytoskeleton. Although the activation of these two GTPases has ways, whereas the equally distributed protein pool contained 5 been correlated with neurite extension (3), a critical question pathways (Fig. 1H and SI Appendix, Fig. 5E). Interestingly, remains as to how they interact with multiple upstream regula- functional annotation of the neurite and soma proteomes re- tors and downstream effectors to achieve spatial regulation of vealed a high degree of spatial separation of signaling pathways the actin cytoskeleton. Therefore, we mined the proteomics between the two different cellular compartments. For example, datasets to identify all possible upstream regulators (GEFs and pathways that regulate guidance, the actin cytoskeleton, GAPs) and downstream effectors that could interact with Rac integrin, and signaling are highly represented in the and Cdc42, using IPKB and the gene ontology resource Babe- neurite, whereas pathways that regulate estrogen receptor sig- lomics (9). This revealed a complex putative Rac/Cdc42 signaling naling, cell cycle entry, and ubiquitination are dominant in the network with 13 GEFs, 7 GAPs, and 16 downstream effectors soma. This asymmetrical representation of signaling networks (Fig. 2; detailed information about each protein is found in SI likely reflects the highly polarized state of the neuron and its Appendix, Table 4). Interestingly, the majority of the GEFs (11 dedication to the neuritogenesis process. Consistent with high of 14) and GAPs (6 of 7) were enriched in the neurite, which is

Erk activity in the neurite (Fig. 1G), an ERK/MAP signaling consistent with the highly elevated Rac and Cdc42 activity in this CELL BIOLOGY pathway was found in the neurite. An unexpected finding was domain (Fig. 1F). Our findings strongly suggest that multiple

Pertz et al. PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 ͉ 1933 Downloaded by guest on September 29, 2021 Fig. 3. Morphodynamics and cytosketetal changes of cells transfected with the indicated GEF and GAP siRNAs. (A) Time-lapse series of control and ArhGAP30 siRNA transfected neurons. Neurite tip trajectory is shown in red. (Scale bar, 50 ␮m.) (B) Representative neurite tip tracks of control, ArhGAP30, Dock4, and Dock10 siRNA transfected cells. (Scale bar, 100 ␮m.) (C) Control, SrGAP2, and Trio siRNA transfected cells were allowed to spread for8honlaminin-coated coverslips and immunostained for actin (green), tubulin (red), and nucleus (blue). (D) Fluorescence micrographs of growth cone morphologies associated with BCR and SrGAP2 knockdowns. Cells were immunostained for actin (green) and tubulin (red). White arrows indicate high-density arrays of filopodia. The bar graph shows the occurrence of growth cones with ‘‘low’’ or ‘‘high’’ filopodia density from neurites longer than one soma length scored on four different regions of the coverslip from a 24-h time point. P values between control and specific knockdowns were computed by using a t test. (E) Time-lapse analysis of neurite formation of SrGAP2 and Trio knockdown cells. Red arrows indicate phase refractile globular structures indicative of retracting neurites. (Scale bar, 20 ␮m.)

