of 25 7C (see www.mih.unibas.ch/Nature/Fotiadis.html), but this is still below physio-logical temperature. Phase separation in a burst disc membrane may slow downunder these conditions but will not be prevented. In osmotically disrupted cattlediscs, for example, phase-separation arte-facts were often visible in early X-ray patterns that required 15 h of exposure atroom temperature4.
We therefore maintain that nativerhodopsin exists in monomeric form in theretinal rods of vertebrates. The double-rowarrays seen in AFM7,8 in isolated and osmoti-cally disrupted mouse discs probably resultfrom separation of the lipid phase from theproteins, which occurred rapidly at 0 7C andmore slowly at 25 7C, during the long incuba-tion and upon osmotic disruption of the discs.
Our contention excludes only consti-tutive oligomerization of non-activatedrhodopsin in dark-adapted rods. After illu-mination, in an arrestin-mediated deacti-vation process, rhodopsin molecules mayundergo pairing9. Oligomerization alsooccurs in other G-protein-coupled recep-tors10 in the b-arrestin-mediated process oftheir inactivation and clustering in coatedpits, but these processes are distinct from theoligomerization of non-activated receptors.Marc Chabre*, Richard Cone†, Helen Saibil‡*Institut de Pharmacologie, CNRS-Université deNice-Sophia Antipolis, 06560 Valbonne, Francee-mail: [email protected]†Department of Biophysics, Johns HopkinsUniversity, Baltimore, Maryland 21218, USA‡Department of Crystallography, Birkbeck College,London WC1E 7HX, UK1. Cone, R. A. Nature New Biol. 236, 39–43 (1972).
2. Poo, M. & Cone, R. A. Nature 247, 438–441 (1974).
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7. Fotiadis, D. et al. Nature 421, 127–128 (2003).
8. Liang, Y. et al. J. Biol. Chem. 278, 21655–21662 (2003).
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Fotiadis et al. reply — Individual biologicalmolecules can be imaged under physiologi-cal conditions by atomic force microscopy.Our results from AFM, supported by electron microscopy, revealed distinct rowsof rhodopsin dimers and paracrystallinearrays in native murine disc membranes1,2.This supramolecular arrangement was alsofound for the light-activated form, opsin1.We counted 30,000–55,000 rhodopsin mol-ecules per square micrometre, a packingdensity comparable to that measured byoptical methods in amphibian discs3. Fromthe lattice vectors describing the paracrys-talline arrays, a maximum possible packingdensity of 62,900 rhodopsin molecules per
square micrometre was calculated. Therhodopsin dimers seen in AFM topographs2
have cytosolic protrusions separated by 3.8nm, providing an ideal docking platformfor arrestin, which has two binding groovesthat are separated by 3.8 nm as well1.
On the basis of data reported some 30years ago, Chabre et al. are challenging ourobservations. Results from their and other3
early experiments,which were all carried outon large ensembles of amphibian disc membranes, indicated that inactivated aswell as activated rhodopsin diffuses freely asa monomer in the lipid bilayer. Chabre et al.now propose that the rhodopsin-packingarrangement that we observe by AFM1,2 isinduced by the segregation of proteins andlipids at low temperatures.
To respond to this criticism, we recordedelectron micrographs as well as AFMtopographs of rhodopsin paracrystals onnative disc membranes that had been pre-pared and imaged at room temperature (seewww.mih.unibas.ch/Nature/Fotiadis.html).Three different surface types could again be distinguished: rhodopsin paracrystals,rhodopsin rafts and the lipid bilayer; atincreased magnification, rhodopsin dimers,the building block of the paracrystal, areclearly seen. This dimerization and higher-order organization of rhodopsin at roomtemperature argues against the claim byChabre et al. that the packing arrangementwe describe1,2 is a result of low-temperature-induced protein–lipid segregation, particu-larly if we consider that the phase-transitiontemperature of the bovine-disc lipids isbelow 3 7C (ref.4).
