Biomedical subjects

Estuardo Robles

Publications and source records attributed to Estuardo Robles.

6 recordsLinked to original sources

PubMed2006

Focal adhesion kinase signaling at sites of integrin-mediated adhesion controls axon pathfinding.

Extracellular matrix (ECM) components regulate neurite outgrowth in tissue culture and in vivo. Live imaging of phosphotyrosine (PY) signals revealed that Xenopus laevis growth cones extending on permissive ECM substrata assemble adhesive point contacts containing enriched levels of tyrosine-phosphorylated proteins. Whereas focal adhesion kinase (FAK) signaling is dispensable for the assembly of focal adhesions in non-neuronal cells, FAK activity is required for the formation of growth cone point contacts. FAK-dependent point contacts promote rapid neurite outgrowth by stabilizing lamellipodial protrusions on permissive ECM substrata. Moreover, local FAK activity is required for ECM-dependent growth cone turning in vitro, suggesting that FAK may control axon pathfinding in vivo. Consistent with this possibility, proper growth and guidance of Rohon-Beard sensory neurons and spinal commissural interneurons requires FAK activity. These findings identify FAK as a key regulator of axon growth and guidance downstream of growth cone-ECM interactions.

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PubMed2005

Src-dependent tyrosine phosphorylation at the tips of growth cone filopodia promotes extension.

Extracellular cues guide axon outgrowth by activating intracellular signaling cascades that control the growth cone cytoskeleton. However, the spatial and temporal coordination of signaling intermediates remains essentially unknown. Live imaging of tyrosine phosphorylation in growth cones revealed dynamic phospho-tyrosine (PY) signals in filopodia that directly correlate with filopodial behavior. Local PY signals are generated at distal tips of filopodia during extension and are lost during retraction. Active Src family kinases localize to the tips of filopodia, and Src activity regulates both filopodial dynamics and local PY signaling. Positive guidance cues stimulate filopodial motility by locally increasing tyrosine phosphorylation in a cell division cycle 42 (Cdc42)-dependent manner. Locally reduced Src activity on one side of the growth cone generates an asymmetry in filopodial motility and PY signaling that promotes repulsive turning, suggesting that local changes in filopodial PY levels may underlie growth cone pathfinding decisions. p21-activated kinase (PAK), a Cdc42 effector whose activity is regulated by Src phosphorylation, also localizes to the tips of extending filopodia and controls filopodial motility. Coordinated activation of cytoskeletal effector proteins by GTPase binding and Src-mediated tyrosine phosphorylation may function to produce specific growth cone behaviors in response to guidance cues.

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PubMed2004

The great escape; phosphorylation of Ena/VASP by PKA promotes filopodial formation.

The Ena/VASP family of proteins consists of adaptor molecules that localize to subcellular sites of actin polymerization. The role of Ena/VASP proteins in the regulation of cell motility and axon outgrowth has been controversial. Recently, these proteins have been proposed to function as "anticapping" factors, which may have differential effects on filopodial versus lammelipodial actin-based protrusions. A study by Lebrand et al. in this issue of Neuron supports this model and identifies PKA as a key regulator of Ena/VASP function downstream of the chemoattractant Netrin.

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PubMed2003

Filopodial calcium transients regulate growth cone motility and guidance through local activation of calpain.

Spontaneous intracellular calcium ([Ca2+](i)) transients in growth cone filopodia reduce filopodial motility, slow neurite outgrowth, and promote turning when generated asymmetrically; however, the downstream effectors of these Ca2+ -dependent behaviors are unknown. We report that Ca2+ transients in filopodia activate the intracellular protease calpain, which slows neurite outgrowth and promotes repulsive growth cone turning upon local activation. Active calpain alters the balance between tyrosine kinase and phosphatase activities in filopodia, resulting in a net decrease in tyrosine phosphorylation, which mediates both filopodial stabilization and reduced lamellipodial protrusion. Our findings indicate that locally generated Ca2+ signals repel axon outgrowth through calpain-dependent regulation of phosphotyrosine signaling at integrin-mediated adhesion sites.

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PubMed2003

Imaging calcium dynamics in developing neurons.

Here we describe the techniques developed to image Ca2+ signals in motile nerve growth cones both in culture and in the developing Xenopus spinal cord. We have used these methods to identify two spatially and temporally distinct classes of Ca2+ transients in growth cones. Imaging Ca2+ in morphologically complex migratory cells allows for analysis and correlation of discrete signals with a wide variety of cellular behaviors. For example, we find that localized Ca2+ changes at the tips of individual filopodia correlate with reduced filopodial motility. Further, rapid fixation after Ca2+ imaging made it possible to determine that transients occur at integrin receptor clusters that may generate and in turn be regulated by these local signals. We describe the use of caged-Ca2+ to locally impose Ca2+ transients in individual filopodia and find this treatment sufficient to repel neurite outgrowth. Calcium signals across broad spatial and temporal dimensions are universal regulators of numerous complex and varied cellular functions. The imaging methods we describe here begin to view growth cones over a range of spatial resolutions and temporal frequencies necessary to detect different types of Ca2+ transients, however it is clear that not all dimensions have been examined. In particular, imaging cells more rapidly and at higher magnification may one day allow us to detect more elemental events such as single-channel openings, as has been achieved in nonneuronal cells. We also describe techniques used to examine Ca2+ signals in growth cones migrating within the spinal cord. These types of studies are ultimately necessary to confirm the relevance of in vitro findings. Although designed for the Xenopus spinal cord, the methods we outline should be applicable to other tissues and organisms. Finally, we use caged Ca2+ as a tool to reproduce very precise changes in cytosolic Ca2+ levels. This is a powerful means to test the function of different types of Ca2+ transients and assess the downstream regulators of those signals. These types of manipulations can also be used with other types of caged compounds, many of which are commercially available (Molecular Probes) or readily synthesized.

Aniline Compounds↗

PubMed2003

Working with Xenopus spinal neurons in live cell culture.

Neurons from the Xenopus spinal cord are highly versatile and easily manipulated, making them an ideal model system to answer questions regarding the cellular and molecular basis of early neural development and function. Xenopus has been a productive model system in studies ranging from axon growth and guidance to synaptic plasticity. Exogenous molecules, such as proteins, fluorescent tracers, and nucleic acids, can be injected into early blastomeres to load tracers in all neurons or into late blastomeres to target specific classes of neurons based on established lineage maps. Xenopus spinal neurons also provide an excellent culture system, as neurons extend processes on a variety of substrata and develop at room temperature in minimal salt solutions. Live fluorescent neurons can be imaged for hours with fluorescence microscopy at room temperature in static cultures without neurotrophic support or serum. This highly reduced culture system minimizes variables that can confound interpretation of results. Cultures can be prepared at various stages of development as dissociated neurons or as spinal cord explants. Both excitatory and inhibitory neurons develop in culture, and synaptic contacts among neurons and between neurons and nonneuronal targets form naturally. The simple anatomy and rapid rostral-to-caudal development of the Xenopus spinal cord also make this an excellent in vivo model system to analyze axon guidance by identifiable classes of neurons. This chapter focuses on techniques that exploit both in vitro and in vivo qualities of this system.

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