signaling mechanisms regulate GTPase activation in the neurite were not affected (data not shown). These findings suggest that compartment. However, of the 16 downstream effectors, only 5 there is a high level of redundancy built into the Rac/Cdc42 were neurite-enriched, whereas 10 were equally distributed, and regulatory network that mediates neurite formation and that the 1 was enriched in the soma. Thus, the neurite enrichment of various GEFs and GAPs play a more subtle role in fine tuning Rac/Cdc42 effectors does not necessarily correlate with the neuritogenesis. However, the possibility remains that the small spatially increased GTPase activity. However, most effectors are residual level of protein present after knockdown is sufficient to regulated through intramolecular interactions independently of drive neurite extension. To investigate this possibility, we exam- changes in protein levels (11). ined the details of neurite formation of the various GEF and GAP knockdown cells with time-lapse video microscopy and Functional Characterization of Rac and Cdc42 GEFs and GAPs. The immunofluorescence staining of the actin-tubulin cytoskeleton. biological role of individual Rac and Cdc42 GEFs and GAPs are These studies revealed distinct phenotypes that affected neuri- beginning to be elucidated, but our understanding of how these togenesis in different ways and are summarized in SI Appendix, molecules are spatially organized within the dynamic cell at the Table 5. systems level has not been addressed. Our proteomic analysis Time-lapse analyses showed that neurons depleted of the GAP suggests that multiple GEF and GAP pathways operate in the ArhGAP30 or of the GEFs Dock4 and Dock10 displayed defects neurite to regulate Rac and Cdc42 activity. This prompted us to in neurite protrusion/retraction events compared with control determine which of the Rac and Cdc42 GEFs and GAPs are siRNA transfected or wild-type cells (Fig. 3A and SI Movies functionally required for neuritogenesis, using RNA interfer- 1–3). Specifically, these neurites displayed a more persistent and ence and cell-based assays of neurite formation. For these straight path leading to an overall increase in neurite length (Fig. studies, we picked the four most neurite-enriched GEFs (Dock4, 3B). A similar phenotype was observed when the GEF Itsn-l or Trio, Itsn1, and Dock10), the four most neurite-enriched GAPs the GAP ArhGAP17 were silenced although the alteration in the (ArhGAP21, ArhGAP30, SrGAP2, and BCR), and (as a con- extension/retraction events was somewhat milder (SI Movies 4 trol) the only soma-enriched GEF (Vav3) (Fig. 2, green arrows). and 5) with some residual retraction events. Although the We also analyzed ArhGAP30, a neurite-enriched, previously extension/retraction kinetics were altered in these cells, we did uncharacterized GAP of which the specificity is unknown. Of not detect changes in the actin-tubulin cytoskeleton by immu- these, the GEFs Trio, Itsn-l, and Vav3 (12) and the GAPs nofluorescence (data not shown). Finally, silencing of the SrGAP2, BCR, and ArhGAP17 have been linked to neurito- neurite-enriched GAP ArhGAP21 did not affect neuritogenesis genesis or other neuronal functions (12–17). The GEFs Dock4 (data not shown) nor did knockdown of the soma-enriched GEF and Dock10 and the GAPs ArhGAP21 and ArhGAP30 have not Vav3 (SI Movie 6). yet been linked to neuronal functions. In contrast to the regulators described above, silencing of the Potent knockdown was observed as measured by Western blot GAPs SrGAP2 and BCR or the GEF Trio strongly impacted the analysis or quantitative PCR (SI Appendix, Fig. 8), and in all actin cytoskeleton as observed by immunofluorescence staining. cases, siRNA transfected cells were viable and readily attached For both SrGAP2 and BCR, a potent defect of protrusion/ to the ECM proteins. Surprisingly, silencing of none of the retraction events leading to more persistent neurite extension different GEFs and GAPs prevented neurite formation. Rather, was again observed (SI Movie 7 and data not shown). Knock- in 7 of 10 knockdowns (all but Trio, ArhGAP21, and Vav3), a down of the Cdc42-specific GAP SrGAP2 increased initial cell potent increase in neurite length was observed (SI Appendix, Fig. spreading on the ECM with the formation of prominent lamel- 9). Other parameters, such as branching and number of neurites, lipodia characterized by a dense array of peripheral filopodia

