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Localized Role of CRMP1 and CRMP2 in Neurite Outgrowth and Growth Cone Steering

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Localized Role of CRMP1 and CRMP2 in Neurite Outgrowth and Growth Cone Steering Localized Role of CRMP1 and CRMP2 in Neurite Outgrowth and Growth Cone Steering Masakazu Higurashi,1,2 Masumi Iketani,1 Kohtaro Takei,1 Naoya Yamashita,1 Reina Aoki,1 Nobutaka Kawahara,2 Yoshio Goshima1 1 Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan 2 Department of Neurosurgery, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan Received 21 December 2011; revised 22 February 2012; accepted 23 February 2012 ABSTRACT: Collapsin response mediator protein 1 domain of the growth cones consistently arrested neurite (CRMP1) and CRMP2 have been known as mediators of outgrowth, whereas micro-CALI of CRMP1 in the same extracellular guidance cues such as semaphorin 3A and region caused significant lamellipodial retraction, followed contribute to cytoskeletal reorganization in the axonal by retardation of neurite outgrowth. Focal inactivation of pathfinding process. To date, how CRMP1 and CRMP2 CRMP1 in its half region of the growth cone resulted in the focally regulate axonal pathfinding in the growth cone has growth cone turning away from the irradiated site. not been elucidated. To delineate the local functions of Conversely, focal inactivation of CRMP2 resulted in the these CRMPs, we carried out microscale-chromophore- growth cone turning toward the irradiated site. These find- assisted light inactivation (micro-CALI), which enables ings suggest different functions for CRMP1 and CRMP2 in investigation of localized molecular functions with highly growth cone behavior and neurite outgrowth. ' 2012 Wiley spatial and temporal resolutions. Inactivation of either Periodicals, Inc. Develop Neurobiol 72: 1528–1540, 2012 CRMP1 or CRMP2 in the neurite shaft led to arrested Keywords: CRMP1; CRMP2; CALI; neurite neurite outgrowth. Micro-CALI of CRMP2 in the central outgrowth; growth cone steering INTRODUCTION located at the distal tip of the growing axon. The growth cone plays a central role in this motility through Development of neural circuitry requires an appropriate reorganization of cellular cytoskeletons. A number of axonal pathfinding process, which consists of elastic studies have demonstrated that coordination of the cy- advance and flexible steering of the growth cone toskeleton, i.e., actin filament (AF) and microtubule (MT), at the leading edge of the growth cone and in filopodia is indispensable to accomplish proper asym- Additional Supporting Information may be found in the online metrical motility across the growth cone in accordance version of this article. Correspondence to: Y. Goshima ([email protected] with the gradient of diverse extracellular guidance mol- cu.ac.jp) or K. Takei ([email protected]). ecules (Challacombe et al., 1997; Nakamura et al., Contract grant sponsors: Grants-in-aid for Scientific Research in 2001; Fukata et al., 2002a; Zhou and Cohan, 2004; a Priority Area from the Ministry of Education, Science, Sports and Culture, Japan Society for the Promotion of Science Research Lowery and Van Vactor, 2009; Tojima et al., 2011). Fellowships for Young Scientists, and the Yokohama Medical Collapsin response mediator protein (CRMP) was Foundation. originally identified as a cytosolic phosphoprotein ' 2012 Wiley Periodicals, Inc. that mediated semaphorin 3A (Sema3A) signaling Published online 29 February 2012 in Wiley Online Library (wileyonlinelibrary.com). (Goshima et al., 1995). CRMP is now known to con- DOI 10.1002/dneu.22017 sist of five homologue proteins CRMP1–5, all of 1528 Localized Roles of CRMP1 and CRMP2 in Neurite Outgrowth 1529 which are phosphoproteins and are highly expressed clonal; Sigma), Cy3-labeled goat anti-Armenian hamster in developing nervous systems (Hamajima et al., 1996; (Jackson ImmunoResearch), Alexa488 or 594-labeled phal- Wang and Strittmatter, 1996; Fukada et al., 2000; loidin, Alexa488 or 594-labeled goat anti-mouse, and Yuasa-Kawada et al., 2003; Uchida et al., 2005; Alexa488-labeled donkey anti-rat (Invitrogen). Yamashita et al., 2006, 2007, 2011). CRMP has been shown to be an intracellular molecule directly or indi- Cell Culture rectly regulating cytoskeletal organization and mem- brane trafficking (Fukata et al., 2002b; Nishimura A primary dissociated cell culture of chick dorsal root gan- et al., 2003; Kawano et al., 2005; Rosslenbroich et al., glion (DRG) neurons was prepared basically as previously described (Takei et al., 1999). DRG neurons from embry- 2005; Schmidt and Strittmatter, 2007; Hensley et al., onic Day 7 (E7) chick embryos were dissociated by trypsi- 2010), which is orchestrated in response to various nization with 0.25% trypsin (Invitrogen) in phosphate-buf- kinds of extracellular guidance