Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit.

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.     

Patient mutations in doublecortin define a repeated tubulin-binding domain

Doublecortin (DCX) missense mutations are found in two clusters in patients with defective cortical neuronal migration. Although DCX can function as a microtubule-associated protein (MAP), the potential relationship between its MAP activity and neuronal migration is not understood. Here we show that the two clusters of patient mutations precisely define an internal tandem repeat. Each repeat alone binds tubulin, whereas neither repeat is sufficient for co-assembly with microtubules. The two tandem repeats are sufficient to mediate microtubule polymerization, and representative patient missense mutations lead to impaired polymerization both in vitro and in vivo as well as impaired microtubule stabilization. Furthermore, each repeat is predicted to have the secondary structure of a beta-grasp superfold motif, a motif not found in other MAPs. The patient mutations are predicted to disrupt the structure of the motif, suggesting that the motif may be critical for the DCX-tubulin interaction. These data provide both genetic and biochemical evidence that the interaction of DCX with microtubules is dependent upon this novel repeated tubulin-binding motif.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

Thirteen is the lucky number for doublecortin

Doublecortin is a microtubule-associated protein that is essential for normal brain development. A recent report published in Molecular Cell shows that doublecortin associates preferentially with microtubules made of 13 protofilaments, by recognizing a novel site between the protofilaments. These findings explain how doublecortin stabilizes microtubules and provide clues about its function during neuronal migration.     

zyg-8, a gene required for spindle positioning in C. elegans, encodes a doublecortin-related kinase that promotes microtubule assembly.

Proper spindle positioning is essential for spatial control of cell division. Here, we show that zyg-8 plays a key role in spindle positioning during asymmetric division of one-cell stage C. elegans embryos by promoting microtubule assembly during anaphase. ZYG-8 harbors a kinase domain and a domain related to Doublecortin, a microtubule-associated protein (MAP) affected in patients with neuronal migration disorders. Sequencing of zyg-8 mutant alleles demonstrates that both domains are essential for function. ZYG-8 binds to microtubules in vitro, colocalizes with microtubules in vivo, and promotes stabilization of microtubules to drug or cold depolymerization in COS-7 cells. Our findings demonstrate that ZYG-8 is a MAP crucial for proper spindle positioning in C. elegans, and indicate that the function of the Doublecortin domain in modulating microtubule dynamics is conserved across metazoan evolution.     

Microtubule association of the neuronal p35 activator of Cdk5

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.     

PACSIN1, a Tau-interacting Protein,Regulates Axonal Elongation and Branching by Facilitating Microtubule Instability

Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.     

Myosin function in nervous and sensory systems

Development of the nervous system requires remarkable changes in cell structure that are dependent upon the cytoskeleton. The importance of specific components of the neuronal cytoskeleton, such as microtubules and neurofilaments, to neuronal function and development has been well established. Recently, increasing focus has been put on understanding the functional role of the actin cytoskeleton in neurons. Important modulators of the actin cytoskeleton are the large family of myosins, many of which (classes I, II, III, V, VI, VII, IX, and XV; Fig. 1) are expressed in developing neurons or sensory cells. Myosins are force-producing proteins that have been implicated in a wide variety of cellular functions in the developing nervous system, including neuronal migration, process outgrowth, and growth cone motility, as well as other aspects of morphogenesis, axonal transport, and synaptic and sensory functions. We review the roles that neuronal myosins play in these functions with particular focus on the first three events listed above, as well as sensory function.     

Fibroblast growth factor 13 is a microtubule-stabilizing protein regulating neuronal polarization and migration

Secretory fibroblast growth factors (FGFs) and their receptors are known for their regulatory function in the early stages of neural development. FGF13, a nonsecretory protein of the FGF family, is expressed in cerebral cortical neurons during development and is a candidate gene for syndromal and nonspecific forms of X-chromosome-linked mental retardation (XLMR). However, its function during development remains unclear. We show that FGF13 acts intracellularly as a microtubule-stabilizing protein required for axon and leading process development and neuronal migration in the cerebral cortex. FGF13 is enriched in axonal growth cones and interacts directly with microtubules. Furthermore, FGF13 polymerizes tubulins and stabilizes microtubules. The loss of FGF13 impairs neuronal polarization and increases the branching of axons and leading processes. Genetic deletion of FGF13 in mice results in neuronal migration defects in both the neocortex and the hippocampus. FGF13-deficient mice also exhibit weakened learning and memory, which is correlated to XLMR patients' intellectual disability.     

Lis1 and doublecortin function with dynein to mediate coupling of the nucleus to the centrosome in neuronal migration

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1+/- neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear "cage" to the centrosome. Lis1+/- neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.     

Colocalization of doublecortin with the microtubules: an ex vivo colocalization study of mutant doublecortin

Doublecortin (DCX) plays an important role in neuronal migration and development, and the participation of DCX in neuronal migration has been demonstrated by intensive mutational analysis for patients with X-linked or sporadic lissencephaly, and/or subcortical laminar heterotopia. Although a previous search for protein similarity showed that DCX has a region homologous to the putative Ca(2+)/calmodulin-dependent protein kinase, the function of the DCX gene (DCX) has remained unknown. We show here that mouse DCX colocalizes with the microtubules and provide evidence that its conformational structure is important for its subcellular localization by means of mutant doublecortin expression study. The results of our study may suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development.     

