Neuroscience Letters 582 (2014) 38–42
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Coronin 1A inhibits neurite outgrowth in PC12 cells Yunus Kasim Terzi, Yusuf Cetin Kocaefe, Sukriye Ayter ∗ Hacettepe University, Faculty of Medicine, Department of Medical Biology, Sihhiye, 06100, Ankara, Turkey
h i g h l i g h t s • Remodeling of actin cytoskeleton plays an important role in axonal sprouting. • Overexpression of Coronin 1A in PC12 cells effects neurite outgrowth. • Coronin 1A has an inhibitory effect on neurite outgrowth in vitro.
a r t i c l e
i n f o
Article history: Received 11 June 2014 Received in revised form 21 August 2014 Accepted 25 August 2014 Available online 30 August 2014 Keywords: Nervous system Regeneration Coronin 1A Neurite outgrowth Cytoskeleton PC12 cell line
a b s t r a c t Regenerative response to central nervous system damage in mammals is limited because of inhibitor signals which consist of myelin associated inhibitor proteins and chondroitin sulfate proteoglycans. Inhibitor signals mainly affect cytoskeleton elements which are important for axonal sprouting and neurite outgrowth. Coronin 1A is an actin cytoskeleton associated protein. Coronin 1A shows its effect on actin cytoskeleton through binding to the Arp2/3 complex which is a key nucleator of actin polymerization and regulates its activation on actin cytoskeleton. Coronin 1A–Arp2/3 interaction is regulated by phosphorylation of Coronin 1A from the C and N terminal region. Thus, Coronin 1A–Arp2/3 complex is one of the targets of inhibitory signaling cascades. The aim of this study was to investigate the effect of Coronin 1A on neurite outgrowth in PC12 cells in vitro. The results showed that Coronin 1A is expressed in differentiated PC12 cells and localized along axonal sprouting region of the neurites. Other results showed that overexpression of Coronin 1A in PC12 cells effects neurite outgrowth. Neurite lengths of the Coronin 1A overexpressing PC12 cells were lower than the untransfected (p < 0.001) and control transfected (p = 0.002) PC12 cells. These results indicate that Coronin 1A has an inhibitory effect on neurite outgrowth in vitro. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cytoskeleton organization and remodeling are essential cellular events during sprouting and neurite outgrowth [1]. Correct spatial and temporal organization of actin cytoskeleton, which is regulated by the assembly and disassembly processes, is important for the establishment of neuronal connections [2–4]. A number of actin binding proteins have been shown to orchestrate actin cytoskeletal dynamics, and these include the coronin family of proteins [5,6]. Coronin was originally identified as an F-actin binding protein enriched at the leading edge and at phagocytic cup structures of the slime mold Dictyostelium discoideum [5]. It was shown that one of the coronin protein family members Coronin 1A directly binds to Arp2/3 protein complex [7], and inhibits F-actin nucleation by freezing the Arp2/3 complex in its inactive “open” conformation
∗ Corresponding author. Tel.: +90 312 292 4434; fax: +90 312 292 4432. E-mail addresses: [email protected], [email protected] (S. Ayter). http://dx.doi.org/10.1016/j.neulet.2014.08.044 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
[8–11]. The interaction between Coronin 1A and Arp2/3 is regulated by phosphorylation of Coronin 1A from both the C and N terminal regions with protein kinase C [7,8,12,13]. However, so far the role of Coronin 1A is only investigated in the immune system, due to the specific expression pattern in the phagocytic cells [14]. On the other hand, it was reported that the expression of Coronin 1A was significantly upregulated in rats following the spinal cord injury [15]. In addition, while this paper was prepared, Jayachandran et al. [16] published their data about the effects of Coronin 1A in nervous system, and showed that Coronin 1A deficiency may cause severe neurobehavioral and cognitive defects in mouse. The above observations prompted us to postulate that Coronin 1A could play a role in regulating cytoskeleton organization during axon formation or neurite outgrowth in neuronal cells. Although the role of Coronin 1A in immune system cells has already been investigated in numerous studies, this is the first study where the effects of Coronin 1A on neurite outgrowth were investigated, and the inhibiting effects on neurite outgrowth were shown.
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
2. Materials and methods 2.1. Cell culture PC12 cells (ATCC CRL-1721) were cultured in RPMI 1640, 15% horse serum, 5% fetal calf serum supplemented (media and serums from Gibco/Invitrogen) with 2 mM l-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, at 37 ◦ C in a humidified and 5% CO2 incubator. The cells were grown on collagen type 4-coated (Sigma) (40 g/l) flasks and glass coverslips. To provide the differentiation of PC12 cells, only 1% horse serum was used in complete medium (differentiation medium). 2.2. Immunofluorescence staining PC12 cells were grown on collagen type 4-coated glass coverslips. The cells were cultured for 24 h in differentiation medium and treated with and without NGF (50 ng/ml) (Millipore) for the times indicated (0, 24, 48, 72, 96, and 120 h). The cells were fixed with 4% (w/v) paraformaldehyde (Sigma) in PBS. The localizations of Coronin 1A and cytoskeletal elements (neuron specific III-tubulin and actin) were documented by incubation with Coronin 1A mouse monoclonal antibody (1:100) (Novus Biologicals), rabbit polyclonal anti-actin antibody (1:100) (Abcam), and mouse monoclonal antiIII tubulin antibody (1:250) (Abcam), respectively. Alexa Fluor 488 and 568 anti-rabbit, and anti-mouse (Molecular Probes/Invitrogen) were used as secondary antibodies (1:500 for both of them). Nuclei staining were carried out by incubation with DAPI. The coverslips were mounted in ProLong Gold antifade reagent (Molecular Probes/Invitrogen) and immunofluorescence images were acquired with trinocular inverted microscope with fluorescent attachment (Leica DMIL, Leica Microsystems, Heidelberg, Germany). 2.3. Western blot analysis PC12 cells were grown on collagen type 4-coated 25 cm2 culture flasks. For the experimental procedure, the cells were cultured for 24 h in differentiation medium and treated with and without NGF (50 ng/ml) (Millipore) for the times indicated (0, 24, 48, 72, 96, and 120 h). Lysed in blending buffer (62.5 mM Tris–HCl (pH 6.8), 5 mM EDTA, 10% SDS (pH 7.2), and mini protease inhibitor tablet). Bicinchoninic acid (BCA) Protein Assay Kit (Pierce) was used to determine the protein concentrations of cell lysates. Proteins (40 g per lane) were separated using SDS-polyacrylamide gels under reducing conditions, and transferred to nitrocellulose membrane (Thermo Scientific). Blots were probed with anti-Coronin 1A antibody (1:500). Goat anti-rabbit IgG HRP-conjugated secondary antibody (1:3000) (Molecular Probes/Invitrogen) was used for protein band detection. The blots were developed by ECL Plus chemiluminescence kit (Amersham Biosciences). To monitor the amount of protein loaded, membranes were stripped and reprobed with mouse anti--actin antibody. For quantitative Western blotting, densitometric analyses of the blots were made by Scion Image software (www.scioncorp.com). Data were calculated as the ratio of arbitrary densitometric units of Coronin 1A immunoreactive bands normalized to values obtained for -actin bands on the same immunoblots.
