Laser inactivation of fasciclin I disrupts axon adhesion of grasshopper pioneer neurons

Abstract

A molecular mechanism for selective axonal adhesion is a central question of neural development. Cell adhesion molecules have been identified, but it has been difficult to ascribe functions for these proteins in vivo. Here we show that the neuronal membrane glycoprotein fasciclin I has a role in the adhesion of sister axons during the development of the grasshopper limb bud. To do this we used a new technique, chromophore-assisted laser inactivation (CALI), which causes the precisely timed thermal denaturation of specific proteins by laser light targeted through a dye-labelled antibody, without any other observable damage to living cells. This can be achieved by relaxation of the laser-excited dye which releases heat to denature the bound protein; the rapid dissipation of heat with distance insulates unbound proteins from damage. CALI is a molecular analogue of cellular laser ablation and provides an unprecedented level of spatial and temporal resolution. Using dye-labelled antibodies that recognize fasciclin I, CALI disrupts fasciculation of the pioneer neurons without affecting their growth or guidance.

Chromophore-assisted laser inactivation of patched protein switches cell fate in the larval visual system of Drosophila.

“…To test whether the supernumerary BO neurons are derived from the pool of optic-lobe precursor cells, we made use of an "enhancer-trap line" (13,29) which causes f3-galactosidase expression in the optic lobe but not in the BO precursors ofwild-type embryos (Fig. 3 a and b).…”

Role of Central Nervous System Periostin in Cerebral Ischemia

“…6 Also, laser inactivation of fasciclin I disrupts axon adhesion of grasshopper pioneer neurons. 7 Thus, periostin might have pivotal roles in the CNS in mammals, although its expression and function have not been clarified in the adult CNS.…”

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

“…Despite the difficulties faced by these techniques, chemical labeling is of great interest because it permits novel types of experiments by targeting chemicals with a wider range of functionality than that of fluorescent proteins. For example, intracellular targeting of a photosensitizing compound to a protein of interest can be used to rapidly inactivate that protein upon irradiation (8). GFP is not an efficient photosensitizer, presumably because the protein shell of GFP prevents the generation or the efficacy of reactive oxygen species (ROS) that mediate the inactivation (9).…”