Cell damage by near-IR microbeams

摘要: SCIENTIFIC CORRESPONDENCE extracellular E1 E2 E3 0 141 intracellular Correlated mutations in rat olfactory receptors (see also ref. 7). Transmembrane ol—helices (denoted by roman numerals) are shown as cylinders, interhelical loops lines. E1, and indicate respective loops. Squares correlated residues; shaded circles predicted ligand-binding open residues of unknown function. recent finding that messenger RNA for putative recep- tors is present the axon termi- nals ORNs within their target glomerulus5qq, suggesting receptor molecules may be involved guiding axons to glomeruli. We propose loop contributes this through axon-to-axon or axon-to-substrate interactions. Sequence motifs loop, especially conserved cys- teines, mediate function; similar integrins growth factors. The mutation anal- ysis7 shows a one-to-one corre- spondence between at r,~~ 316 457 0.973 430 505 509 0.997 Significant pairwise correlations with residues. r,-,~ correlation coefficient comparison sequence positions i j. Correlations were calculated CORMUT option WHATIF15. potential importance has already been suggested nonrandom amino-acid substitutions resulting from positive selectionq), implying function unique each subtype. Also, survey family hydrophobicity profiles“ contains approximately 35 residues, substantially more than most other members G- protein-coupled superfamily. Among possible functions E2, interactions odour seem less likely because hydrophilic aqueous environment provide poor binding site molecules, which terrestrial mammals typically hydro- phobicz. Interactions proteins (OBPS) chaperonins they appear inconsistent extreme diversity apparent functional specificity subtype 10). A possibility raised 1. Chess, A., Simon, l., Cedar, H. & Axel, R. Cell 78, 823-834 (1994). 2. Lancet, D. A. Rev. Neurosci. 9, 329-355 (1986). 3. Ressler, K. J., Sullivan, 5. L. Buck, B. 73, 597-609 (1993). 4. Vassar, R., Ngai, J. 74, 309-318 et al. 79, 981-991 6. 8. 1245-1255 7. Singer, M. S., Olivelra, L., Vriend, G. Shepherd, Receptors Channels (in press). S. NeuroRepoit 5, 1297-1300 9. Kuipers, W. Membrane Protein Models: Experiment, Theory Speculation (ed. Findlay, J.) (Oxford, BIOS, (loop E2) (predicted site; see table figure legend). Thus, residue position carries suf- ficient information infer (Lys:His, Gln:Gln, Asn:Lys Valzlle, respectively). Positions form another such pair. anticipate additional pairs will revealed analysis further receptors. Loop can therefore cell-surface identifier subtype, reflect- ing odour-binding pocket. Consistent our observa- tion no two sub- types share same sequence”’”. variation across subtypes contribute abilities ORN one subset find common glomerulus, related subsets neighbouring This could basis function- al topography bulb. homophilic axons, bind preferentially specific receptors, adhesion, chemotac- tic molecules. Other domains (E1 E3) participate these interactions, but do not special properties namely hypervariability, selection, uncommon length cysteine motifs. Immunocytochemistry presence pro- tein (R. Reed, personal communication). Furthermore, mRNA evident before reach bulb estab- lish synapses”, consistent role development. Olfactory perform dual role, sensory cilia guidance axons. represent highly mechanism any previously identified implies novel property some G-protein-coupled Michael Gordon Shepherd Charles Greer Sections Neurobiology Neurosurgery, Yale University School Medicine, New Haven, Connecticut 06510, USA damage near-IR microbeams SIR — Continuous-wave laser micro- beams generally used diagnostic tools scanning microscopes or, case near—infrared microbeams, optical traps cell manipulation force characterization”. Because single- beam created objectives high numerical aperture (NA), typical trapping intensities photon flux densi- ties are, respectively, order MW cm'2 1027 cmq sq. These extremely fields induce two-photon absorption processes” anom- alous biological effects. have investigated phenomena studying optically trapped motile human spermatozoa maintained HEPES—buffered fresh tubal fluid labelled live/dead fluorescence assay kit (Molecular Probes). Trapping effects 760 800 nm (70 mW) compared using tunable, cence (515 nm) corresponding mem- brane-permeable live-cell probe SYBR 14 (Fig. 1). Damaged cells showed red fluo- rescence (636 nm), due intranuclear accumulation membrane-imperme- able dead-cell propidium iodide. Figure 1a brightfield image illus- trating sperm structure, flagellar motion an intense green spot centre head. Without illumi- nation, sub-micrometre persisted 10. 11. 12. 13. 14. 15. 20 Hughes, Hughes mo/ec. Evol. 36, 249-254 Engelman M., Steitz, T.A. Goldman, biophys. Chem. 15, 321-353 CeII65, 175-187 (1991). Ben-Arie, N. Hum. Genet. 3, 229-235 Strotmann, Wanner, Helfrich, T. Breer, Eur. 7, 492-500 (1995). Graph. 8, 52-56 (1990). continuous-wave Ti : sapphire coupled inverted microscope. Visible two-photon- excited was detected cooled CCD camera. Viable fluores- FIG. 1a, Brightfield confined 760-nm trap. Two-photon- fluorescence fluorescent clearly visible submicrometre 5pm-long head despite halogen lamp illumination. b, two-photon-excited absence NATURE - VOL 377 7 SEPTEMBER 1995