Cell damage by near-IR microbeams

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 as lines. E1, E2 and E3 indicate respective extracellular loops. Squares indicate correlated E2 residues; shaded circles indicate predicted ligand-binding residues; open circles indicate correlated residues of unknown function. recent finding that messenger RNA for putative olfactory recep- tors is present in the axon termi- nals of ORNs within their target glomerulus5qq, suggesting that the receptor molecules may be involved in guiding axons to their target glomeruli. We propose that loop E2 contributes to this through axon-to-axon or axon-to-substrate interactions. Sequence motifs in the loop, especially conserved cys- teines, may mediate this function; similar motifs are present in integrins and growth factors. The correlated mutation anal- ysis7 shows a one-to-one corre- spondence between the residues at Correlated residues r,~~ 316 457 0.973 430 457 0.973 505 509 0.997 Significant pairwise correlations with E2 residues. r,-,~ is the correlation coefficient for pairwise comparison of sequence positions i and j. Correlations were calculated by the CORMUT option of WHATIF15. The potential importance of loop E2 has already been suggested by the finding of nonrandom amino-acid substitutions resulting from positive selectionq), implying that loop E2 has a function unique to each receptor subtype. Also, a survey of the olfactory receptor family by hydrophobicity proﬁles“ has shown that loop E2 contains approximately 35 residues, substantially more than most other members of the G- protein-coupled receptor superfamily. Among possible functions for loop E2, interactions with odour molecules seem less likely because the hydrophilic loop and aqueous environment provide a poor binding site for odour molecules, which for terrestrial mammals are typically hydro- phobicz. Interactions with odour binding proteins (OBPS) or chaperonins are also less likely because they appear inconsistent with the extreme sequence diversity in the loop and the apparent functional specificity of each subtype (see ref. 10). A more likely possibility is raised by the 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, L. B. Cell 73, 597-609 (1993). 4. Vassar, R., Ngai, J. & Axel, R. Cell 74, 309-318 (1993). 5. Vassar, R. et al. Cell 79, 981-991 (1994). 6. Ressler, K. J., Sullivan, 8. L. & Buck, L. B. Cell 79, 1245-1255 (1994). 7. Singer, M. S., Olivelra, L., Vriend, G. & Shepherd, G. M. Receptors and Channels (in the press). 8. Singer, M. S. & Shepherd, G. M. NeuroRepoit 5, 1297-1300 (1994). 9. Kuipers, W. et al. in Membrane Protein Models: Experiment, Theory and Speculation (ed. Findlay, J.) (Oxford, BIOS, in the press). positions 457 (loop E2) and 430 (predicted odour binding site; see table in figure legend). Thus, the residue at position 457 carries suf- ficient information to infer the residue at position 430 (Lys:His, Gln:Gln, Asn:Lys and Valzlle, respectively). Positions 505 and 509 form another such pair. We anticipate that additional correlated pairs will be revealed by analysis of further receptors. Loop E2 can therefore function as a cell-surface identifier for the receptor subtype, reﬂect- ing the specificity of the odour-binding pocket. Consistent with this is our observa- tion that no two olfactory receptor sub- types share the same loop E2 sequence”’”. The variation of loop E2 residues across subtypes may contribute to the abilities of ORN axons of one subset to find their common target glomerulus, and of related subsets to target neighbouring glomeruli. This could provide a basis for the function- al topography of the olfactory bulb. The loops could form homophilic interactions between axons, or bind preferentially to specific receptors, adhesion, or chemotac- tic molecules. Other extracellular domains (E1 and E3) could participate in these interactions, but they do not seem to share the special properties of loop E2, namely the hypervariability, positive selection, uncommon length and cysteine motifs. Immunocytochemistry has suggested the presence of the olfactory receptor pro- tein in the ORN axons (R. Reed, personal communication). Furthermore, receptor mRNA is evident in ORNs before their axons reach the olfactory bulb and estab- lish synapses”, consistent with a role for the receptor in development. Olfactory receptors may perform a dual role, as odour receptors in the sensory cilia and as guidance molecules in the axons. This may represent a more highly specific mechanism of axon guidance than any previously identified and implies a novel property for some members of the G-protein-coupled receptor superfamily. Michael 5. Singer, Gordon M. Shepherd Charles A. Greer Sections of Neurobiology and Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06510, USA Cell damage by near-IR microbeams SIR — Continuous-wave laser micro- beams are generally used as diagnostic tools in laser scanning microscopes or, in the case of near—infrared microbeams, as optical traps for cell manipulation and force characterization”. Because single- beam traps are created with objectives of high numerical aperture (NA), typical trapping intensities and photon ﬂux densi- ties are, respectively, of the order of MW cm&#39;2 and 1027 cmq sq. These extremely high fields may induce two-photon absorption processes” and anom- alous biological effects. We have investigated these phenomena by studying optically trapped motile human spermatozoa maintained in HEPES—buffered fresh human tubal ﬂuid labelled with a live/dead ﬂuorescence assay kit (Molecular Probes). Trapping effects at 760 and 800 nm (70 mW) were compared using a tunable, cence (515 nm) corresponding to the mem- brane-permeable live-cell probe SYBR 14 (Fig. 1). Damaged cells showed red ﬂuo- rescence (636 nm), due to intranuclear accumulation of the membrane-imperme- able dead-cell probe propidium iodide. Figure 1a shows a brightfield image illus- trating sperm structure, ﬂagellar motion and an intense green spot in the centre of the sperm head. Without brightfield illumi- nation, the sub-micrometre spot persisted 10. 11. 12. 13. 14. 15. 20 Hughes, A. L. & Hughes M. K. J. mo/ec. Evol. 36, 249-254 (1993). Engelman D. M., Steitz, T.A. & Goldman, A. A. Rev. biophys. Chem. 15, 321-353 (1986). Buck, L. & Axel, R. CeII65, 175-187 (1991). Ben-Arie, N. et al. Hum. mo/ec. Genet. 3, 229-235 (1993). Strotmann, J., Wanner, l., Helfrich, T. & Breer, H. Eur. J. Neurosci. 7, 492-500 (1995). Vriend, G. J. mo/ec. Graph. 8, 52-56 (1990). continuous-wave Ti : sapphire laser coupled to an inverted ﬂuorescence microscope. Visible two-photon- excited intracellular ﬂuorescence was detected with a cooled CCD camera. Viable cells showed green ﬂuores- FIG. 1a, Brightfield image of a motile human sperm cell confined in the 760-nm optical trap. Two-photon- excited fluorescence of a live/dead fluorescent probe is clearly visible as an intense submicrometre spot in the 5pm-long sperm head despite the presence of halogen lamp illumination. b, The two-photon-excited fluorescent spot is more clearly visible in the absence of brightfield illumination. NATURE - VOL 377 - 7 SEPTEMBER 1995