APC nuclear membrane association and microtubule polarity.

BACKGROUND INFORMATION: Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. RESULTS: We previously showed, using primary fibroblasts and astrocytes in in vitro scratch-induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus-ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. CONCLUSIONS: We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. Unlike other Cdks that promote cell cycle, Cdk5 is activated in postmitotic neurons and critically regulates neuronal migration by phosphorylating its substrates during brain development. Recently, we found that Cdk5 phosphorylates focal adhesion kinase (FAK) at Serine 732 in vitro and is responsible for this phosphorylation in the developing brain. Our experiments using a phospho-specific antibody and an S732-unphosphorylatable mutant FAK suggest that S732 phosphorylation may regulate a centrosome-associated microtubule structure to promote nuclear translocation, a critical step in neuronal migration. S732 phosphorylation does not directly impact on the kinase activity of FAK, but appears to prevent the accumulation of FAK at the centrosome. Our study reveals a similarity between Cdk5 and Cdk1 in the regulation of neuronal migration and cell division, respectively. In addition, our study implicates FAK in a signaling pathway that directly regulates microtubules.     

TAC-1, a regulator of microtubule length in the C. elegans embryo

Regulation of microtubule growth is critical for many cellular processes, including meiosis, mitosis, and nuclear migration. We carried out a genome-wide RNAi screen in Caenorhabditis elegans to identify genes required for pronuclear migration, one of the first events in embryogenesis requiring microtubules. Among these, we identified and characterized tac-1 a new member of the TACC (Transforming Acidic Coiled-Coil) family [1]. tac-1(RNAi) embryos exhibit very short microtubules nucleated from the centrosomes as well as short spindles. TAC-1 is initially enriched at the meiotic spindle poles and is later recruited to the sperm centrosome. TAC-1 localization at the centrosomes is regulated during the cell cycle, with high levels during mitosis and a reduction during interphase, and is dependent on aurora kinase 1 (AIR-1), a protein involved in centrosome maturation. tac-1(RNAi) embryos resemble mutants of zyg-9, which encodes a previously characterized centrosomal protein of the XMAP215 family and was also found in our screen. We show that TAC-1 and ZYG-9 are dependent on one another for their localization at the centrosome, and this dependence suggests that they may function together as a complex. We conclude that TAC-1 is a major regulator of microtubule length in the C. elegans embryo.     

Local activation of Rap1 contributes to directional vascular endothelial cell migration accompanied by extension of microtubules on which RAPL, a Rap1-associating molecule, localizes

Endothelial cell migration is promoted by chemoattractants and is accompanied with microtubule extension toward the leading edge. Cytoskeletal microtubules polarize to function as rails for delivering a variety of molecules by motor proteins during cell migration. It remains, however, unclear how directional migration with polarized extension of microtubules is regulated. Here we report that Rap1 controls the migration of vascular endothelial cells. We found that Rap1-associating molecule, RAPL, which belongs to the Ras association domain family (Rassf), localized on microtubules and that activated Rap1 induced dissociation of RAPL from microtubules. A Rap1 activation-monitoring probe based on the fluorescence resonance energy transfer enabled us to demonstrate that local Rap1 activation occurs at the leading edge of the cells under the two types of cell migration, chemotaxis and wound healing. Time lapse imaging of microtubules marked by enhanced green fluorescent protein-RAPL showed the directional growth of microtubules toward the leading edge of the migrating cells. Using adenovirus, inactivation of Rap1 by expression of rap1GAPII inhibited wound healing. In addition, disconnection of Rap1 and RAPL by expression of a RAPL mutant also perturbed wound healing. Collectively, the locally activated Rap1 and its association with RAPL controls the directional migration of vascular endothelial cells.     

APC binds intermediate filaments and is required for their reorganization during cell migration

Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.     

The chemokine CX3CL1 reduces migration and increases adhesion of neurons with mechanisms dependent on the beta1 integrin subunit

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the beta(1) and alpha(6) integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the beta(1) subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.     

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.     

Cytoskeleton of retinular cells from the stomatopod,Gonodactylus oerstedii: possible roles in pigment granule migration

Retinular cells of the compound eyes of stomatopods (mantis shrimps) contain screening pigment granules that migrate radially in response to light. To clarify the role of the cytoskeleton in these movements, we have performed light microscopy and ultrastructural analyses of cytoskeletal organelles in retinular cells. Rhodamine phalloidin staining indicates that filamentous actin is a component of microvillar rhabdomeres and zonula adherens between retinular cells. Ultrastructural studies reveal three populations of microtubules in retinular cells that differ in their orientations and labilities to fixation. Two of these populations are oriented longitudinally in cells: the soma microtubules, found primarily in a column in the cell soma, and the more labile palisade microtubules, which extend alongside the palisade vacuole near the rhabdomere. The third, most labile microtubule population, and filaments 9–30 nm in diameter, are oriented radially in retinular cells, some within cytoplasmic bridges that span the palisade. The radial microtubules and filaments are appropriately oriented for participating in pigment granule migration. Determination of microtubule polarities in retinular cells by decoration with endogenous tubulin indicates that palisade and soma microtubules contain subpopulations having opposite polarity orientations, as has been observed in neuronal dendrites. In contrast, neighboring pigment cells contain microtubules uniformly oriented with minus ends towards the nucleus, as has been observed in most cell types studied.