Differential phosphoproteome of the regenerating radial nerve cord of the sea star M. glacialis Franco C.1, Soares R.1, Pires E.1, Santos R.2, and Coelho A.V.1 1 Laboratório de Espectrometria de Massa, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. 2 Unidade de Investigação em Ciências Orais e Biomédicas, Faculdade de Medicina Dentária, Universidade de Lisboa, Portugal Echinoderms, as invertebrate deuterostomes, have and amazing neuronal intrinsic growth capability, which can be triggered at any time point during the animal lifespan, leading to a successful functional tissue re-growth, a trait well known to be in opposition of their mammal close phylogenic relatives, which have completely lost their ability to regenerate their central nervous system. Although this intrinsic echinoderm trait is of a promising nature, only recently this complex jigsaw has started to be assembled. In this study, we used a 2-DE gel based phosphoproteomics approach to investigate injury related changes is the phosphorylation of proteins from a soluble fraction of the injured sea star radial nerve cord collected 48h and 13 days following arm tip ablation. Over 500 spots were resolved in 3.0-5.6 NL pH strips of which 190 and approximately 140 spots had a phosphoprotein signal in the controls and in the injury experimental groups respectively. A total of 47 different proteins were identified with MALDI-TOF/TOF, several of which presenting an injury correlated phosphorylation. Altogether, several intervenients of important injury signalling pathways, which seem to be modulated through phosphorylation, were identified in the sea star radial nerve cord early regeneration events and never before reported in the echinoderm nervous system regeneration machinery. These include cytoskeleton re-organization towards the formation of the neuronal growth cones; membrane rearrangements; actin filaments and microtubules dynamics; mRNA binding and transport; lipid signalling; Notch pathway; calcium activated pathways regulated through calmodulin binding and; neuropeptide processing. Experimental approach 1| Experimental groups 2| Arm tip ablation 3| Protein extraction and solubilization 4| 2DE and phosphoprotein analysis 5| Protein identification with MADLI-TOF/TOF 6 regenerating animals 6 control 48h post arm tip ablation Accumulation of cells at wound site Little proliferation 3 pI 5.6 NL ProQ Diamond Protein extraction buffer 8M Urea 2M Thiorea 4% CHAPS 6 regenerating animals 6 control 10-15 days post arm tip ablation Cell proliferation Increases Re-growth of lost tissues Sypro Ruby Radial nerve cord collection Statistical analysis was carried out on the determined phosphorylation ratio, namely mean±SE and student's t-test. Four technical replicates were preformed per experimental group Protein identification was performed using the MOWSE (Mascot software v.2.2) and the Paragon (Protein Pilot software v. 3.0) D B 1 Control nerves Fig. 1 - Sea star radial nerve cord 2D-PAGE gel (1: 7cm IPG strip, pI 3-10 and 2: 7cm IPG strip, pI 3-5.6) stained with A) Colloidal Coomassie, B) Pro-Q Diamond. C) Overlay image of the two staining procedures (Blue: Colloidal Coomassie; Red: Pro-Q Diamond), D) Sypro Ruby Regenerated nerves 2 Fig. 2 – Comparison of phosphorylation ratio between sea star regenerated nerve tissue and control sample Protein id Accession no. Phosphorylation status Number of id peptides Biological function Spectrin gi|115920116|ref|XP_785949.2| Phosphorylated in control (unique) 2 AXON GUIDANCE/DEVELOPMENTAL/CYTOSKELETON DYNAMICS Calmodulin gi|115960363; gi|115728588; sp|A8CEP3|CALM_SACJA; UniRef100_P62184 Phosphorylated in 48 h, 13d, and control 10 CALCIUM/DEVELOPMENT/CYTOSKELETON DYNAMICS/SYNAPTIC TRANSMISSION/PHOSPHATASE ACTIVITY/KINASE REGULATION START domain-containing protein A9ZT01 7 CYTOSKELETON DYNAMICS/DEVELOPMENTAL Calpain-like protease 1 sp|Q03792|RIM13_YEAST Phosphorylated in 48h and 13 d 1 CYTOSKELETON DYNAMICS/SIGNALING/RETROGRADE TRANSPORT Notch homolog gi|115941626|ref|XP_001187416.1| DEVELOPMENTAL/REGENERATION/ACTIVATED BY PROTEOLYSIS Neuroendocrine convertase sp|Q5REC2|NEC2_PONAB Phosphorylated in 48h and control 3 GO biological function: PROTEIN AUTOPROCESSING; NERVOUS SYSTEM DEVELOPMENT; ISLET AMYLOID POLYPEPTIDE PROCESSING; INSULIN PROCESSING; ENKEPHALIN PROCESSING Table 1 – Examples of Sea star radial nerve cord phosphorylated proteins identified from 2D-PAGE gels Results: 72 spots were only observed in the control sample and 8 spots in the regenerated tissue. 32 spots only in the regenerated tissue after 48h, and 17 spots only in the regenerated tissue after 13d when comparing with the control groups Proteins involved in several important biological processes, like cytoskeleton dynamics, signaling, phosphatase/kinase regulation and regeneration were identified. Several proteins were found to be phosphorylated in the injured radial nerve cord included for example serine protease calpain, START domain protein, being involved in activity modulation and signaling during regeneration events Dephosphorylation of proteins also occurred in the injured nerve cord, namely spectrin, Notch homologue and neuroendocrine convertase, being involved in biological processes such as degradation for cytoskeleton rearrangements, cell differentiation and maturation of signaling neuropeptides during radial nerve cord regeneration events. Dephosphorylation/phosphorylation events are critical to the regenerative responses during radial nerve cord wound healing events Future work: Confirmation of phosphorylated proteins and identification of the phosphorylation site using MSn tandem experiments Acknowledgements To Fundação para a Ciência e Tecnologia for grants (PhD grant of Catarina Franco, ref. SFRH/BD/29799/2006; Post-Doc grant of Romana Santos, ref. SFRH/BPD/33091/2007, and project grant ref. PTDC/MAR/104058/2008), Ciência 2008 of Renata Soares, and for the financial support on equipment acquisition within the framework of the National Re-equipment Program — National Network Mass Spectrometry (REDE/1504/REM/2005). Angela Barreto for the staining procedures.