Myosin-X provides a motor-based link between integrins and the cytoskeleton.

Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.

The septate junction protein caspr is required for structural support and retention of KCNQ4 at calyceal synapses of vestibular hair cells.

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed presynaptic and postsynaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K(+) channel KCNQ4 at the postsynaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knock-out mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the postsynaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses.

Myosin-X: a molecular motor at the cell's fingertips.

Research in several areas, including unconventional myosins and deafness genes, has converged recently on a group of myosins whose tails contain myosin tail homology 4 (MyTH4) and band 4.1, ezrin, radixin, moesin (FERM) domains. Although these 'MyTH-FERM' myosins are not present in yeast and plants, they are present in slime molds, worms, flies and mammals, where they mediate interactions between the cytoskeleton and the plasma membrane. The most broadly distributed MyTH-FERM myosin in vertebrate cells appears to be myosin-X (Myo10). This myosin can act as a link to integrins and microtubules, stimulate the formation of filopodia and undergo a novel form of motility within filopodia.

Corrigendum: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

This corrects the article DOI: 10.1038/ncomms10833.

Organization and function of septate junctions: an evolutionary perspective.

In most cell types, distinct forms of intercellular junctions have been visualized at the ultrastructural level. Among these, the septate junctions are thought to seal the neighboring cells and thus to function as the paracellular barriers. The most extensively studied form of septate junctions, referred to as the pleated septate junctions, is ultrastructurally distinct with an electron-dense ladder-like arrangement of transverse septa present in invertebrates as well as vertebrates. In invertebrates, such as the fruit fly Drosophila melanogaster, septate junctions are present in all ectodermally derived epithelia, imaginal discs, and the nervous system. In vertebrates, septate junctions are present in the myelinated nerves at the paranodal interface between the myelin loops and the axonal membrane. In this review, we present an evolutionary perspective of septate junctions, especially their initial identification across phyla, and discuss many common features of their morphology, molecular organization, and functional similarities in invertebrates and vertebrates.

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.

Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours.

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy.

Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

A novel form of motility in filopodia revealed by imaging myosin-X at the single-molecule level.

Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow, it is less clear how molecules move forward in filopodia. Myosin-X (Myo10) is an actin-based motor hypothesized to use its motor activity to move forward along actin filaments to the tips of filopodia. Here we use a sensitive total internal reflection fluorescence (TIRF) microscopy system to directly visualize the movements of GFP-Myo10. This reveals a novel form of motility at or near the single-molecule level in living cells wherein extremely faint particles of Myo10 move in a rapid and directed fashion toward the filopodial tip. These fast forward movements occur at approximately 600 nm/s over distances of up to approximately 10 microm and require Myo10 motor activity and actin filaments. As expected for imaging at the single-molecule level, the faint particles of GFP-Myo10 are diffraction limited, have an intensity range similar to single GFP molecules, and exhibit stepwise bleaching. Faint particles of GFP-Myo5a can also move toward the filopodial tip, but at a slower characteristic velocity of approximately 250 nm/s. Similar movements were not detected with GFP-Myo1a, indicating that not all myosins are capable of intrafilopodial motility. These data indicate the existence of a novel system of long-range transport based on the rapid movement of myosin molecules along filopodial actin filaments.

Cytoskeletal transition at the paranodes: the Achilles' heel of myelinated axons.

Myelination organizes axons into distinct domains that allow nerve impulses to propagate in a saltatory manner. The edges of the myelin sheath are sealed at the paranodes by axon-glial junctions that have a crucial role in organizing the axonal cytoskeleton. Here we propose a model in which the myelinated axons depend on the axon-glial junctions to stabilize the cytoskeletal transition at the paranodes. Thus paranodal regions are likely to be particularly susceptible to damage induced by demyelinating diseases such as multiple sclerosis.

No effect of genetic deletion of contactin-associated protein (CASPR) on axonal orientation and synaptic plasticity.

