New and Notable 1755

proteins are considerably less likely tobe trapped in smaller membrane do-mains (Edidin et al., 1991) and SPT oflipid analogs shows no evidence of con-finement to plasma membrane domains(Lee et al., 1993).

There is a significant discrepancy be-tween the SPT and photobleaching re-sults: membrane proteins in the station-ary mode (6%) cannot account for thelarger immobile fraction (25%) meas-ured by photobleaching. Kusumi et al(1993) argue that a portion of the con-fined diffusion mode could account forthe rest of the immobile fraction. Thisis because the area bleached by the laser(0.63 pLm2) is significant larger than theputative membrane domains, so thatsome bleached regions won't have acontiguous reservoir to sustain fluores-cence recovery. However, if all the pro-teins in the confined diffusion mode areassumed to contribute to the immobilefraction, it becomes far greater than thevalue measured by photobleaching.This problem can be resolved if abouthalf of the proteins in the confined dif-fusion mode could escape to adjacentdomains. The authors propose a modelin which the fences that bound a mem-brane domain are dynamic, having un-specified "gates" that open temporarily.In this way a single protein, which isconfined much of the time, may movelong distances in the membrane plane.Indeed, unpublished studies indicatethat the labeled E-cadherin shows inter-compartmental movements (Kusumiet al., personal communication).Whether a "gate" allows "escape" of agiven protein to an adjacent domainwill depend on the size of its cytoplas-mic moiety, so that the effective do-main size may depend on the protein.This would explain why domain sizesdiffer for various proteins.

Those who are fascinated by thecomplex dynamics of plasma mem-branes will eagerly wait additional de-tailed SPT studies of other membraneproteins, for they will most certainlyenhance our understanding of this or-ganelle which is so pivotal in modemcell biology.

REFERENCESAnderson, C. A., G. N. Georgion, I. E. G. Mor-

rison, G. V. W. Stevenson, and R. Cherry.

1992. Tracking of cell surface receptors byfluorescence digital imaging microscopy usingcharge-coupled device camera. Low density li-poprotein and influenza virus receptor mobilityat 4 C. J. Cell Sci. 101:415-425.

Cherry, R. J. 1992. Keeping track of cell surfacereceptors. Trends Cell Biol. 2:242-244.

De Brabander, M., R. Nuydens, A. Ishihara, B.Holifield, K Jacobson, and H. Greets. 1991.Lateral diffusion and retrograde movements ofindividual cell surface components on singlemotile cells observed with nanovid micros-copy. J. Cell BioL 112:111-124.

Edidin, M., S. C. Kuo, and M. P. Sheetz. 1991.Lateral movements of membrane glycopro-teins restricted by dynamic cytoplasmic barri-ers. Science (Wash. DC). 254:1379-1382.

Ghosh, R. N., and W. W. Webb. 1990. Evidencefor intra-membrane constraints to cell surfaceLDL receptor motion. Biophys. J. 57:286a.(Abstr.)

Lee, G. M., F. Zhang, A. Ishihara, C. L. McNeil,and K. A. Jacobson 1993. Unconfined lateraldiffusion and an estimate of periceilular matrixviscosity revealed by measuring the mobilityof gold-tagged lipids. J. Cell Biol. 120:25-35.

Kusumi, A., Y. Sako, and M. Yamamoto. 1993.Confined lateral diffusion of membrane recep-tors as studied by single particle tracking(nanovid microscopy). Biophys. J. 65:2021-2040.

Pumplin, D. W., and R. J. Bloch. 1993. The mem-brane skeleton. Trends Cell BioL 3:113-117.

Sako, Y., A. Nagafuchi, M. Takeichi, and A. Ku-sumi. 1992. Intracellular regulation of themovements of E-cadherin on the cell surface.Mol. Bio. Cell 3:219a. (Abstr.)

Saxton, M. J. 1990. The membrane skeleton oferythrocytes:models of its effect on lateral dif-fusion. Int. J. Biochem. 22:801-809.

Saxton, M. J. 1993. Lateral diffusion in an ar-chipelago: single particle diffusion. Biophys. J.64:1766-1780.

Sheetz, M. P., M. Schindler, and D. E. Koppel.1980. Lateral mobility of integral membraneproteins is increased in spherocytic erythro-cytes. Nature (Lond.). 285:510-512.

Sheetz, M. P. 1983. Membrane skeletal dynam-ics: role in modulation of red cell deformabil-ity, mobility of transmembrane proteins, andshape. Semin. Hemat. 20:175-188.

Sheetz, M. P., S. Tumey, H. Qian, and E. L. El-son. 1989. Nanometre-level analysis demon-strations that lipid flow does not drive mem-brane glycoprotein movements Nature(Lond.). 340:284-288.

Tsuji, A., and S. Ohnishi. 1986. Restriction of thelateral motion of band 3 in the erythrocytemembrane by the cytoskeletal network: depen-dence on spectrin association state. Biochem-istry. 25:6133-6139.

Tsuji, A., K. Kawasaki, S. Ohnishi, H. Merkle,and A. Kusumi. 1988. Regulation of band 3mobilities in erythrocyte ghost membranes byprotein association and cytoskeletal mesh-work. Biochemistry. 27:7447-7452.

Zhang, F., G. M. Lee, and K. Jacobson. 1993.Protein lateral mobility as a reflection of mem-brane microstructure. BioEssays. 15:579-588.