Freeze-fracture electron microscopy hasalso revealed paracrystalline rhodopsinarrays in Drosophila photoreceptive mem-branes5 and in the plasma membrane ofbovine-rod outer segments6. The concept ofoligomerization in the presence or absenceof ligands is generally accepted for many G-protein-coupled receptors7,and rhodopsinis not an exception.Dimitrios Fotiadis*, Yan Liang†,Slawomir Filipek‡, David A. Saperstein†,Andreas Engel*, Krzysztof Palczewski†§¶*M.E. Müller Institute for Microscopy, Biozentrum,University of Basel, Basel 4056, SwitzerlandDepartments of †Ophthalmology, §Pharmacology,and ¶Chemistry, University of Washington, Seattle,Washington 98195, USAe-mail:[email protected]‡International Institute of Molecular and CellBiology, and Department of Chemistry, Universityof Warsaw, Warsaw 02109, Poland1. Liang, Y. et al. J. Biol. Chem. 278, 21655–21662 (2003).
2. Fotiadis, D. et al. Nature 421, 127–128 (2003).
3. Liebman, P. A. & Entine, G. Science 185, 457–459 (1974).
4. Miljanich, G. P., Brown, M. F., Mabrey-Gaud, S., Dratz, E. A. &
Sturtevant, J. M. J. Membr. Biol. 85, 79–86 (1985).
5. Suzuki, E., Katayama, E. & Hirosawa, K. J. Electron Microsc.
(Tokyo) 42, 178–184 (1993).
6. Kajimura, N., Harada, Y. & Usukura, J. J. Electron Microsc.
(Tokyo) 49, 691–697 (2000).
7. Bouvier, M. Nature Rev. Neurosci. 2, 274–286 (2001).
from the conditions used in sample prepa-ration for AFM.
Fast, transient, flash-induced photo-dichroism1 first revealed the rapid rotationaldiffusion of rhodopsin in situ, and the kinet-ics of flash-bleaching recovery2 indicatedthat rhodopsin undergoes rapid lateral dif-fusion in intact rods.Its random distributionin the membrane was evident from X-rayand neutron diffraction3–5 and was con-firmed by electron microscopy6. We there-fore question whether the structures seen byFotiadis et al.7 in an osmotically burst discmembrane represent a native disc structure,or whether they can be explained by separa-tion of the proteins and lipids during the 15-h incubation at 0 7C in 2 mM Tris–HCl inthe preparation of samples for AFM8.
A long exposure at low temperature willalter the native disc membrane structure inrods isolated from a warm-blooded verte-brate. In-plane X-ray diffraction revealedlong ago that cooling cattle rods to 5 7C foronly 15 min induces a partial phase transi-tion of the lipids and a correlated segregationof the proteins4. By contrast, no phase sepa-ration in frog rods was ever observed by X-ray4 or neutron5 diffraction.
Low-temperature investigation of flash-induced transient photodichroism (R.C.,unpublished results) has confirmed that frogrhodopsin can still undergo rapid rotationaldiffusion at temperatures close to 0 7C.Transient photodichroism cannot reliablydistinguish between freely rotating dimersand monomers, but the packed rows ofrhodopsin seen in the AFM images ofFotiadis et al.7 are incompatible with rapidrotational diffusion. And if rhodopsin wereoligomerized into rows in vivo, a stable orslowly decaying flash-induced dichroismwould have been detected long ago in humanvision and in isolated vertebrate retinae1.
Electron-microscope images of snap-frozen, freeze-etched frog rods6 were able toresolve rhodopsin monomers in a randomarray, with no evidence of dimers, althoughthe rods had been incubated at 4 7C for 1 h.After digestion of these fragmented rods withphospholipase C, however, disc-membranephospholipids were seen to segregate intodroplets, whereas the packing of rhodopsinchanged into concentrated, row-like arrays6.It is possible that these could be the equivalent of the double-row arrays thatwere observed by AFM7.
All of these biophysical data are old,but remain valid. Until now, they have notbeen experimentally challenged or refuted.They exclude the occurrence of rhodopsinoligomers in native,unactivated,frog rods andprovide evidence for the rapid phase separa-tion of rhodopsin and lipids in mammaliandisc membranes kept at low temperature.
Fotiadis et al. observe the same separa-tion of rhodopsin and lipids when their sam-ples are incubated at the higher temperature
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NATURE | VOL 426 | 6 NOVEMBER 2003 | www.nature.com/nature 31© 2003 Nature Publishing Group
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