1934 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706545105 Pertz et al. Downloaded by guest on September 29, 2021 (Fig. 3C). Time-lapse analysis showed that this occurred Ϸ3h served for ArhGAP21). Furthermore, many proteins could be after plating (SI Movie 7) and resulted in a 2.3-fold increase in activated locally by posttranslational modifications but still have cell surface area when compared with control cells (n ϭ 25 cells homogeneous subcellular distribution. This approach comple- each condition, P Ͻ 0.01, measured3hafterplating). This ments traditional imaging methodologies used to examine pro- increase in spreading then rapidly resolved. Also associated with tein compartmentalization and should readily transfer to other this phenotype was an increase in the formation of large cell types, guidance and collapsing agents. fan-shaped growth cones with prominent arrays of dense filop- Our findings provide a spatial view of the neurite and soma odia (Fig. 3 D and E and SI Movie 7). BCR knockdown led to proteomes of neuroblastoma cells extending neurite-like pro- a similar but less potent phenotype. However, this response was cesses in response to a directional cue. This revealed a prominent not associated with changes in cell spreading (data not shown). asymmetry in the signaling pathways present in both subcellular Silencing of the GEF Trio led to a defect in cell spreading (20% domains with pathways involved in sensing extracellular cues, decrease in cell surface area compared with control cells at the regulation of actin, and adhesion dynamics being enriched in the 24-h time point, n ϭ 140 cells for each condition, P Ͻ 0.01) with neurite. Our findings that the neurite contains a highly com- the formation of long and poorly organized filopodia emanating partmentalized and complex set of potential Cdc42 and Rac from the neurites (Fig. 3C). Time-lapse analysis revealed that GEFs and GAPs highlight an important question in this field. these neurites were highly unstable and frequently collapsed, but Why are there multiple GEFs and GAPs present in the cell to immediately extended again (Fig. 3E and SI Movie 8). However, regulate the same GTPase? Our functional analyses show that these neurites were eventually able to compensate for the defect these regulators control different cellular functions during neu- and by 24 h displayed neurites as long as those from control cells ritogenesis that cooperate to fine tune neurite extension. Thus, (SI Appendix, Fig. 9). Together, these findings indicate that these rather than Rac and Cdc42 regulating neurite extension per se, GEFs and GAPs play an important role in regulating various Rac our results suggest a more complex scenario in which different and Cdc42 dependent cellular functions that cooperate to fine GEFs and GAPs control the activity of multiple GTPase pools tune neurite formation. with distinct functions in time and space. The finding of up- The fact that knockdown of Rac and Cdc42-specific GEFs and stream regulators that impact directly on different aspects of the GAPs only led to impairment of subtle cellular functions during cytoskeleton or that act independently of it suggests that such neuritogenesis, prompted us to investigate whether Rac and spatiotemporal GTPase pools couple with distinct downstream Cdc42 were necessary for neurite formation. It is important to effectors to regulate the cytoskeleton, membrane trafficking, or note that inhibition of Rac and Cdc42 activity has been com- generation of polarity at precise subcellular localizations. It is monly achieved by using overexpression of dominant negative notable that most studies of neuritogenesis have relied on (DN) GTPase constructs. These constructs act by sequestering overexpression of DN GTPase mutants to link a specific GTPase multiple GEFs, which can affect nonspecifically multiple GT- to a particular cellular function (3). However, this approach can Pase pathways (18). Thus, we silenced endogenous Rac1 and titrate out GEFs indiscriminately, which could globally inhibit Cdc42, using RNA interference, and then evaluated neurite various neurite functions. This may explain the discrepancy with extension (summarized in SI Appendix, Table 5). In undifferen- the more subtle phenotypes observed in our study. This is tiated cells (i.e., in the presence of serum), knockdown of Rac or consistent with gene targeting studies, which show that Rac1 Cdc42 proteins (SI Appendix, Fig. 10A) reduced cell spreading knockout does not impair cell migration (21) and that Cdc42 (SI Appendix, Fig. 10 B and C) without any impairment in cell knockout does not impair filopodium formation and cell migra- viability (data not shown). Surprisingly, unlike with expression of tion (18). These findings, together with our results, suggest the DN Rac or Cdc42, which inhibits neurite extension (7), siRNA possible involvement of compensatory mechanisms involving knockdown of Rac or Cdc42 protein did not significantly inhibit structurally related GTPases with redundant functions such as neurite outgrowth. In fact, Cdc42 knockdown actually led to a Rac3, which is known to be expressed in the developing brain significant increase in neurite length (SI Appendix, Fig. 10 D and (20), and the Cdc42-related proteins TC10 and TCL, which E). Also, no obvious changes in the actin cytoskeleton were control filopodium formation during N1E-115 neuronal differ- observed in cells with reduced Rac or Cdc42 protein levels. entiation (19). These GTPases might also respond to the differ- Indeed, these cells exhibited normal growth cones and filopodial ent GEFs and GAPs reported here. Alternatively, GEFs and structures when compared with control cells (SI Appendix, Fig. GAPs might also regulate signaling mechanisms that operate 10 F and G). The increase in neurite length in Cdc42 knockdown independently of Rho family GTPases during neuritogenesis. In cells was due to a loss of neurite protrusion and retraction cycles any case, the lack of neurite protrusion/retraction events asso- (SI Movie 9) as described above for knockdown of the Cdc42 ciated with Cdc42 knockdown is consistent with the phenotypes specific regulators Itsn-1, Dock10, and ArhGAP17. Rac knock- obtained from the knockdown of the Cdc42-specific GEFs down did not significantly alter neurite dynamics (SI Movie 10). Dock10 and Itsn-1 and the GAP ArhGAP17. These results indicate that reducing Rac and Cdc42 protein Although our findings provide initial insight into the signaling levels does not significantly impair neuritogenesis under these complexity of this system, additional work will be necessary to conditions. This may be due to functional compensation by the fully appreciate the various levels of regulation of the Rac/Cdc42 structurally related GTPases, Rac3, Tc10, and TcL (19, 20). signaling module containing various GEFs, GAPs, and related GTPases. In this regard, it will not be possible to rescue our Discussion siRNA defects by using overexpression of mutant active forms of The present study introduces a simple method to selectively Rho family GTPases, because this would lead to global GTPase isolate neurites from the somata of neuron-like neuroblastoma activation throughout the entire cell. Rather, this will require cells in large scale for biochemical analyses. This opens up methods that reveal signaling in space and time, including unique possibilities to not only globally monitor the spatial biochemical approaches, like the neurite purification assay or the organization of proteins in these two different cellular compart- use of fluorescent probes that measure spatiotemporal dynamics ments but also directly examine protein regulatory activities of GTPase activation. Finally, the finding that the soma-enriched involving posttranslational modifications (e.g., phosphorylation, GEF Vav3 is not involved in neurite extension in our model but ubiquitination) and protein–protein interactions that dictate is essential for -induced neuritogenesis (12) neuritogenesis. This will be important in the future, because suggests that different GTPase signaling programs can be acti-

neurite localization of a protein does not necessarily mean that vated depending on the extracellular cue that triggers neurito- CELL BIOLOGY it should be functionally important for neuritogenesis (as ob- genesis. Our neurite purification system provides a model to

