|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2009) 33, 578585 (Printed in Great Britain)
Involvement of headless myosin X in the motility of immortalized gonadotropin-releasing hormone neuronal cells
Jun‑Jie Wanga, Xiu‑Qing Fua, Yu‑Guang Guoa, Lin Yuana, Qian‑Qian Gaoa, Hua‑Li Yua, Heng‑Liang Shia, Xing‑Zhi Wanga, Wen‑Cheng Xiongb and Xiao‑Juan Zhua*
aInstitute of Cytology and Genetics, Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, 5268 Renmin Street, Changchun 130024, Jilin, PR China
bProgram of Developmental Neurobiology, IMMAG and Department of Neurology, Medical College of Georgia, Augusta, GA 30912, USA
Myosin X (Myo X), an unconventional myosin with a tail homology 4-band 4.1/ezrin/radixin/moesin (MyTH4-FERM) tail, is expressed ubiquitously in various mammalian tissues. In addition to the full-length Myo X (Myo X FL), a headless form is synthesized in the brain. So far, little is known about the function of this motor-less Myo X. In this study, the role of the headless Myo X was investigated in immortalized gonadotropin-releasing hormone (GnRH) neuronal cells, NLT. NLT cells overexpressing the headless Myo X formed fewer focal adhesions and spread more slowly than the wild-type NLT cells and GFP-expressing NLT cells. In chemomigration assays, the NLT cells overexpressing the headless Myo X migrated shorter distances and had fewer migratory cells compared with the control NLT cells.
Keywords: Filopodia, Overexpression, Adhesion, Migration.
*Corresponding author. Tel.: +86 431 85099769; fax: +86 431 85099822.
Myosins are actin-based motors that play a critical role in diverse cellular motile events (Berg et al., 2001; Sellers, 2000). Myosin X (Myo X) is one of the least well-understood unconventional myosins expressed at low concentrations in most vertebrate cells. The heavy chain of full-length Myo X (Myo X FL) can be divided into head, neck and tail (Berg et al., 2000). The head of Myo X is the motor domain, which binds to actin, hydrolyzes ATP and generates movement toward the barded end of the actin filament. The segment of Myo X tail consists of a coiled coil, three PEST motifs, three pleckstrin homology (PH) domains, a myosin tail homology 4 (MyTH4) domain and a band 4.1/ezrin/radixin/moesin (FERM) domain.
Myo X FL is critical for the initiation and extension of filopodia, and the transport of the cargo molecules by intra-filopodial motility (Berg and Cheney, 2002; Tokuo et al., 2007). Myo X transports Mena/VASP to the tip of filopodia, and the Mena/VASP promotes the elongation of actin filaments by interacting with the plus ends, shielding from capping proteins (Tokuo and Ikebe, 2004). The FERM domain of Myo X interacts with integrins and relocalizes integrins to the tip of filopodia (Zhang et al., 2004). This relocalization of integrins might serve to form adhesive structures and promote filopodial extension. Filopodia are motile structures linking to the enhancement of directed cell migration (Mattila and Lappalainen, 2008). In addition to actin-based motility, this unconventional myosin has a role in signal transduction. A unique feature of Myo X is the presence of three PH domains in its tail (Sellers, 2000). The second PH domain in Myo X binds phosphatidylinositol-3, 4, 5-trisphosphate [PI(3,4,5)P3] (Tacon and Peckham, 2004). Because PI(3,4,5)P3 is a product of the PI3-kinase, Myo X is likely to function downstream of this enzyme, an important signaling molecule in cancer and cell motility (Cantley, 2002; Comer and Parent, 2002; Ridley, 2001). A MyTH4 domain of Myo X which binds to microtubule is located next to the PH domains, and the tail of Myo X contains a C-terminal FERM domain. In addition to binding with β-integrin, Myo X also interacts with the two netrin receptors, deleted in colorectal cancer (DCC) and neogenin, an interaction dependent on the FERM domain. Myo X redistributes DCC to the cell periphery or the tips of neurites and functions in neurite outgrowth and axonal path-finding (Zhu et al., 2007). Pi et al. (2007) have reported a novel role for Myo X in endothelial cell migration, which is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial formation and amplification of BMP6 signals.
