RESEARCH ARTICLE Cellular processes underlying maturation of P19 neurons: Changes in protein folding regimen and cytoskeleton organization Alex Inberg * , Yoel Bogoch * , Yaniv Bledi and Michal Linial Department of Biological Chemistry, Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along dif- ferentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm 2 ) or dense (4000 cells/mm 2 ) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite out- growth. The protein expression levels of the sparse and dense cultures were compared 48 h fol- lowing RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (.four- fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, a-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions. Received: July 21, 2006 Revised: September 29, 2006 Accepted: November 13, 2006 Keywords: Differential expression / Embryonic cancinoma / Neurite outgrowth / Neuronal differ- entiation / Synaptic plasticity 910 Proteomics 2007, 7, 910–920 1 Introduction P19 cells are embryonal carcinoma (EC) cells that serve as a model for studying differentiation processes including com- mitment to cell lineage [1]. Following cell aggregation in the presence of retinoic acid (RA), P19 cells differentiate into a neuroectodermal lineage of neurons and glial cells. In a procedure that eliminates all dividing glial cells a homo- genous postmitotic neuronal culture is maintained [2, 3]. This culture is thus suitable for studying neuronal differ- entiation by genomic and proteomic approaches. Undifferentiated P19 cells are fast dividing cells. In the presence of RA, morphogenesis is induced by activation of critical signaling pathways including the Wnt [4, 5] and BMP [6]. Gene expression changes associated with early stages of differentiation in P19 have been reported [7]. Interestingly, changes in gene expression along differentiation of P19 neurons resemble those of embryonal development in the mammalian CNS [8]. Correspondence: Professor Michal Linial, Department of Biologi- cal Chemistry, Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel E-mail: michall@cc.huji.ac.il Fax: 1972-2-6586448 Abbreviations: HSP, heat shock protein; IF, intermediate filament; NF-M, medium-sized neurofilament; RA, retinoic acid; TCP-1, T- complex 1 * Both authors contributed equally. DOI 10.1002/pmic.200600547  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com