Distribution of Voltage Sensorsin Mammalian Outer Hair Cells

Daniel L. Alkon,*Rene Etcheberrigaray,*and Eduardo Rojas** Laboratory of Adaptive Systems, NationalInstitute of Neurological Disorders andStroke, and Laboratory of Cell Biologyand Genetics, National Institute of Diabetesand Digestive and Kidney Diseases,National Institutes of Health, Bethesda,Maryland 20852, USA

It is now widely accepted that outer haircells (OHC) play a fundamental rolein normal cochlear transduction(Brownell et al., 1985; Ashmore, 1987;Kalinec et al., 1992). Understandinghow the behavior of the OHC deter-mine the critical frequency selectivityof the mammalian cochlea, however,has been a more recent development towhich the article in this issue by Huangand Santos-Sacchi (p. 2228) makes anoutstanding contribution.

In mammals, the cochlea separatessound frequencies by controlling themotion of the basilar membrane. It hasbecome increasingly clear that a meta-bolically labile process involving theOHC greatly contributes to this motioncontrol of the basilar membrane (Hol-ley and Ashmore, 1988; Iwasa andKachar, 1988; Santos-Sacchi, 1991).The OHC exhibit electrically inducedelongation and contraction movementsthat enhance the frequency selectivityand sensitivity of the basilar membranemovement wave. Several lines of evi-dence have shown that the OHC move-ments depend on or are sensitive tochanges in membrane potential (Iwasaand Kachar, 1988). This discovery sug-gested the presence of a voltage-actingmolecule within the plasma membraneof the OHC. The presence of a voltage-dependent nonlinear charge movementas manifest by a voltage-dependentcapacitance has provided additionalevidence for the existence of such amolecule (Santos-Sacchi, 1991).

Electrophysiological and micros-copy studies (Kalinec et al., 1992) haveshown that OHC elongation in responseto hyperpolarization and depolariza-

Received for publicationX) 1993 by the Biophysical Society0006-3495/93/11/1755/08 $2.00

1756 Biophysical Journal Volume 65 November 1993

tion-induced shortening, occur at thelateral plasma membrane. These highfrequency movements would involve avoltage-sensitive force generator lo-cated in the lateral membrane and a spe-cialized cytoskeleton beneath thatmaintains the cylindrical shape of thecell, ensuring that forces produced inthe membrane cause changes in celllength. It has also been determined thatthe mechanical responses of the OHCoccur only in the central region of thecell (Dallos et al., 1991). The presentstudy in this issue of Biophysical Jour-nal mainly focuses on the voltage sen-sor and its localization.OHC were acutely isolated by a com-

bination of mechanical and enzymatictreatment. For recordings, cells weremaintained in saline solution with ioncurrent blockers (tetraethylammonium,tetrodotoxin-, and CoCl2) to avoid inter-ference with capacitive current mea-surements. A protocol involving the useof a partitioning microchamber and adouble-voltage clamp was used to elec-trically amputate and measure cell ca-pacitance in different regions of theOHC.By analogy with the two classical

models of signal transduction which in-

volve confined displacements ofcharged regions of a protein molecule(the voltage sensor), namely Na+-channel gating (Armstrong and Beza-nilla, 1974; Keynes and Rojas, 1974)and excitation-contraction coupling(Schneider and Chandler, 1973), theauthors concluded that in OHCs thephysical domain of the nonlinearcharge movement corresponds to thatof the mechanical effector. This physi-cal domain apparently occurs in theplasmalemma of the cell's central por-tion and not the apical and basalportions.

Mechanosensory frequency filtering,thus provides a new example of signaltransduction involving confined dis-placements of charged regions of pro-tein molecules. Multiple applicationsfor this single type of molecular trans-duction strategy provide another re-markable instance of nature's economyof function.

REFERENCES1. Armstrong, C. M., and F. Bezanilla. 1974.

Charge movement associated with the open-ing and closing of the activation gates of Nachannels. J. Gen. Physiol. 63:533-552.

2. Ashmore, J. F. 1987. A fast motile response

in ginea-pig outer hair cells: the cellular basisof the cochlear amplifier. J. Phisiol. (Lond.).388:323-347.

3. Brownell, W. E., C. R. Bader, D. Bertrand,and Y. de Ribaupierre. 1986. Evoked me-chanical responses of isolated cochlear outerhair cells. Science (Wash. DC).227:194-196.

4. Holley, M. C., and J. F. Ashmore. 1988. Onthe mechanism of a high-frequency forcegenerator in outer hair cells isolated from theguinea pig cochlea. Proc. R. Soc. Lond. B.232:413-429.

5. Huang, G., and J. Santos-Sacchi. 1993. Map-ping the distribution of the outer hair cell mo-tility voltage-sensor by electrical amputa-tion. Biophys. J. 65:2228-2236.

6. Iwasa, K. H., and B. Kachar. 1989. Fast invitro movement of outer hair cells in an ex-ternal electric field: effect of digitonin, amembrane permeabilizing agent. HearingRes. 40:247-254.

7. Kalinec, F., M. C. Holley, K. H. Iwasa, D. L.Limb, and B. Kachar. 1992. A membrane-based force generation mechanism in audi-tory sensory cells. Proc. Natl. Acad. Sci.USA. 89:8671-8675.

8. Keynes, R. D., and E. Rojas. 1974. Kineticsand steady-state properties of the chargedsystem controlling sodium conductance inthe squid giant axon.J. Physiol. (Lond.). 239:393-434.

9. Schneider, M. F., and W. K. Chandler. 1973.Voltage-dependent charge movement inskeletal muscle: a possible step in excitation-contraction coupling. Nature (Lond.). 242:244-246.