Brain synthesizes a shorter form of Myo X which lacks the myosin head domain but retains all other domains (&007E;165
The immortalized GnRH cells are widely used in studying cellular mechanisms underlying neuronal migration in vitro (Nielsen-Preiss et al., 2007; Cariboni et al., 2005, 2004; Allen et al., 2002; Giacobini et al., 2002; Ronnekleiv and Resko, 1990). GnRH cells are obtained from a mouse tumor in the olfactory bulb; they retain a high migratory activity (Radovick et al., 1991; Zhen et al., 1997a). We found that overexpression of the headless Myo X in vitro reduces the adhesion and migratory abilities of GnRH cells in vitro, and suggests that the headless Myo X plays a role in the neuronal migratory process.
2 Materials and methods
2.1 Antibodies and expression vectors
Antibodies were purchased as follows: anti-vinculin antibody from Sigma (St Louis, MO, USA); Rabbit polyclonal antibody anti-Myo X was generated as described (Zhu et al., 2007), as also the expression vector. pEGFP-headless Myo X contains Myo X amino acids 727-2058.
2.2 Immunofluorescence staining procedures
NLT-type GnRH cells were cultured with high-glucose Dulbecco's minimum essential medium (DMEM) containing 10% newborn calf serum (NCS) (Gibco; New York, USA) at 37
2.3 Selection of stably transfected cell lines
NLT cells were transfected using Lipofectamine 2000. The transfected cells were selected with G418 (Gibco; New York, USA, 500
Cells were washed once in PBS at 4
2.5 Cell adhesion assay
For these assays, 24-well plates (Corning, New York, USA) were used. The wells were coated with 0.1
2.6 Cell aggregates and collagen gel assay
Rat tail collagen solution was prepared as described by McAteer and Cavanagh (1982). Cell aggregates were prepared by the ‘hanging drop’ technique (Maggi et al., 2000). The collagen solution (30
2.7 Boyden's chamber migration assays
NLT and the stably transfected cells, growing in complete media until subconfluence, were rinsed once in PBS and removed from cell culture plates by trypsin digestion. The cell suspension was centrifuged at 1000
3.1 Expression of endogenous Myo X and an establishment of stably transfected NLT cells
Expression of endogenous Myo X in NLT cells was investigated by immunofluorescence analysis with the anti-Myo X antibody. Myo X was distributed throughout the cytosol of NLT cells and conspicuously concentrated at the tip of the filopodia (Fig. 1a). Two bands of 240 and 165
Expressions of endogenous Myo X proteins in NLT cells and establishments of stably transfected cell lines. a. Immunocytofluorescence analysis of endogenous Myo X distribution in NLT cells. Endogenous Myo X is distributed throughout the cytosol and concentrated at the tips of the filopodia. White arrow indicates the filopodia. Myo X labeled with 488 green probe (Molecular Probes, Invitrogen), bar
Cell adhesion assay. a. Immunocytochemical localization of adhesion complex in spread untransfected NLT cells (upper panel), NLT-GFP cells (middle panel) and NLT-HL cells (bottom panel), and focal adhesion plaques were stained with anti-vinculin antibody (red). NLT-HL cells had less adhesion plaques than wild NLT cells and NLT-GFP cells, bar
3.2 Overexpression of the headless Myo X reduced the cell adhesion
To see whether headless Myo X plays a role in NLT cell adhesion, focal adhesion plaques were identified with anti-vinculin antibody. Immunofluorescence assay demonstrated that NLT-HL cells contained fewer and smaller focal adhesions along the cell edges than NLT or NLT-GFP cells (Fig. 2a). The adhesion properties of NLT-HL were evaluated by the ‘stick and wash’ assay (Mokhtari et al., 2008). The cells were suspended and allowed to attach for 2
3.3 Migration ability is inhibited by overexpression of headless Myo X
