Biomedical subjects

Aurea D Sousa

Publications and source records attributed to Aurea D Sousa.

7 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2001

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.

5-Hydroxytryptophan↗