39
as follows: 2 (g):10 (l) DNA to Fugene HD ratios. The transfection complex medium was removed 48 h later and replaced with differentiation medium with or without addition of 50 ng/ml NGF. The efficiency was calculated by observing pEGFP expression 24 h after transfection. 2.5. Neurite outgrowth analysis We performed neurite outgrowth analyses in four groups: The first group was untransfected (T(−)) PC12 cells, the second groups was Coronin 1A overexpressing (T(+)) PC12 cells, the third and the fourth groups were control groups where transfection agent (Fugene HD), and pEGFP vector were applied alone to cells to evaluate the effects of transfection procedure, and also expression vector on neurite outgrowth, respectively. Neurite outgrowth analysis was performed as described by Fournier et al. [17] with some modifications. Briefly, the induction of differentiation in PC12 cells was evaluated by counting the proportion of cells bearing neurites and measuring the length of the longest neurite in individual cells. Cells bearing neurites with length greater than twice the cell body diameter were considered positive and neurite lengths were measured by using Leica Application Suite program (V2.4.0 R1). Tissue culture plates were photographed with 20× magnification, and 120 ± 20 cells were analyzed per tissue culture plate. The experiments were performed in biological triplicates. Mean values (SD) were determined for each time point and the length of the Coronin 1A overexpressing neurite cones were compared to that of control cultures. 2.6. Statistics Nonparametric Kruskal–Wallis test was used for analysis of difference between groups. Significant differences between groups were determined using Mann–Whitney U test. A p value less than 0.05 was considered as statistically significant. All statistical analyses and tests were performed with the SPSS statistical package (SPSS 11.0 for Windows, Chicago, IL, USA). 3. Results 3.1. Localization and expression of Coronin 1A in PC12 cells Localization of Coronin 1A and its association with cytoskeleton (actin and microtubules) were documented by using immunofluorescence (Fig. 1). Immunofluorescence staining (Fig. 1A) showed that Coronin 1A has been localized at the cortical cytoskeleton, cytoplasm, neurites and the neurite outgrowth cone overlapping with the actin cytoskeleton. To determine the expression of Coronin 1A in the course of PC12 cell differentiation, immunoblotting was performed (Fig. 2). We determined the Coronin 1A specific band at 57 kDa by immunoblotting. Coronin 1A expression level was evaluated with densitometric analysis by using Scion Image program during differentiation of PC12 cells. 3.2. Localization and expression of Coronin 1A in Coronin 1A/pEGFP co-transfected PC12 cells
2.4. Transfection PC12 cells were plated 24 h before the transfection with antibiotic free medium at a density of 72,000 cells/cm2 and transfected using Fugene HD (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s recommendations using the following constructs: pQE-TriSystem-6-Coronin 1A (Qiagen) and pEGFP C2 (Clontech, Palo Alto, CA). Transfection mix was prepared
Coronin 1A overexpressing (T(+)) PC12 cells were analyzed by immunofluorescence staining to document any changes of Coronin 1A localization. Results of immunofluorescence staining revealed that intracellular distribution of Coronin 1A was similar to T(−) PC12 cells and was localized at the cortical cytoskeleton, cytoplasm, neurites and the neurite outgrowth cone overlapping with the actin cytoskeleton (Fig. 3).
40
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
Fig. 1. Immunofluorescence staining of untransfected PC12 cells after 96-h differentiation. Coronin 1A, is stained in red (a and d), actin and neuron specific III tubulin are stained in green (b and e, respectively) and nuclei are stained in blue by DAPI. Coronin 1A and actin immunofluorescence staining patterns have similar localizations in the cell, at the cortical cytoskeleton, cytoplasm, neurites and neurite outgrowth cone (a–c). Overlapping staining of Coronin 1A and actin cytoskeleton are shown by white arrows (c). Neuron specific III tubulin and Coronin 1A reveal different staining localizations in the cell (d–f). Bar is 10 m for a–d, and 20 m for e–f. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Immunoblot analysis of Coronin 1A expression during PC12 differentiation. Specific immunoreactive band for Coronin 1A was observed at 57 kDa. Expression level was evaluated by densitometric analysis and no change was observed.
Coronin 1A overexpressing PC12 cells were differentiated for indicated time intervals (24, 48, 72, 96, and 120 h) via NGF treatment. The time-course cell lysates of the T(+) PC12 cells were analyzed by Western blot analysis in order to determine Coronin 1A protein (Supplement Fig. S1). The presence of a 57 kDa band was regarded as Coronin 1A. Coronin 1A transgene exhibited a stable expression up to 120 h in the differentiated PC12 cells. In addition, expression vector carries a poly-histidine tag within the open reading frame of Coronin 1A and probing the same membrane with Penta-His primary antibody indicated that signals from the Western blot analysis were specific to Coronin 1A which is expressed from the expression vector.
Fig. 3. Immunofluorescence staining of PC12 cells overexpressing Coronin 1A, after 96-h differentiation (a, d). Coronin 1A, is stained in red (a and d), actin and neuron specific III tubulin are stained in green (b and e respectively) and nuclei are stained in blue by DAPI. Overlapping Coronin 1A and actin localization in the cell is not affected by Coronin 1A overexpression (a–c). Intense staining at the branching regions is indicated by white arrows (d). Coronin 1A and neuron specific III tubulin show overlapping staining in Coronin 1A overexpressing cells in neurite body due to the excess amount of protein in the cell. Scale bar is 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
41
Fig. 4. Comparison of the average neurite lengths of the 120 h differentiated Coronin 1A overexpressing (T(+)) and control (untransfected (T(−)), Fugene HD applied only, and transfected with pEGFP) PC12 cells. Average neurite lengths of T(+) PC12 cells were significantly shorter than control cells (*p < 0.001 and **p = 0.002). Mann–Whitney U test has been performed to show differences between groups. Tr(−), untransfected PC12 cells; Tr(+), Coronin 1A/pEGFP co-transfected PC12 cells; Fugene HD, Fugene HD applied only; pEGFP, transfected with pEGFP.
Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2014.08. 044. 3.3. Neurite outgrowth analyses in transfected and untransfected PC12 cells PC12 cells’ neurite lengths were measured after 120 h of differentiation (Supplement Table S1). Measurements showed that neurite lengths were variable among cells in the same tissue culture plate resulted in increased standard deviation during statistical analyses (Supplement Table S1). Mann–Whitney U test was performed to show differences between groups. Statistical analyses showed that average neurite length of T(+) PC12 cells’ (n = 102) were significantly shorter than T(−) PC12 cells (n = 140) (p < 0.001), and Fugene HD (n = 108) (p < 0.001) and pEGFP (n = 102) (p = 0.002) applied control PC12 cells (Fig. 4). On the other hand, there was not a statistically significant difference among Fugene HD applied, pEGFP applied and T(−) cells. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2014. 08.044. 4. Discussion According to our knowledge, this is the first study to investigate the effect of Coronin 1A on neurite outgrowth in PC12 cells. The first part of this study was to analyze the expression and localization of Coronin 1A in PC12 cells. Coronin 1A is known to be involved in the regulation of dynamic structure of the actin cytoskeleton within the cell [18]. Our immunofluorescence staining results revealed that Coronin 1A co-localizes with actin cytoskeleton in PC12 cells (Fig. 1a–c). In differentiated PC12 cells, especially at the branching regions of the neurite extensions more intense staining was observed (Fig. 3d). It is expected that actin cytoskeleton shows branching in parallel with branching of the neurite extensions. To ensure this, Arp2/3 protein complex is accumulated at the branching region. Because of the effective regulation of the function of Arp2/3 protein complex, accumulation of Coronin 1A is expected at the branching region in parallel with Arp2/3 protein complex.
Results of immunofluorescence staining were consistent with this information. On the other hand, localization of Coronin 1A was distinct from the staining patterns of the microtubules at the neurites and neurite outgrowth cones (Fig. 1d–f). This finding supports the assumption that Coronin 1A shows similar distribution with actin cytoskeleton but not with microtubules. Expression level of the Coronin 1A was analyzed by Western blot analysis in PC12 cells and the results showed that Coronin 1A protein was expressed ubiquitously in PC12 cells (Fig. 2), and the expression level did not change during the differentiation. The binding of Coronin 1A to the actin related protein complex is controlled by phosphorylation of Coronin 1A [7,8,19] and Coronin 1A activity may be suppressed without any change at the expression level in the course of differentiation. Due to lack of a specific antibody against the phosphorylated isoform, we were unable to document the phosphorylation status of Coronin 1A. The second part of the study, addresses the effect of Coronin 1A overexpression on neurite outgrowth. First, the intracellular localization of Coronin 1A was assessed. We tried to find out if the Coronin 1A localization was changed due to its overexpression. Immunofluorescence of overexpressed Coronin 1A showed that localization did not change (Fig. 3). In this context, PC12 cells, co-transfected with Coronin 1A/pEGFP and controls, were differentiated for indicated time periods (24, 48, 72, 96, and 120 h), and neurite lengths were measured. Our study showed that, one of the members of the Coronin protein family, Coronin 1A, caused a decrease in the length of the neurites. Although, it has been previously reported that Coronin 1B, which has similar characteristics with Coronin 1A, provides axonal plasticity in vitro and in vivo [20]. Coronin 1A showed opposite effect on neurite outgrowth. This result supports the finding that different members of coronin family may have different functions [19]. This study is the first to address the impact of Coronin 1A in neuronal differentiation and its role in neurite outgrowth. Suppressive effect of Coronin 1A on neurite outgrowth is consistent with its relationship with dynamic nature of the actin cytoskeleton. Due to the dynamic nature of the actin cytoskeleton, polymerization and depolymerization of the actin filaments take place simultaneously within a cell. Actin binding proteins, Actin depolymerizing factor (ADF)/cofilin family proteins play a key role
42
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
on polymerization of the actin filaments in eukaryotic cells [21]. However, several other proteins have been shown to assist cofilin in actin depolymerization. One of these proteins, actin-interacting protein 1 (Aip1) acts as a cofactor of cofilin. Another one is Coronin 1A [21,22]. It was suggested that the presence of Coronin 1A and/or Aip1 facilitates cofilin function resulting in an increased cofilin activity to induce actin depolymerization at a determined level. In line with this knowledge, the overexpression of Coronin 1A might facilitate the depolymerizing effect of cofilin on actin cytoskeleton. The longest neurite length was 348.9 m for the T(+) PC12 cells, whereas in the control groups, longest neurite lengths were 873.5 m, 1070.8 m, and 1111.8 m for pEGFP, Fugene HD applied cells and T(-) PC12 cells, respectively (Fig. 4, Supplement Table S1). In addition to this finding, minimum neurite lengths were similar in all groups. These data show us that although standard deviation values varied in a high range in all groups, because of the difference between minimum and maximum neurite lengths, neurite length of the T(+) PC12 cells was significantly shorter than all the control groups. Comparing the average neurite lengths of T(+) and control PC12 cells showed that average neurite length in T(+) PC12 cells was limited and was not able to reach beyond a certain neurite size (Supplement Table S1). In addition, when the average neurite length of the Fugene HD, and pEGFP applied cells were compared to T(-) PC12 cells, a decrease on average neurite lengths was observed especially in pEGFP applied cells, and as well as Fugene HD applied cells. These results suggested that the transfection procedure itself had an effect on cells during neurite outgrowth, however, overexpression of Coronin 1A had a greater effect on neurite outgrowth, and the reduction of the average neurite length was significantly shorter in all groups (Fig. 4). The maximum effect of Coronin 1A overexpression was observed when the neurite lengths reached above the 75th percentile of the group. Neurite lengths at the 75th percentile of T(+) PC12 cells (157.6 m) were shorter than the control cells (T(-): 362.6 m, Fugene HD: 323.4 m, pEGFP: 199.5 m). Taken all these together, cells in all groups were able to produce neurite lengths, however, maximum neurite length of the T(+) cells barely reached 75th percentile of control group cells. We concluded that this was due to the disruption of the cytoskeleton. It is likely that the reduction in the average length of the neurites in T(+) PC12 cells are associated with increasing depolymerization of the actin filaments. Furthermore, differentiation signals increase actin polymerization and in this way leads to neurite outgrowth. However, overexpression of Coronin 1A leads to increased actin depolymerization. Our results show that the balance between polymerization and depolymerization was shifted to depolymerization by Coronin 1A overexpression. According to these findings we suggest that overexpression of Coronin 1A exhibits a suppressive effect on neurite outgrowth by inhibiting polymerization of the actin filaments. 5. Conclusion As a conclusion, it was shown that Coronin 1A has a suppressive effect on neurite outgrowth. However, these findings were obtained from in vitro studies. To prove inhibitory effect of Coronin 1A on neurite outgrowth, its function should also be tested in vivo. Further dissection of the role of Coronin 1A can help to understand the regeneration processes in axonal outgrowth that may contribute to develop novel treatment approaches in axonal regeneration.