Pertz et al. PNAS ͉ February 12, 2008 ͉ vol. 105 ͉ no. 6 ͉ 1935 Downloaded by guest on September 29, 2021 explore the different signaling mechanisms that drive neurito- the pores for 24 h in serum-free media. The microporous filters were then fixed genesis in response to various biological cues. Although neuro- by a 20-min incubation in ice-cold methanol, and the neurites on the filter blastoma cells and the filter purification system recapitulate bottom or the somata on the filter top or where scraped away, using a cotton many aspects of neuritogenesis, it will be important to confirm swab. The remaining structures were then solubilized in the appropriate lysis buffer. For routine Western blot analysis, a 1% SDS buffer containing protease these findings in primary neurons. Further optimization of the inhibitors and 2 mM Vanadate was used. This typically yielded 30–40 ␮gof sensitivity of the large-scale proteomic methods described here neurite versus 800 ␮g of soma sample, necessitating the pooling of multiple should make it possible to achieve similar results with smaller filters for any biochemical experiment. For proteomics studies, a buffer con- amounts of cell lysate obtained from isolated primary neurons. taining 40 mM Tris (pH 8.4), 7 M urea, 2 M thiourea, 0.5% Nonidet P-40, 2 mM In summary, the ability to purify the neurite and its integration Vanadate, 50 mM Calyculin A, and protease inhibitors were used. Five milli- with contemporary proteomics allowed us to spatially map the grams of neurite and soma each were generated for the proteomics analysis. neurite and soma proteomes, creating a comprehensive database Multiple soma and neurite preparations were pooled to generate the 5 mg of each fraction needed here. The samples were then digested with trypsin, and for investigation of neuritogenesis and cell polarization. Pro- the resulting peptides were desalted and quantified. For active Rac and Cdc42 teomic and functional analyses revealed a complex Rac and pulldowns, which necessitates native conditions, the fixation step was omitted Cdc42 regulatory network consisting of multiple GEFs and and the microporous filters were allowed to cool to 4°C. After the scraping GAPs that regulate distinct cellular processes during neurito- procedure, solubilization and pulldowns were then performed as described by genesis. Future work to integrate our findings with emerging the manufacturer (Millipore–Upstate). The pulldown experiments were per- genomic and proteomic datasets representing human disease formed on 200 ␮g of lysate. Note that the native conditions used here were states will provide valuable insight into neuropathology and provide not able to solubilize all Rac1 and Cdc42 as with the 1% SDS buffer. Thus, a GTPase pool is not soluble under these conditions and is not accessible for important resources to develop disease-related diagnostics. immunoprecipitation. See SI Appendix, SI Text for additional information. Materials and Methods ACKNOWLEDGMENTS. We thank Monica Holcomb for early contributions to Neurite Sample Preparation and Biochemical Analysis. N1E-115 neuroblastoma this research project; Konstantin Stoletov for help with Imaris software; and cells (American Tissue Culture Collection) were cultured in Dulbecco’s modi- Dr. H. Katoh, M. Umeda, and W. Balch for providing antibodies. This work was fied Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% FBS, 1% supported by Susan G. Komen Foundation Grant PDF0503999 (to Y.W.), Gln, and 1% gentamycin. For differentiation, N1E-115 cells were starved for National Institutes of Health Grants GM068487 (to R.L.K.) and CA097022 (to 24 h in serum-free media optimized for neuroblastoma neuronal differenti- R.L.K.), and Cell Migration Consortium Grants GM064346 (to R.L.K.) and RR018522 (to R.D.S). The Environmental Molecular Sciences Laboratory is a ation (Millipore–Chemicon). Cells were then detached, and 1.2 ϫ 106 cells U.S. Department of Energy national scientific user facility located at Pacific ␮ were replated on a 2.4-cm wide 3- m microporous filter that had previously Northwest National Laboratory, a multiprogram national laboratory oper- been coated on the bottom surface with 10 ␮g/ml laminin (Millipore– ated by Battelle for the U.S. Department of Energy under Contract DE-AC05- Chemicon) for2hat37°C. Cells were then allowed to extend neurites through 76RL01830.

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