cues, including sema- fered saline (PBS) for 18 min. The neurons were then cul- phorins, Reelin, neurotrophins, myelin-derived axonal tured with Leibovitz-15 medium (Invitrogen) containing growth inhibitors, and lysophosphatidic acid (Goshima nerve growth factor (Wako), glucose, L-glutamine, gentami- et al., 1995; Arimura et al., 2000; Quach et al., 2004; cin, and fetal bovine serum (Biowest) on a glass base dish Mimura et al., 2006; Uchida et al., 2005, 2009; Yoshi- (Iwaki) coated with poly-L-lysine (100 lg/mL, Wako) and mura et al., 2005, 2006, 2011). laminin (10 lg/mL, Invitrogen). The cultured cells were Our previous RNAi knockdown or dominant-nega- incubated at 378C for at least 2 h, by which time most neu- tive experiments in cultured sensory neurons revealed rons had initiated neurite outgrowth. that both CRMP1 and CRMP2 mediate Sema3A- induced growth cone collapse through their phospho- Immunocytochemistry rylation (Uchida et al., 2005). However, respective role of each CRMP family protein in growth cone behavior Chick DRG cultures were incubated at 378C for 3 h. The cells were fixed in warmed 4% paraformaldehyde in culture have not been addressed. To elucidate the local medium at 378C for 10 min followed by exposure to room functions of CRMP1 and CRMP2, we conducted a temperature for 10 min. After being rinsed with PBS, the light-mediated acute protein-ablation method called cells were permeabilized with 0.1% Triton X-100 (Sigma) microscale-chromophore-assisted light inactivation in PBS for 5 min, blocked with 1% normal goat serum (micro-CALI). This method of acute protein ablation (Vector) in 0.1% Triton X-100 for 1 h. In case of CRMP2, uses laser-light irradiation to direct spatially restricted the neurons were probed primarily with anti-CRMP2 photogenerated hydroxyl radical damage to target pro- (1:1000) and anti-tyrosinated-tubulin (1:2000) antibodies teins through malachite green isothiocyanate (MG)- overnight at 48C. They were then probed secondarily with conjugated antibodies (Jay, 1988; Liao et al., 1994). fluorescence-conjugated antibodies (1:1000) or rhodamine- Micro-CALI allows preferential and acute inactivation conjugated phalloidin (1:250) for 1 h at room temperature. of protein functions with highly spatial and temporal For CRMP1, the cells were additionally altered in 4% form- amide at room temperature for 10 min after paraformalde- resolutions without affecting other cellular functions. hyde fixation. After the blocking process, the cells were Here, we found that CRMP1 and CRMP2 play different probed primarily with anti-CRMP1 (1:100) and with anti-b- roles in neurite outgrowth and growth cone steering. actin (1:1000) or anti-tyrosinated-tubulin antibodies, then probed secondarily with fluorescence-conjugated antibodies MATERIALS AND METHODS (1:1000). The cells were observed at a water-immersed objective of 363 (C-apochromat/1.2 W korr) with a laser- scanning microscope (LSM510, Carl Zeiss) equipped with Antibodies an Axioplan 2 imaging microscope (Carl Zeiss). In all Hamster anti-CRMP1 monoclonal antibody (2C6G) was cases, matched control immunostainings were carried out raised as previously described (Yamashita et al., 2006, with nonimmune immunoglobulin G (IgG) or without a pri- 2007). Mouse anti-CRMP2 monoclonal antibody (9F) was mary antibody. The quantitative analyses of cellular distri- raised by injection of a C-terminal region of human bution were measured with the Metamorph software pro- CRMP2 (amino acid 486–528) into a Balb/c mouse. Their gram (Molecular Device). binding specificity was confirmed by immunocytochemistry and immunoblot analysis using COS7 cells and HEK293T Immunoblot Analysis cells transfected with each CRMP-Myc (CRMP1–5) expressing vector [Supporting Information Fig. 1(A,B)]. Sample lysate was prepared and homogenized in immunopre- Each CRMP in chick cells were also detected specifically cipitation buffer [20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 by these antibodies [Supporting Information Fig. 1(C)]. mM EDTA, 10 mM NaF, 1 mM Na3VO4, 1% Nonidet P-40, Other antibodies used were anti-tyrosinated-tubulin (rat 50 lM q-amidinophenylmethanesulfonyl fluoride, and monoclonal; Harlan Sera-Lab), anti-b-actin (mouse mono- 10 lg/mL of aprotinin]. The lysates were centrifuged at Developmental Neurobiology 1530 Higurashi et al. 1200 rpm for 15 min at 48C, and the supernatants were cytochemistry by simultaneously staining actin or normalized for total protein concentrations. Proteins were tubulin. Although both CRMP1 and CRMP2 were subjected to sodium dodecyl sulfate-polyacrylamide gel elec- distributed throughout the growth cone, quantitative trophoresis (8% gel) in the Laemmli buffer system. After elec- data showed some differences between them. trophoretic transfer to polyvinylidene fluoride membranes CRMP1 was uniformly localized
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