Conflict of interest We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Acknowledgements This study was supported by The Scientific Research Council of Turkey (Project Number: 108S195) and Hacettepe University Scientific Research and Development Office (Project Numbers: H.U. BAB. 010 T02 102 001 and H.U. BAB. 03662). We thank to Dr. Mutlu Hayran for his assistance in statistical analysis. References [1] E.W. Dent, F.B. Gertler, Cytoskeletal dynamics and transport in growth cone motility and axon guidance, Neuron 40 (2003) 209–227. [2] J.E. Bear, M. Krause, F.B. Gertler, Regulating cellular actin assembly, Curr. Opin. Cell Biol. 13 (2001) 158–166. [3] A.E. Engqvist-Goldstein, D.G. Drubin, Actin assembly and endocytosis: from yeast to mammals, Annu. Rev. Cell Dev. Biol. 19 (2003) 287–332. [4] T.D. Pollard, G.G. Borisy, Cellular motility driven by assembly and disassembly of actin filaments, Cell 112 (2003) 453–465. [5] E.L. De Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, G. Gerisch, Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits, EMBO J. 10 (1991) 4097–4104. [6] T.H. Millard, S.J. Sharp, L.M. Machesky, Signalling to actin assembly via the WASP (Wiskott–Aldrich syndrome protein) – family proteins and the Arp2/3 complex, Biochem. J. 380 (2004) 1–17. [7] C.L. Humphries, H.I. Balcer, J.L. D’Agostino, B. Winsor, D.G. Drubin, G. Barnes, B.J. Andrews, B.L. Goode, Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin, J. Cell Biol. 159 (2002) 993–1004. [8] A.A. Rodal, O. Sokolova, D.B. Robins, K.M. Daugherty, S. Hippenmeyer, H. Riezman, N. Grigorieff, B.L. Goode, Conformational changes in the Arp2/3 complex leading to actin nucleation, Nat. Struct. Mol. Biol. 12 (2005) 26–31. [9] E.L. De Hostos, The coronin family of actin-associated proteins, Trends Cell Biol. 9 (1999) 345–350. [10] M. Okumura, C. Kung, S. Wong, M. Rodgers, M.L. Thomas, Definition of family of coronin-related proteins conserved between humans and mice: close genetic linkage between coronin-2 and CD45-associated protein, DNA Cell Biol. 17 (1998) 779–787. [11] V. Rybakin, C.S. Clemen, Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking, Bioessays 27 (2005) 625–632. [12] A. Markus, T.D. Patel, W.D. Snider, Neurotrophic factors and axonal growth, Curr. Opin. Neurobiol. 12 (2002) 523–531. [13] N. Foger, L. Rangell, D.M. Danilenko, A.C. Chan, Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis, Science 313 (2006) 839–842. [14] B. Nal, P. Carroll, E. Mohr, C. Verthuy, M.I. Da Silva, O. Gayet, X.J. Guo, H.T. He, A. Alcover, P. Ferrier, Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation, Int. Immunol. 16 (2004) 231–240. [15] A. De Biase, S.M. Knoblach, S. Di Giovanni, C. Fan, A. Molon, E.P. Hoffman, A.I. Faden, Gene expression profiling of experimental traumatic spinal cord injury as a function of distance from impact site and injury severity, Physiol. Genom. 22 (2005) 368–381. [16] R. Jayachandran, X. Liu, S. Bosedasgupta, P. Müller, C.L. Zhang, D. Moshous, V. Studer, J. Schneider, C. Genoud, C. Fossoud, F. Gambino, M. Khelfaoui, C. Müller, D. Bartholdi, H. Rossez, M. Stiess, X. Houbaert, R. Jaussi, D. Frey, R.A. Kammerer, X. Deupi, J.P. de Villartay, A. Lüthi, Y. Humeau, J. Pieters, PLoS Biol. 12 (2014) e1001820. [17] A.E. Fournier, B.T. Takizawa, S.M. Strittmatter, Rho kinase inhibition enhances axonal regeneration in the injured CNS, J. Neurosci. 23 (2003) 1416–1423. [18] A.C. Uetrecht, J.E. Bear, Coronins: the return of the crown, Trends Cell Biol. 16 (2006) 421–426. [19] K. Tsujita, T. Itoh, A. Kondo, M. Oyama, H. Kozuka-Hata, Y. Irino, J. Hasegawa, T. Takenawa, Proteome of acidic phospholipid-binding proteins: spatial and temporal regulation of Coronin 1A by phosphoinositides, J. Biol. Chem. 285 (2010) 6781–6789. [20] S. Di Giovanni, A. de Biase, A. Yakovlev, T. Finn, J. Beers, E.P. Hoffman, A.I. Faden, In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury, J. Biol. Chem. 280 (2005) 2084–2091. [21] W.M. Brieher, H.Y. Kueh, B.A. Ballif, T.J. Mitchison, Rapid actin monomer-insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1, J. Cell Biol. 175 (2006) 315–324. [22] H.Y. Kueh, G.T. Charras, T.J. Mitchison, W.M. Brieher, Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers, J. Cell Biol. 182 (2008) 341–353.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Coronin 1A inhibits neurite outgrowth in PC12 cells Yunus Kasim Terzi, Yusuf Cetin Kocaefe, Sukriye Ayter ∗ Hacettepe University, Faculty of Medicine, Department of Medical Biology, Sihhiye, 06100, Ankara, Turkey
h i g h l i g h t s • Remodeling of actin cytoskeleton plays an important role in axonal sprouting. • Overexpression of Coronin 1A in PC12 cells effects neurite outgrowth. • Coronin 1A has an inhibitory effect on neurite outgrowth in vitro.
