ABSTRACT
Doublecortin (DCX) is a microtubule-associated protein widely expressed in the developing mammalian nervous system and important for neuronal migration. DCX is known to belong to a novel protein family defined by sequence homology and the presence of a conserved microtubule-binding domain, but the functions of other members of this family are still undefined. In this study, we describe the cloning of the chick ortholog of doublecortin-like kinase (DCLK), a member of this family, and assess the expression of DCX and DCLK in the layered regions of the developing chick brain. DCX and DCLK are widely expressed in pallial and subpallial structures, including the telencephalon, optic tectum, and cerebellum, in similar distribution patterns. In addition to their expression in migrating cells, both proteins were also detected in the ventricular zone and in postmigratory Purkinje cells. Finally, DCX and DCLK were found to be coexpressed in all areas examined. In postmigratory Purkinje cells, DCX and DCLK both colocalized to the cell membrane, although DCLK was also distributed more generally throughout the cell soma. These data are consistent with multiple roles for DCX and DCLK in the developing chicken brain and suggest that the chick cerebellum will be an intriguing system to explore the effects of DCX and DCLK on postmigratory neuronal function.
Subject(s)
Brain/embryology , Gene Expression Regulation, Developmental , Microtubule-Associated Proteins/biosynthesis , Neuropeptides/biosynthesis , Protein Serine-Threonine Kinases/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cell Movement , Chick Embryo , Cloning, Molecular , Doublecortin Domain Proteins , Doublecortin Protein , Doublecortin-Like Kinases , Immunohistochemistry , In Situ Hybridization , Microscopy, Confocal , Microtubule-Associated Proteins/physiology , Molecular Sequence Data , Neurons/metabolism , Protein Serine-Threonine Kinases/pharmacology , Purkinje Cells/cytology , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Microtubule-Associated Proteins/metabolism , Neuropeptides/metabolism , Amino Acid Substitution/physiology , Animals , Cloning, Molecular/methods , Cyclin-Dependent Kinase 5 , Doublecortin Domain Proteins , Doublecortin Protein , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Mutation/physiology , Neuropeptides/chemistry , Neuropeptides/genetics , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
An in vitro coculture system is described to study the avian Purkinje neuron and the interactions occurring with astrocytes and granule cells during development in the cerebellum. Astrocytes initially and granule cells later regulate Purkinje neuron morphology. The coculture system presented here provides an excellent system for investigating the morphological, immunocytochemical, and electrophysiological differentiation of Purkinje neurons under controlled conditions and for studying cell-cell interactions and extrinsic factors, e.g., glutamate in normal and neuropathological conditions.
Subject(s)
Astrocytes/cytology , Cells, Cultured/cytology , Glutamic Acid/pharmacology , Purkinje Cells/cytology , Purkinje Cells/drug effects , Animals , Astrocytes/drug effects , Astrocytes/physiology , Cell Communication/drug effects , Cell Communication/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured/drug effects , Cells, Cultured/physiology , Chick Embryo , Coculture Techniques/instrumentation , Coculture Techniques/methods , Excitatory Amino Acid Transporter 2/antagonists & inhibitors , Excitatory Amino Acid Transporter 2/metabolism , Glutamic Acid/metabolism , Purkinje Cells/physiologyABSTRACT
The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5(NM1)), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5(NM1) was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.