Because the overexpression of headless Myo X reduced the adhering capability of GnRH cell, we asked whether the migration ability of these cells is also affected. The collagen gel assay is a widely used assay in analyzing cell migration in 3-D matrix (Cariboni et al., 2005, 2004; Giacobini et al., 2002). Both the morphology and migrated distance of migrating cells can reliably be evaluated. The cell aggregates embedded in the collagen gel were covered by DMEM containing 1% NCS, which acts as stimulus for migration of GnRH cells. GnRH cells are able to migrate from cell aggregates into a matrix of collagen gel after the exposure to NCS. Numerous NLT and NLT-GFP cells migrated radially in chains out of the aggregate into the collagen matrix after 48
The migration of NLT cell into a matrix of collagen gel was suppressed by overexpressing the headless Myo X. Bar
3.4 Chemotaxis of GnRH cells is also reduced by overexpression of GFP-headless Myo X
Subsequently, the Boyden's chamber assay was performed to investigate the role of the headless Myo X in the chemotaxis of the GnRH cells. NLT, NLT-GFP and NLT-HL cells were separately plated into the upper chamber of Transwells and allowed to migrate in the presence of 1% NCS into the lower chamber. Four hours later, the numbers of NLT, NLT-GFP and NLT-HL cells that had passed through the pores and adhered to the underside of the membrane were counted. Fewer NLT-HL cells were observed in the lower chamber (NLT cells, 221.4
Cell chemotaxis was inhibited by overexpressing the headless Myo X. a. Photomicrographs of migrated cells cultured for 4
With the aid of nucleotide sequence analysis, northern blots and immunoblots, Sousa and coworkers discovered the headless form of Myo X synthesized in the brain from an alternative transcription start site (Sousa et al., 2006). Recent progress has been made in identifying domain functions of the Myo X FL for the initiation, development and transport activity of filopodia, and its critical roles in directed endothelial cell migration and axon growth have been identified (Pi et al., 2007; Zhu et al., 2007). We have investigated the role of its headless form of Myo X in the adherence and migratory ability of the immortalized GnRH neurons. The headless Myo X was expressed in NLT-type GnRH cells, and overexpression of headless Myo X impaired the adhesive and migration capacities in vitro. This suggests that the headless Myo X may be implicated in the GnRH neuron migration during the brain development.
Headless Myo X with &007E;165
Integrins are directly engaged in focal adhesion assembly, cell adhesion and the reshaping of dynamic cellular structures. Myo X interacts with integrin through the FERM domain (Zhang et al., 2004). Headless X was expressed in NLT cells and overexpression of headless Myo X in NLT cells impaired their adhesion and spreading activities. The headless Myo X contains the whole tail domain, overexpression of the headless Myo X might interferers the binding activity between integrins and Myo X FL or disturb the interaction of integrins with other FERM domain-containing proteins, such as talin that is also required for cell migration (Zhang et al., 2004). The assembly and disassembly of integrins in response to extracellular cues are essential for cell migration (Ridley et al., 2003), and thus it is likely that overexpression of headless Myo X impairs the binding activity between Myo X and integrins, and leads to reduced capabilities of adhesion and spreading of GnRH cells, which in turn contributed to impaired migration of GnRH cells (see below).
Collagen gel assay is widely used in the characterization of GnRH cell migration in vitro. Many factors such as HGF/SF, reelin and KAL1 play a role in guiding GnRH neuron migration (Cariboni et al., 2005, 2004; Giacobini et al., 2002). Under the same experimental conditions, the NLT-HL cells had a shorter distance relative to NLT-GFP and NLT cells, indicating that the headless Myo X suppressed cell motility in 3-D collagen gel. The Boyden's chamber assay is a procedure to evaluate cell chemotaxis. Fewer NLT-HL cells traversed the pores to the target side of the membrane than NLT-GFP or NLT cells upon stimulation with NCS, showing that the overexpression of headless Myo X inhibits the chemotaxis of GnRH cells. Thus, both spontaneous and chemotactic molecule-directed migration capabilities are reduced by the overexpression of headless Myo X.