a r t i c l e
i n f o
Article history: Received 11 June 2014 Received in revised form 21 August 2014 Accepted 25 August 2014 Available online 30 August 2014 Keywords: Nervous system Regeneration Coronin 1A Neurite outgrowth Cytoskeleton PC12 cell line
a b s t r a c t Regenerative response to central nervous system damage in mammals is limited because of inhibitor signals which consist of myelin associated inhibitor proteins and chondroitin sulfate proteoglycans. Inhibitor signals mainly affect cytoskeleton elements which are important for axonal sprouting and neurite outgrowth. Coronin 1A is an actin cytoskeleton associated protein. Coronin 1A shows its effect on actin cytoskeleton through binding to the Arp2/3 complex which is a key nucleator of actin polymerization and regulates its activation on actin cytoskeleton. Coronin 1A–Arp2/3 interaction is regulated by phosphorylation of Coronin 1A from the C and N terminal region. Thus, Coronin 1A–Arp2/3 complex is one of the targets of inhibitory signaling cascades. The aim of this study was to investigate the effect of Coronin 1A on neurite outgrowth in PC12 cells in vitro. The results showed that Coronin 1A is expressed in differentiated PC12 cells and localized along axonal sprouting region of the neurites. Other results showed that overexpression of Coronin 1A in PC12 cells effects neurite outgrowth. Neurite lengths of the Coronin 1A overexpressing PC12 cells were lower than the untransfected (p < 0.001) and control transfected (p = 0.002) PC12 cells. These results indicate that Coronin 1A has an inhibitory effect on neurite outgrowth in vitro. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cytoskeleton organization and remodeling are essential cellular events during sprouting and neurite outgrowth [1]. Correct spatial and temporal organization of actin cytoskeleton, which is regulated by the assembly and disassembly processes, is important for the establishment of neuronal connections [2–4]. A number of actin binding proteins have been shown to orchestrate actin cytoskeletal dynamics, and these include the coronin family of proteins [5,6]. Coronin was originally identified as an F-actin binding protein enriched at the leading edge and at phagocytic cup structures of the slime mold Dictyostelium discoideum [5]. It was shown that one of the coronin protein family members Coronin 1A directly binds to Arp2/3 protein complex [7], and inhibits F-actin nucleation by freezing the Arp2/3 complex in its inactive “open” conformation
∗ Corresponding author. Tel.: +90 312 292 4434; fax: +90 312 292 4432. E-mail addresses: [email protected], [email protected] (S. Ayter). http://dx.doi.org/10.1016/j.neulet.2014.08.044 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
[8–11]. The interaction between Coronin 1A and Arp2/3 is regulated by phosphorylation of Coronin 1A from both the C and N terminal regions with protein kinase C [7,8,12,13]. However, so far the role of Coronin 1A is only investigated in the immune system, due to the specific expression pattern in the phagocytic cells [14]. On the other hand, it was reported that the expression of Coronin 1A was significantly upregulated in rats following the spinal cord injury [15]. In addition, while this paper was prepared, Jayachandran et al. [16] published their data about the effects of Coronin 1A in nervous system, and showed that Coronin 1A deficiency may cause severe neurobehavioral and cognitive defects in mouse. The above observations prompted us to postulate that Coronin 1A could play a role in regulating cytoskeleton organization during axon formation or neurite outgrowth in neuronal cells. Although the role of Coronin 1A in immune system cells has already been investigated in numerous studies, this is the first study where the effects of Coronin 1A on neurite outgrowth were investigated, and the inhibiting effects on neurite outgrowth were shown.
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
2. Materials and methods 2.1. Cell culture PC12 cells (ATCC CRL-1721) were cultured in RPMI 1640, 15% horse serum, 5% fetal calf serum supplemented (media and serums from Gibco/Invitrogen) with 2 mM l-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, at 37 ◦ C in a humidified and 5% CO2 incubator. The cells were grown on collagen type 4-coated (Sigma) (40 g/l) flasks and glass coverslips. To provide the differentiation of PC12 cells, only 1% horse serum was used in complete medium (differentiation medium). 2.2. Immunofluorescence staining PC12 cells were grown on collagen type 4-coated glass coverslips. The cells were cultured for 24 h in differentiation medium and treated with and without NGF (50 ng/ml) (Millipore) for the times indicated (0, 24, 48, 72, 96, and 120 h). The cells were fixed with 4% (w/v) paraformaldehyde (Sigma) in PBS. The localizations of Coronin 1A and cytoskeletal elements (neuron specific III-tubulin and actin) were documented by incubation with Coronin 1A mouse monoclonal antibody (1:100) (Novus Biologicals), rabbit polyclonal anti-actin antibody (1:100) (Abcam), and mouse monoclonal antiIII tubulin antibody (1:250) (Abcam), respectively. Alexa Fluor 488 and 568 anti-rabbit, and anti-mouse (Molecular Probes/Invitrogen) were used as secondary antibodies (1:500 for both of them). Nuclei staining were carried out by incubation with DAPI. The coverslips were mounted in ProLong Gold antifade reagent (Molecular Probes/Invitrogen) and immunofluorescence images were acquired with trinocular inverted microscope with fluorescent attachment (Leica DMIL, Leica Microsystems, Heidelberg, Germany). 2.3. Western blot analysis PC12 cells were grown on collagen type 4-coated 25 cm2 culture flasks. For the experimental procedure, the cells were cultured for 24 h in differentiation medium and treated with and without NGF (50 ng/ml) (Millipore) for the times indicated (0, 24, 48, 72, 96, and 120 h). Lysed in blending buffer (62.5 mM Tris–HCl (pH 6.8), 5 mM EDTA, 10% SDS (pH 7.2), and mini protease inhibitor tablet). Bicinchoninic acid (BCA) Protein Assay Kit (Pierce) was used to determine the protein concentrations of cell lysates. Proteins (40 g per lane) were separated using SDS-polyacrylamide gels under reducing conditions, and transferred to nitrocellulose membrane (Thermo Scientific). Blots were probed with anti-Coronin 1A antibody (1:500). Goat anti-rabbit IgG HRP-conjugated secondary antibody (1:3000) (Molecular Probes/Invitrogen) was used for protein band detection. The blots were developed by ECL Plus chemiluminescence kit (Amersham Biosciences). To monitor the amount of protein loaded, membranes were stripped and reprobed with mouse anti--actin antibody. For quantitative Western blotting, densitometric analyses of the blots were made by Scion Image software (www.scioncorp.com). Data were calculated as the ratio of arbitrary densitometric units of Coronin 1A immunoreactive bands normalized to values obtained for -actin bands on the same immunoblots.
