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Spatial Mapping of the Neurite and Soma Proteomes Reveals a Functional Cdc42/Rac Regulatory Network

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Spatial Mapping of the Neurite and Soma Proteomes Reveals a Functional Cdc42/Rac Regulatory Network Spatial mapping of the neurite and soma 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 growth cone 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 cytoskeleton 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 axons or an elaborate dendritic arbor. This and have been well characterized for their neuron-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 neurons (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 cytoskeletons (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
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