Mechanism of microtubule stabilization by doublecortin.

Neurons undertake an amazing journey from the center of the developing mammalian brain to the outer layers of the cerebral cortex. Doublecortin, a component of the microtubule cytoskeleton, is essential in postmitotic neurons and was identified because its mutation disrupts human brain development. Doublecortin stabilizes microtubules and stimulates their polymerization but has no homology with other MAPs. We used electron microscopy to characterize microtubule binding by doublecortin and visualize its binding site. Doublecortin binds selectively to 13 protofilament microtubules, its in vivo substrate, and also causes preferential assembly of 13 protofilament microtubules. This specificity was explained when we found that doublecortin binds between the protofilaments from which microtubules are built, a previously uncharacterized binding site that is ideal for microtubule stabilization. These data reveal the structural basis for doublecortin's binding selectivity and provide insight into its role in maintaining microtubule architecture in maturing neurons.

[1]  R A Milligan,et al.  Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy , 1987, The Journal of cell biology.

[2]  C. Walsh,et al.  The DCX-domain tandems of doublecortin and doublecortin-like kinase , 2003, Nature Structural Biology.

[3]  C. Walsh,et al.  Doublecortin Is a Microtubule-Associated Protein and Is Expressed Widely by Migrating Neurons , 1999, Neuron.

[4]  S. Pietrokovski,et al.  Doublecortin mutations cluster in evolutionarily conserved functional domains. , 2000, Human molecular genetics.

[5]  I. Scheffer,et al.  doublecortin , a Brain-Specific Gene Mutated in Human X-Linked Lissencephaly and Double Cortex Syndrome, Encodes a Putative Signaling Protein , 1998, Cell.

[6]  K. Kalil,et al.  Reorganization and Movement of Microtubules in Axonal Growth Cones and Developing Interstitial Branches , 1999, The Journal of Neuroscience.

[7]  R. Wade,et al.  Characterization of microtubule protofilament numbers. How does the surface lattice accommodate? , 1990, Journal of molecular biology.

[8]  G. Eichele,et al.  Identification of neurabin II as a novel doublecortin interacting protein , 2003, Mechanisms of Development.

[9]  C. Walsh,et al.  Protein–Protein interactions, cytoskeletal regulation and neuronal migration , 2001, Nature Reviews Neuroscience.

[10]  K. Kizhatil,et al.  A New Activity of Doublecortin in Recognition of the Phospho-FIGQY Tyrosine in the Cytoplasmic Domain of Neurofascin , 2002, The Journal of Neuroscience.

[11]  S. Mcconnell,et al.  Doublecortin Is a Developmentally Regulated, Microtubule-Associated Protein Expressed in Migrating and Differentiating Neurons , 1999, Neuron.

[12]  F Metoz,et al.  Lattice defects in microtubules: protofilament numbers vary within individual microtubules , 1992, The Journal of cell biology.

[13]  L. Cassimeris,et al.  Regulation of microtubule-associated proteins. , 2001, International review of cytology.

[14]  O Reiner,et al.  Interaction between LIS1 and doublecortin, two lissencephaly gene products. , 2000, Human molecular genetics.

[15]  Patrick A. Curmi,et al.  Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain , 2004, Nature.

[16]  S. Mcconnell,et al.  Doublecortin Microtubule Affinity Is Regulated by a Balance of Kinase and Phosphatase Activity at the Leading Edge of Migrating Neurons , 2004, Neuron.

[17]  D. H. Snyder,et al.  MICROTUBULES: EVIDENCE FOR 13 PROTOFILAMENTS , 1973, The Journal of cell biology.

[18]  M. Kirschner,et al.  Tau protein binds to microtubules through a flexible array of distributed weak sites , 1991, The Journal of cell biology.

[19]  O. Reiner,et al.  DCX, a new mediator of the JNK pathway , 2004, The EMBO journal.

[20]  C. Walsh,et al.  Patient Mutations in Doublecortin Define a Repeated Tubulin-binding Domain* , 2000, The Journal of Biological Chemistry.

[21]  J. Gleeson Classical lissencephaly and double cortex (subcortical band heterotopia): LIS1 and doublecortin. , 2000, Current opinion in neurology.

[22]  L. Amos,et al.  How Taxol stabilises microtubule structure. , 1999, Chemistry & biology.

[23]  P. Gordon-Weeks Microtubules and growth cone function. , 2004, Journal of neurobiology.

[24]  E. Nogales,et al.  Refined structure of alpha beta-tubulin at 3.5 A resolution. , 2001, Journal of molecular biology.

[25]  A. Yvon,et al.  Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells. , 1997, Journal of cell science.

[26]  J. Winkler,et al.  Molecular mechanisms of neuronal migration disorders, quo vadis? , 2001, Current molecular medicine.

[27]  O. Reiner,et al.  Doublecortin-like Kinase Is Associated with Microtubules in Neuronal Growth Cones , 2000, Molecular and Cellular Neuroscience.

[28]  A. Koulakoff,et al.  Doublecortin functions at the extremities of growing neuronal processes. , 2003, Cerebral cortex.

[29]  S. Halpain,et al.  MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments , 2002, The Journal of cell biology.

[30]  R. Milligan,et al.  Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice , 2003, The Journal of cell biology.

[31]  T. Mitchison,et al.  Microtubule polymerization dynamics. , 1997, Annual review of cell and developmental biology.

[32]  T. Svitkina,et al.  Cytoplasmic assembly of microtubules in cultured cells. , 1997, Journal of cell science.

[33]  L. Tsai,et al.  Cdk5 Phosphorylation of Doublecortin Ser297 Regulates Its Effect on Neuronal Migration , 2004, Neuron.

[34]  R A Milligan,et al.  Kinesin follows the microtubule's protofilament axis , 1993, The Journal of cell biology.

[35]  D. Agard,et al.  γ-Tubulin complexes and microtubule nucleation , 2001 .

[36]  James Q. Zheng,et al.  Growth Cone Turning Induced by Direct Local Modification of Microtubule Dynamics , 2002, The Journal of Neuroscience.

[37]  C. Walsh,et al.  DCAMKL1 Encodes a Protein Kinase with Homology to Doublecortin that Regulates Microtubule Polymerization , 2000, The Journal of Neuroscience.

[38]  S. Mcconnell,et al.  Doublecortin Interacts with μ Subunits of Clathrin Adaptor Complexes in the Developing Nervous System , 2001, Molecular and Cellular Neuroscience.

[39]  Y. Berwald‐Netter,et al.  A Novel CNS Gene Required for Neuronal Migration and Involved in X-Linked Subcortical Laminar Heterotopia and Lissencephaly Syndrome , 1998, Cell.

[40]  A. Hyman,et al.  Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. , 1992, Molecular biology of the cell.

[41]  E. Nogales,et al.  High-Resolution Model of the Microtubule , 1999, Cell.

[42]  E. Egelman,et al.  Salmonella SipA Polymerizes Actin by Stapling Filaments with Nonglobular Protein Arms , 2003, Science.

[43]  C. Walsh,et al.  Smooth, rough and upside-down neocortical development. , 2002, Current opinion in genetics & development.

[44]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[45]  L. Tsai,et al.  Life is a journey: a genetic look at neocortical development , 2002, Nature Reviews Genetics.

[46]  O. Reiner,et al.  Doublecortin, a stabilizer of microtubules. , 1999, Human molecular genetics.