39
as follows: 2 (g):10 (l) DNA to Fugene HD ratios. The transfection complex medium was removed 48 h later and replaced with differentiation medium with or without addition of 50 ng/ml NGF. The efficiency was calculated by observing pEGFP expression 24 h after transfection. 2.5. Neurite outgrowth analysis We performed neurite outgrowth analyses in four groups: The first group was untransfected (T(−)) PC12 cells, the second groups was Coronin 1A overexpressing (T(+)) PC12 cells, the third and the fourth groups were control groups where transfection agent (Fugene HD), and pEGFP vector were applied alone to cells to evaluate the effects of transfection procedure, and also expression vector on neurite outgrowth, respectively. Neurite outgrowth analysis was performed as described by Fournier et al. [17] with some modifications. Briefly, the induction of differentiation in PC12 cells was evaluated by counting the proportion of cells bearing neurites and measuring the length of the longest neurite in individual cells. Cells bearing neurites with length greater than twice the cell body diameter were considered positive and neurite lengths were measured by using Leica Application Suite program (V2.4.0 R1). Tissue culture plates were photographed with 20× magnification, and 120 ± 20 cells were analyzed per tissue culture plate. The experiments were performed in biological triplicates. Mean values (SD) were determined for each time point and the length of the Coronin 1A overexpressing neurite cones were compared to that of control cultures. 2.6. Statistics Nonparametric Kruskal–Wallis test was used for analysis of difference between groups. Significant differences between groups were determined using Mann–Whitney U test. A p value less than 0.05 was considered as statistically significant. All statistical analyses and tests were performed with the SPSS statistical package (SPSS 11.0 for Windows, Chicago, IL, USA). 3. Results 3.1. Localization and expression of Coronin 1A in PC12 cells Localization of Coronin 1A and its association with cytoskeleton (actin and microtubules) were documented by using immunofluorescence (Fig. 1). Immunofluorescence staining (Fig. 1A) showed that Coronin 1A has been localized at the cortical cytoskeleton, cytoplasm, neurites and the neurite outgrowth cone overlapping with the actin cytoskeleton. To determine the expression of Coronin 1A in the course of PC12 cell differentiation, immunoblotting was performed (Fig. 2). We determined the Coronin 1A specific band at 57 kDa by immunoblotting. Coronin 1A expression level was evaluated with densitometric analysis by using Scion Image program during differentiation of PC12 cells. 3.2. Localization and expression of Coronin 1A in Coronin 1A/pEGFP co-transfected PC12 cells
2.4. Transfection PC12 cells were plated 24 h before the transfection with antibiotic free medium at a density of 72,000 cells/cm2 and transfected using Fugene HD (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s recommendations using the following constructs: pQE-TriSystem-6-Coronin 1A (Qiagen) and pEGFP C2 (Clontech, Palo Alto, CA). Transfection mix was prepared
Coronin 1A overexpressing (T(+)) PC12 cells were analyzed by immunofluorescence staining to document any changes of Coronin 1A localization. Results of immunofluorescence staining revealed that intracellular distribution of Coronin 1A was similar to T(−) PC12 cells and was localized at the cortical cytoskeleton, cytoplasm, neurites and the neurite outgrowth cone overlapping with the actin cytoskeleton (Fig. 3).
40
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
Fig. 1. Immunofluorescence staining of untransfected PC12 cells after 96-h differentiation. Coronin 1A, is stained in red (a and d), actin and neuron specific III tubulin are stained in green (b and e, respectively) and nuclei are stained in blue by DAPI. Coronin 1A and actin immunofluorescence staining patterns have similar localizations in the cell, at the cortical cytoskeleton, cytoplasm, neurites and neurite outgrowth cone (a–c). Overlapping staining of Coronin 1A and actin cytoskeleton are shown by white arrows (c). Neuron specific III tubulin and Coronin 1A reveal different staining localizations in the cell (d–f). Bar is 10 m for a–d, and 20 m for e–f. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Immunoblot analysis of Coronin 1A expression during PC12 differentiation. Specific immunoreactive band for Coronin 1A was observed at 57 kDa. Expression level was evaluated by densitometric analysis and no change was observed.
Coronin 1A overexpressing PC12 cells were differentiated for indicated time intervals (24, 48, 72, 96, and 120 h) via NGF treatment. The time-course cell lysates of the T(+) PC12 cells were analyzed by Western blot analysis in order to determine Coronin 1A protein (Supplement Fig. S1). The presence of a 57 kDa band was regarded as Coronin 1A. Coronin 1A transgene exhibited a stable expression up to 120 h in the differentiated PC12 cells. In addition, expression vector carries a poly-histidine tag within the open reading frame of Coronin 1A and probing the same membrane with Penta-His primary antibody indicated that signals from the Western blot analysis were specific to Coronin 1A which is expressed from the expression vector.
Fig. 3. Immunofluorescence staining of PC12 cells overexpressing Coronin 1A, after 96-h differentiation (a, d). Coronin 1A, is stained in red (a and d), actin and neuron specific III tubulin are stained in green (b and e respectively) and nuclei are stained in blue by DAPI. Overlapping Coronin 1A and actin localization in the cell is not affected by Coronin 1A overexpression (a–c). Intense staining at the branching regions is indicated by white arrows (d). Coronin 1A and neuron specific III tubulin show overlapping staining in Coronin 1A overexpressing cells in neurite body due to the excess amount of protein in the cell. Scale bar is 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
41
Fig. 4. Comparison of the average neurite lengths of the 120 h differentiated Coronin 1A overexpressing (T(+)) and control (untransfected (T(−)), Fugene HD applied only, and transfected with pEGFP) PC12 cells. Average neurite lengths of T(+) PC12 cells were significantly shorter than control cells (*p < 0.001 and **p = 0.002). Mann–Whitney U test has been performed to show differences between groups. Tr(−), untransfected PC12 cells; Tr(+), Coronin 1A/pEGFP co-transfected PC12 cells; Fugene HD, Fugene HD applied only; pEGFP, transfected with pEGFP.
Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2014.08. 044. 3.3. Neurite outgrowth analyses in transfected and untransfected PC12 cells PC12 cells’ neurite lengths were measured after 120 h of differentiation (Supplement Table S1). Measurements showed that neurite lengths were variable among cells in the same tissue culture plate resulted in increased standard deviation during statistical analyses (Supplement Table S1). Mann–Whitney U test was performed to show differences between groups. Statistical analyses showed that average neurite length of T(+) PC12 cells’ (n = 102) were significantly shorter than T(−) PC12 cells (n = 140) (p < 0.001), and Fugene HD (n = 108) (p < 0.001) and pEGFP (n = 102) (p = 0.002) applied control PC12 cells (Fig. 4). On the other hand, there was not a statistically significant difference among Fugene HD applied, pEGFP applied and T(−) cells. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2014. 08.044. 4. Discussion According to our knowledge, this is the first study to investigate the effect of Coronin 1A on neurite outgrowth in PC12 cells. The first part of this study was to analyze the expression and localization of Coronin 1A in PC12 cells. Coronin 1A is known to be involved in the regulation of dynamic structure of the actin cytoskeleton within the cell [18]. Our immunofluorescence staining results revealed that Coronin 1A co-localizes with actin cytoskeleton in PC12 cells (Fig. 1a–c). In differentiated PC12 cells, especially at the branching regions of the neurite extensions more intense staining was observed (Fig. 3d). It is expected that actin cytoskeleton shows branching in parallel with branching of the neurite extensions. To ensure this, Arp2/3 protein complex is accumulated at the branching region. Because of the effective regulation of the function of Arp2/3 protein complex, accumulation of Coronin 1A is expected at the branching region in parallel with Arp2/3 protein complex.
Results of immunofluorescence staining were consistent with this information. On the other hand, localization of Coronin 1A was distinct from the staining patterns of the microtubules at the neurites and neurite outgrowth cones (Fig. 1d–f). This finding supports the assumption that Coronin 1A shows similar distribution with actin cytoskeleton but not with microtubules. Expression level of the Coronin 1A was analyzed by Western blot analysis in PC12 cells and the results showed that Coronin 1A protein was expressed ubiquitously in PC12 cells (Fig. 2), and the expression level did not change during the differentiation. The binding of Coronin 1A to the actin related protein complex is controlled by phosphorylation of Coronin 1A [7,8,19] and Coronin 1A activity may be suppressed without any change at the expression level in the course of differentiation. Due to lack of a specific antibody against the phosphorylated isoform, we were unable to document the phosphorylation status of Coronin 1A. The second part of the study, addresses the effect of Coronin 1A overexpression on neurite outgrowth. First, the intracellular localization of Coronin 1A was assessed. We tried to find out if the Coronin 1A localization was changed due to its overexpression. Immunofluorescence of overexpressed Coronin 1A showed that localization did not change (Fig. 3). In this context, PC12 cells, co-transfected with Coronin 1A/pEGFP and controls, were differentiated for indicated time periods (24, 48, 72, 96, and 120 h), and neurite lengths were measured. Our study showed that, one of the members of the Coronin protein family, Coronin 1A, caused a decrease in the length of the neurites. Although, it has been previously reported that Coronin 1B, which has similar characteristics with Coronin 1A, provides axonal plasticity in vitro and in vivo [20]. Coronin 1A showed opposite effect on neurite outgrowth. This result supports the finding that different members of coronin family may have different functions [19]. This study is the first to address the impact of Coronin 1A in neuronal differentiation and its role in neurite outgrowth. Suppressive effect of Coronin 1A on neurite outgrowth is consistent with its relationship with dynamic nature of the actin cytoskeleton. Due to the dynamic nature of the actin cytoskeleton, polymerization and depolymerization of the actin filaments take place simultaneously within a cell. Actin binding proteins, Actin depolymerizing factor (ADF)/cofilin family proteins play a key role
42
Y.K. Terzi et al. / Neuroscience Letters 582 (2014) 38–42
on polymerization of the actin filaments in eukaryotic cells [21]. However, several other proteins have been shown to assist cofilin in actin depolymerization. One of these proteins, actin-interacting protein 1 (Aip1) acts as a cofactor of cofilin. Another one is Coronin 1A [21,22]. It was suggested that the presence of Coronin 1A and/or Aip1 facilitates cofilin function resulting in an increased cofilin activity to induce actin depolymerization at a determined level. In line with this knowledge, the overexpression of Coronin 1A might facilitate the depolymerizing effect of cofilin on actin cytoskeleton. The longest neurite length was 348.9 m for the T(+) PC12 cells, whereas in the control groups, longest neurite lengths were 873.5 m, 1070.8 m, and 1111.8 m for pEGFP, Fugene HD applied cells and T(-) PC12 cells, respectively (Fig. 4, Supplement Table S1). In addition to this finding, minimum neurite lengths were similar in all groups. These data show us that although standard deviation values varied in a high range in all groups, because of the difference between minimum and maximum neurite lengths, neurite length of the T(+) PC12 cells was significantly shorter than all the control groups. Comparing the average neurite lengths of T(+) and control PC12 cells showed that average neurite length in T(+) PC12 cells was limited and was not able to reach beyond a certain neurite size (Supplement Table S1). In addition, when the average neurite length of the Fugene HD, and pEGFP applied cells were compared to T(-) PC12 cells, a decrease on average neurite lengths was observed especially in pEGFP applied cells, and as well as Fugene HD applied cells. These results suggested that the transfection procedure itself had an effect on cells during neurite outgrowth, however, overexpression of Coronin 1A had a greater effect on neurite outgrowth, and the reduction of the average neurite length was significantly shorter in all groups (Fig. 4). The maximum effect of Coronin 1A overexpression was observed when the neurite lengths reached above the 75th percentile of the group. Neurite lengths at the 75th percentile of T(+) PC12 cells (157.6 m) were shorter than the control cells (T(-): 362.6 m, Fugene HD: 323.4 m, pEGFP: 199.5 m). Taken all these together, cells in all groups were able to produce neurite lengths, however, maximum neurite length of the T(+) cells barely reached 75th percentile of control group cells. We concluded that this was due to the disruption of the cytoskeleton. It is likely that the reduction in the average length of the neurites in T(+) PC12 cells are associated with increasing depolymerization of the actin filaments. Furthermore, differentiation signals increase actin polymerization and in this way leads to neurite outgrowth. However, overexpression of Coronin 1A leads to increased actin depolymerization. Our results show that the balance between polymerization and depolymerization was shifted to depolymerization by Coronin 1A overexpression. According to these findings we suggest that overexpression of Coronin 1A exhibits a suppressive effect on neurite outgrowth by inhibiting polymerization of the actin filaments. 5. Conclusion As a conclusion, it was shown that Coronin 1A has a suppressive effect on neurite outgrowth. However, these findings were obtained from in vitro studies. To prove inhibitory effect of Coronin 1A on neurite outgrowth, its function should also be tested in vivo. Further dissection of the role of Coronin 1A can help to understand the regeneration processes in axonal outgrowth that may contribute to develop novel treatment approaches in axonal regeneration.