GnRH neurons migrate in the nasal compartment intermittently in association with vomeronasal nerve fibers and increase their frequency of movement upon entering the brain, and the migration is terminated when they reach their final destinations (Tobet and Schwarting, 2006; Bless et al., 2005). The migratory route of GnRH neurons proceeds in close apposition with blood vessels, suggesting that serum factors might play distinct roles in guiding the movement of these neurons (Maggi et al., 2000). Schwarting et al. (2001) showed that DCC, the receptor of guidance factor netrin-1, regulates the trajectories of vomeronasal axons that guide the migration of GnRH neurons. Loss of DCC function results in the migration of many GnRH neurons to inappropriate destinations (Deiner and Sretavan, 1999). However, the mechanism of GnRH migration is still not well known. Myo X is believed to interact with the DCC (Zhu et al., 2007). Expression of Myo X redistributes endogenous DCC to the filopodia, and Myo X mediated filopodium formation and extension could be further enhanced by coexpression of DCC in GnRH neurons (Zhu et al., 2007). The question raised is whether interaction between Myo X and DCC is involved in GnRH migration. Because headless Myo X decreases adhesion and migratory ability of GnRH cells in vitro, we speculate that it might regulate the moving state of GnRH neurons during their migratory process. Headless Myo X may promote the neurons to dissociate from the fibers by reducing the neuron adhesive ability when they come close to their target regions. Certainly, further investigations are needed to elucidate the details of these regulatory events.
Headless Myo X has the capacity to inhibit neurite outgrowth and formation of axon projections in vivo (Zhu et al., 2007). Migration of GnRH neurons in vivo is largely dependent on their interactions with vomeronasal nerve axons (Marin and Rubenstein, 2003). To distinguish the function of headless Myo X on GnRH neurons motility from their role in nerve axon projections, in vitro migration assays were done. Migration of GnRH cells were reduced by the overexpression of headless Myo X. Myo X FL and headless Myo X bind to several molecules that are important in neuronal development, including PI(3,4,5)P3 (Isakoff et al., 1998), microtubules (Weber et al., 2004), β-integrins (Zhang et al., 2004) and DCC (Zhu et al., 2007). Evidence from nervous and non-nervous systems suggests that headless Myo X can modify the function of Myo X FL (Zhu et al., 2007; Cox et al., 2002; Weber et al, 2004). Thus, it is likely that headless Myo X serve as a negative factor in regulating Myo X-involved GnRH neuron migration from the nasal cavity into the brain during the development. Determining the nature of this relationship of headless Myo X with Myo X FL in neuron migration is required.
In conclusion, headless Myo X suppresses adherence of GnRH cells to the matrix and decreases their migratory activity in vitro. These observations support a significant role of Myo X in the migration of GnRH neurons from the periphery into the brain during nervous development.
This work was supported by the National Natural Science Foundation of China (30670689) and the Program for New Century Excellent Talents in University (NCET-07-0173), Specialized Research Fund for the Doctoral Program of High Education (20060200008) and Scientific Research Foundation for the Returned Overseas Chinese Scholars from the Ministry of Education of China.
