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Biomedical subjects

Daniel G Jay

Publications and source records attributed to Daniel G Jay.

7 recordsLinked to original sources
PubMed2003

Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration.

CNS regeneration in higher vertebrates is a long sought after goal in neuroscience. The lack of regeneration is attributable in part to inhibitory factors found in myelin (Caroni and Schwab, 1988a). Myelin-associated glycoprotein (MAG) is an abundant myelin protein that inhibits neurite outgrowth in vitro (McKerracher et al., 1994; Mukhopadhyay et al., 1994), but its role in regeneration remains controversial. To address this role, we performed nerve crush on embryonic day 15 chick retina-optic nerve explants and then acutely eliminated MAG function along the nerve using chromophore-assisted laser inactivation (CALI). CALI of MAG permitted significant regrowth of retinal axons past the site of lesion containing CNS myelin in contrast to various control treatments. Electron microscopy of the site of nerve crush shows abundant regenerating axons crossing the gap. When crushed optic nerve was retrogradely labeled at the nerve stump, no labeling of retinal neurons was observed. In contrast, labeling of CALI of MAG-treated crushed optic nerve showed significant retinal labeling (89 +/- 16 cells per square millimeter), a value indistinguishable from that seen with non-crushed nerve (98 +/- 13 cells per square millimeter). These findings implicate MAG as an important component of the myelin-derived inhibition of nerve regeneration. The acute loss of MAG function can promote significant axon growth across a site of CNS nerve damage.

Animals↗
PubMed2002

Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone.

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin I beta) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

Actins↗
PubMed2002

Ephrin-A5 restricts topographically specific arborization in the chick retinotectal projection in vivo.

The retinotectal map is the best characterized model system to study how axons respond to guidance cues during the formation of the nervous system. Recent studies have shown that the critical event in forming this map is topographic-specific axon branching. To elucidate the in vivo role of the repulsive cue ephrin-A5 in this event, we used chromophore-assisted laser inactivation (CALI) to generate acute loss of ephrin-A5 function in localized areas of the posterior tectum of chick embryos in ovo and analyzed the resulting changes of retinal projections during initial outgrowth (E11) and when retinal axons arborize in the deep layers in the tectum (E12). We confirmed that ephrin-A5 functions to restrict initial axon outgrowth at E11. At E12, CALI of ephrin-A5 did not affect the extent of axon outgrowth on the tectal surface but instead caused ectopic arborization posterior to the topographically correct site in deeper layers of the tectum. This shows that ephrin-A5 restricts arborization during this critical process for developing the retinotopic map. CALI provides an approach to inactivate in vivo function in higher vertebrates with high temporal and spatial specificity that may have wide application.

Animals↗
PubMed2002

Fluorophore-assisted light inactivation: a high-throughput tool for direct target validation of proteins.

To exploit advances in proteomics for drug discovery, high-throughout methods for target validation that directly address the cellular roles of proteins are required. To do this, we have characterized fluorophore-assisted light inactivation (FALI) which uses coherent or diffuse light targeted by fluorescein-labeled probes to inactivate specific proteins. We have shown that it is spatially restricted and tested its efficacy in living cells. FALI is efficient using conventional antibodies and single chain variable fragment phage display antibodies (that are compatible with high-throughput applications). We have shown that singlet oxygen is one of the major components required for FALI-mediated damage. The half-maximal radius of damage is approximately 40 A. FALI causes the specific loss of function of beta 1 integrin in HT-1080 fibrosarcoma cells resulting in a reduction in invasiveness. The efficacy of diffuse light sources (such as a desk lamp) with FALI to inactivate many samples in parallel provides an inexpensive, high-throughput method of wide general applicability for functional proteomics.

Animals↗
PubMed2002

pp60(c-src) is a negative regulator of laminin-1-mediated neurite outgrowth in chick sensory neurons.

Multiple protein tyrosine kinases regulate neurite outgrowth in the developing nervous system. To begin to unravel the complexity of this regulation, we addressed the role of one specific kinase, pp60(c-src), in chick dorsal root ganglion (DRG) neurons grown on laminin-1, a well-characterized system to study neurite outgrowth. Pharmacological inhibition of all tyrosine kinases by genestein treatment of chick DRG neurons significantly increased neurite number and length by approximately 50%. Similar increases in these parameters occurred when src-family kinases were inhibited using PP2. To implicate pp60(c-src) directly in neurite outgrowth, we inactivated it in DRG neuronal growth cones using Chromophore-Assisted Laser Inactivation (CALI). CALI of pp60(c-src) resulted in an 85% inactivation of its kinase activity and a 63% reduction in phosphotyrosine immunofluorescence in neurons. Microscale CALI of pp60(c-src) in DRG growth cones caused a significant and acute two-fold increase in neurite extension rate during irradiation. These findings demonstrate that pp60(c-src) is a negative regulator of laminin-1-mediated neurite outgrowth in chick sensory neurons.

Animals↗
PubMed2002

Global high-throughput screens for cellular function.

There is a critical need for global methods that allow for high-throughput assessment of cellular function for clinical and basic scientists working in both academia and the pharmaceutical industry. These methods typically couple systematic inactivation strategies with high-throughput cell-based assays that facilitate rapid target validation. We present here a survey of these technologies and their applications. We discuss their promise and limitations in addressing the vast number of candidate molecules of disease relevance that are emerging from genomics and proteomics.

Animals↗