Conflict of interest We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Acknowledgements This study was supported by The Scientific Research Council of Turkey (Project Number: 108S195) and Hacettepe University Scientific Research and Development Office (Project Numbers: H.U. BAB. 010 T02 102 001 and H.U. BAB. 03662). We thank to Dr. Mutlu Hayran for his assistance in statistical analysis. References [1] E.W. Dent, F.B. Gertler, Cytoskeletal dynamics and transport in growth cone motility and axon guidance, Neuron 40 (2003) 209–227. [2] J.E. Bear, M. Krause, F.B. Gertler, Regulating cellular actin assembly, Curr. Opin. Cell Biol. 13 (2001) 158–166. [3] A.E. Engqvist-Goldstein, D.G. Drubin, Actin assembly and endocytosis: from yeast to mammals, Annu. Rev. Cell Dev. Biol. 19 (2003) 287–332. [4] T.D. Pollard, G.G. Borisy, Cellular motility driven by assembly and disassembly of actin filaments, Cell 112 (2003) 453–465. [5] E.L. De Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, G. Gerisch, Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits, EMBO J. 10 (1991) 4097–4104. [6] T.H. Millard, S.J. Sharp, L.M. Machesky, Signalling to actin assembly via the WASP (Wiskott–Aldrich syndrome protein) – family proteins and the Arp2/3 complex, Biochem. J. 380 (2004) 1–17. [7] C.L. Humphries, H.I. Balcer, J.L. D’Agostino, B. Winsor, D.G. Drubin, G. Barnes, B.J. Andrews, B.L. Goode, Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin, J. Cell Biol. 159 (2002) 993–1004. [8] A.A. Rodal, O. Sokolova, D.B. Robins, K.M. Daugherty, S. Hippenmeyer, H. Riezman, N. Grigorieff, B.L. Goode, Conformational changes in the Arp2/3 complex leading to actin nucleation, Nat. Struct. Mol. Biol. 12 (2005) 26–31. [9] E.L. De Hostos, The coronin family of actin-associated proteins, Trends Cell Biol. 9 (1999) 345–350. [10] M. Okumura, C. Kung, S. Wong, M. Rodgers, M.L. Thomas, Definition of family of coronin-related proteins conserved between humans and mice: close genetic linkage between coronin-2 and CD45-associated protein, DNA Cell Biol. 17 (1998) 779–787. [11] V. Rybakin, C.S. Clemen, Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking, Bioessays 27 (2005) 625–632. [12] A. Markus, T.D. Patel, W.D. Snider, Neurotrophic factors and axonal growth, Curr. Opin. Neurobiol. 12 (2002) 523–531. [13] N. Foger, L. Rangell, D.M. Danilenko, A.C. Chan, Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis, Science 313 (2006) 839–842. [14] B. Nal, P. Carroll, E. Mohr, C. Verthuy, M.I. Da Silva, O. Gayet, X.J. Guo, H.T. He, A. Alcover, P. Ferrier, Coronin-1 expression in T lymphocytes: insights into protein function during T cell development and activation, Int. Immunol. 16 (2004) 231–240. [15] A. De Biase, S.M. Knoblach, S. Di Giovanni, C. Fan, A. Molon, E.P. Hoffman, A.I. Faden, Gene expression profiling of experimental traumatic spinal cord injury as a function of distance from impact site and injury severity, Physiol. Genom. 22 (2005) 368–381. [16] R. Jayachandran, X. Liu, S. Bosedasgupta, P. Müller, C.L. Zhang, D. Moshous, V. Studer, J. Schneider, C. Genoud, C. Fossoud, F. Gambino, M. Khelfaoui, C. Müller, D. Bartholdi, H. Rossez, M. Stiess, X. Houbaert, R. Jaussi, D. Frey, R.A. Kammerer, X. Deupi, J.P. de Villartay, A. Lüthi, Y. Humeau, J. Pieters, PLoS Biol. 12 (2014) e1001820. [17] A.E. Fournier, B.T. Takizawa, S.M. Strittmatter, Rho kinase inhibition enhances axonal regeneration in the injured CNS, J. Neurosci. 23 (2003) 1416–1423. [18] A.C. Uetrecht, J.E. Bear, Coronins: the return of the crown, Trends Cell Biol. 16 (2006) 421–426. [19] K. Tsujita, T. Itoh, A. Kondo, M. Oyama, H. Kozuka-Hata, Y. Irino, J. Hasegawa, T. Takenawa, Proteome of acidic phospholipid-binding proteins: spatial and temporal regulation of Coronin 1A by phosphoinositides, J. Biol. Chem. 285 (2010) 6781–6789. [20] S. Di Giovanni, A. de Biase, A. Yakovlev, T. Finn, J. Beers, E.P. Hoffman, A.I. Faden, In vivo and in vitro characterization of novel neuronal plasticity factors identified following spinal cord injury, J. Biol. Chem. 280 (2005) 2084–2091. [21] W.M. Brieher, H.Y. Kueh, B.A. Ballif, T.J. Mitchison, Rapid actin monomer-insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1, J. Cell Biol. 175 (2006) 315–324. [22] H.Y. Kueh, G.T. Charras, T.J. Mitchison, W.M. Brieher, Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers, J. Cell Biol. 182 (2008) 341–353.