Allen MP, Linseman, DA, Udo, H, Xu, M, Schaack, JB, Varnum, B. Novel mechanism for Gonadotropin-releasing hormone neuronal migration involving Gas6/Ark signaling to p38 mitogen-activated protein kinase. Mol Cell Biol 2002:22:2:599-613
Berg JS, Derfler, BH, Pennisi, CM, Corey, DP, Cheney, RE. Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin. J Cell Sci 2000:113:3439-51
Bless EP, Walker, HJ, Yu, KW, Knoll, JG, Moenter, SM, Schwarting, GA. Live view of Gonadotropin-releasing hormone containing neuron migration. Endocrinology 2005:146:1:463-8
Cariboni A, Rakic, S, Liapi, A, Maggi, R, Goffinet, A, Parnavelas, JG. Reelin provides an inhibitory signal in the migration of gonadotropin-releasing hormone neurons. Development 2005:132:4709-18
Cariboni A, Pimpinelli, F, Colamarino, S, Zaninetti, R, Piccolella, M, Rumio, C. The product of X-linked Kallmann's syndrome gene (KAL1) affects the migratory activity of gonadotropin-releasing hormone (GnRH)-producing neurons. Hum Mol Genet 2004:13:22:2781-91
Giacobini P, Giampietro, C, Fioretto, M, Maggi, R, Cariboni, A, Peroteau, I. Hepatocyte growth factor/scatter factor facilitates migration of GN-11 immortalized LHRH neurons. Endocrinology 2002:143:9:3306-15
Huang X, Cheng, HJ, Tessier-Lavigne, M, Jin, Y. MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion. Neuron 2002:34:563-76
Isakoff SJ, Cardozo, T, Andreev, J, Li, Z, Ferguson, KM, Abagyan, R. Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast. EMBO J 1998:17:5374-87
Maggi R, Pimpinelli, F, Molteni, L, Milani, M, Martini, L, Piva, F. Immortalized luteinizing hormone-releasing hormone neurons show a different migratory activity in vitro. Endocrinology 2000:141:6:2105-12
McAteer JA, Cavanagh, TJ. Medium hydrated collagen gel as an explant support in organ culture. J Tissue Culture Methods 1982:1:3:117-22
Mokhtari MJ, Motamed, N, Shokrgozar, MA. Evaluation of silibinin on the viability, migration and adhesion of the human prostate adenocarcinoma (PC-3) cell line. Cell Biol Int 2008:32:888-92
Nielsen-Preiss SM, Allen, MP, Xu, M, Linseman, DA, Pawlowski, JE, Bouchard, RJ. Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and Ras stimulation of Rac activity in immortalized gonadotropin-releasing hormone neuronal cells. Endocrinology 2007:148:6:2806-14
Pi X, Ren, R, Kelley, R, Zhang, C, Moser, M, Bohil, AB. Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors. J Cell Biol 2007:179:7:1569-82
Radovick S, Wray, S, Lee, E, Nicols, DK, Nakayama, Y, Weintraub, BD. Migratory arrest of gonadotropin-releasing hormone neurons in transgenic mice. Proc Natl Acad Sci USA 1991:88:3402-6
Schwarting GA, Kostek, C, Bless, EP, Ahmad, N, Tobet, SA. Deleted in colorectal cancer (DCC) regulates the migration of luteinizing hormone-releasing hormone neurons to the basal forebrain. J Neurosci 2001:21:3:911-9
Sousa AD, Berg, JS, Robertson, BW, Meeker, RB, Cheney, RE. Myo10 in brain: developmental regulation, identification of a headless isoform and dynamics in neurons. J Cell Sci 2006:119:184-94
Tokuo H, Mabuchi, K, Ikebe, M. The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation. J Cell Biol 2007:179:2:229-38
Zhang H, Berg, JS, Li, Z, Wang, Y, Lang, P, Sousa, AD. Myosin-X provides a motor-based link between integrins and the cytoskeleton. Nat Cell Biol 2004:6:523-31
Zhen S, Dunn, IC, Wray, S, Liu, Y, Chappell, PE, Levine, JE. An alternative gonadotropin-releasing hormone (GnRH) RNA splicing product found in cultured GnRH neurons and mouse hypothalamus. J Biol Chem 1997:272:12620-5
Zhen S, Zakaria, M, Wolf, A, Radovick, S. Regulation of Gonadotropin-releasing hormone (GnRH) gene expression by insulin-like growth factor I in a cultured GnRH-expressing neuronal cell line. Mol Endocrinol 1997:11:8:1145-55
Zhu X, Wang, C, Dai, P, Xie, Y, Song, N, Liu, Y. Myosin X regulates netrin receptors and functions in axonal path-finding. Nat Cell Biol 2007:9:184-92
Received 18 November 2008/16 January 2009; accepted 20 February 2009doi:10.1016/j.cellbi.2009.02.006