Myelinated axons are endowed with a specialized domain structure that is essential for saltatory action potential conduction. The paranodal domain contains the axoglial junctions and displays a unique ultrastructure that resembles the invertebrate septate junctions (SJs). Biochemical characterizations of the paranodal axoglial SJs have identified several molecular components that include Caspr and contactin (Cont) on the axonal side and neurofascin 155 kDa (NF155) isoform on the glial side. All these proteins are essential for the formation of the axoglial SJs. Based on the interactions between Caspr and Cont and their colocalization in the CA1 synaptic areas, it was proposed that the synaptic function of Cont requires Caspr. Here we have extended the phenotypic analysis of CASPR mutants to address further the role of Caspr at the axoglial SJs and also in axonal orientation and synaptic plasticity. We report that, in CASPR mutants, the smooth endoplasmic reticulum (SER) forms elongated membranous complexes that accumulate at the nodal/paranodal region and stretch into the juxtaparanodal region, a defect that is consistent with the paranodal disorganization. We show that the cerebellar microorganization is unaffected in CASPR mutants. We also demonstrate that Caspr function is not essential for normal CA1 synaptic transmission and plasticity. Taken together with previous findings, our results highlight that the Caspr/Cont complex is essential for the formation of axoglial SJs, whereas Cont may regulate axonal orientation and synaptic plasticity independent of its association with Caspr.

Myo10 in brain: developmental regulation, identification of a headless isoform and dynamics in neurons.

Although Myo10 (myosin-X) is an unconventional myosin associated with filopodia, little is known about its isoforms and roles in the nervous system. We report here that, in addition to full-length Myo10, brain expresses a shorter form of Myo10 that lacks a myosin head domain. This ;headless' Myo10 is thus unable to function as a molecular motor, but is otherwise identical to full-length Myo10 and, like it, contains three pleckstrin homology (PH) domains, a myosin-tail homology 4 (MyTH4) domain, and a band-4.1/ezrin/radixin/moesin (FERM) domain. Immunoblotting demonstrates that both full-length and headless Myo10 exhibit dramatic developmental regulation in mouse brain. Immunofluorescence with an antibody that detects both isoforms demonstrates that Myo10 is expressed in neurons, such as Purkinje cells, as well as non-neuronal cells, such as astrocytes and ependymal cells. CAD cells, a neuronal cell line, express both full-length and headless Myo10, and this endogenous Myo10 is present in cell bodies, neurites, growth cones and the tips of filopodia. To investigate the dynamics of the two forms of Myo10 in neurons, CAD cells were transfected with GFP constructs corresponding to full-length or headless Myo10. Only full-length Myo10 localizes to filopodial tips and undergoes intrafilopodial motility, demonstrating that the motor domain is necessary for these activities. Live cell imaging also reveals that full-length Myo10 localizes to the tips of neuronal filopodia as they explore and interact with their surroundings, suggesting that this myosin has a role in neuronal actin dynamics.

Disruption of axo-glial junctions causes cytoskeletal disorganization and degeneration of Purkinje neuron axons.

Axo-glial junctions (AGJs) play a critical role in the organization and maintenance of molecular domains in myelinated axons. Neurexin IV/Caspr1/paranodin (NCP1) is an important player in the formation of AGJs because it recruits a paranodal complex implicated in the tethering of glial proteins to the axonal membrane and cytoskeleton. Mice deficient in either the axonal protein NCP1 or the glial ceramide galactosyltransferase (CGT) display disruptions in AGJs and severe ataxia. In this article, we correlate these two phenotypes and show that both NCP1 and CGT mutants develop large swellings accompanied by cytoskeletal disorganization and degeneration in the axons of cerebellar Purkinje neurons. We also show that alphaII spectrin is part of the paranodal complex and that, although not properly targeted, this complex is still formed in CGT mutants. Together, these findings establish a physiologically relevant link between AGJs and axonal cytoskeleton and raise the possibility that some neurodegenerative disorders arise from disruption of the AGJs.

Quantitative analysis of tropomyosin linear polymerization equilibrium as a function of ionic strength.

Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken alpha-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin.

A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization.

Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as alpha-amino acetylation of the N-terminus of the muscle protein. Nalpha-acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.