and 40-50 nm long (Holley and Ashmore, 1988a, 1990a; ..... new measurements show that cross-links can be at least ..... York: Elsevier Applied Science.
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Journal of Cell Science 102, 569-580 (1992) Printed in Great Britain © The Company of Biologists Limited 1992
Structure of the cortical cytoskeleton in mammalian outer hair cells
M. C. HOLLEY Department of Physiology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK
F. KALINEC and B. KACHAR* Laboratory of Cell Biology, N1DCD, Building 10, Room 5D46, 9000 Rockville Pike, Bethesda, MD 20892, USA *Author to whom reprint requests should be addressed
Summary The cortical cytoskeletal lattice in outer hair cells is a two-dimensional actin-based structure, which can be labelled with rhodamine/phalloidin and disrupted by the enzyme deoxyribonuclease I. Structural information from thin sectioned, freeze-etched and negatively stained preparations shows that it is based upon two types of filament that form a cross-linked lattice of circumferential filaments. The cross-links are 70-80 nm long. Measurements of the spacing between circumferential filaments suggest that the lattice is stiffer circumferen-
tially than it is longitudinally. Analysis of the orientation of circumferential filaments shows that it is composed of discrete domains of up to 10 fan2. Relative movements between domains could allow substantial changes of cell shape without disrupting the unit structure of the lattice, thus allowing the cell cortex to retain its elastic responses to high-frequency deformations.
Introduction
shape (Holley and Ashmore, 1990b) but it is also an important component of at least two entirely different mechanisms for changing cell length. The first is driven electrically and can generate length changes of up to 5% at frequencies of at least 10 kHz (Brownell et al., 1984; Kachar et al., 1986; Ashmore, 1987; Dallos et al., 1991). It is not directly dependent upon either ATP or calcium. It has been suggested that the lattice might drive these length changes (Bannister et al., 1988; Holley and Ashmore, 1990a). Recent experiments, however, show that mechanical forces are more likely to be generated within the plasma membrane (Holley et al., 1991) and that the lattice ensures that the forces lead to directed changes of cell length. The structural basis of the second mechanism is unknown. Much slower length changes of as much as 30% can be initiated by a variety of stimuli, all of which depend upon ATP and calcium (Zenner, 1986; Flock et al., 1986; Canlon et al., 1988; Brundin et al., 1989; Canlon and Brundin, 1991; Dulon et al., 1991). How is the cytoskeletal lattice adapted to these two different mechanisms for changing cell shape? A fundamental property of the mechanics of the cell cortex can be illustrated experimentally. Cells inflated rapidly by fluid injection via a patch pipette shorten to about 70% of their length, burst and return to their original shape within 1-2 seconds. Under these circumstances the cortex behaves as a visco-elastic solid. If
An outer hair cell is a self-supporting cylinder that is attached to adjacent epithelial cells only at the extreme base and apex (Kimura, 1975; Gulley and Reese, 1976; Lim, 1986). Cell shape is maintained by cortical structures, particularly by the cortical lattice, a twodimensional cytoskeleton that lies beneath the lateral plasma membrane. One interpretation of the structure of this lattice is that it is an anisotropic network composed of long, circumferential filaments, 6-7 nm thick, which are cross-linked by filaments 3-4 nm thick and 40-50 nm long (Holley and Ashmore, 1988a, 1990a; Arima et al., 1991). Neither of these filaments has been identified although immunofluorescence labelling suggests that they may be composed of actin and spectrin (Holley and Ashmore, 1990a). Another interpretation is that the cortical lattice is based primarily upon the thin filaments linked together by short fragments of actin (Bannister et al., 1988), thus resembling the spectrin skeleton in erythrocytes (Byers and Branton, 1985; Shen et al., 1986). The cortex of outer hair cells is further specialised by a system of concentric endoplasmic membranes that lies immediately beneath the cytoskeletal lattice (Ekstrom von Lubitz, 1981; Saito, 1983; Flock et al., 1986; Raphael and Wroblewski, 1986; Evans, 1990). The cortical lattice is well adapted to maintaining cell
Key words: outer hair cells, cytoskeleton, cell cortex, cell shape, cell motility.
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cells are inflated over periods of 60 seconds or more then they irreversibly form spheres but they do not burst (Holley and Ashmore, 1988b). Under these circumstances the cortex slowly deforms like a viscous liquid. Cell shape changes are thus dependent upon the rate of cell deformation, an important feature in a cell that generates both high- and low-frequency shape changes. In this paper we provide further evidence that the cortical cytoskeletal lattice is an anisotropic structure that is dependent upon actin for its structural integrity. We also examine large areas of the lattice and provide evidence for a mechanism by which it can accommodate large cell-shape changes whilst still acting as a coherent elastic support in the cell cortex.
Materials and methods Preparation of isolated cells Guinea pigs were killed by cervical dislocation and their cochleae were removed. The organs of Corti were then dissected from the cochlear spirals in L-15 (Liebovitz, Gibco) and the cells were dissociated by passing them repeatedly through a fine pipette tip. Dissociated cells were allowed to settle in a 50 fA droplet of L-15 for 30 min on a glass coverslip. Preparation of cytoskeletons and solutions Cells were extracted for 60 min at 20cC in either of two buffers, which differed only in the concentration of NaCl. (a) A low salt buffer (LS): 2.5 mM Na2HPO4, 2.5 mM NaH2PO4 (pH 7.2), 0.1 mM MgCl2; and (b) a high salt buffer (HS): as (a) with the addition of 150 mM NaCl. The detergents Triton X-100 or Saponin were added to these buffers as required. For selected experiments the extracted cytoskeletons were both stabilised and labelled with 33 nM rhodamine/phalloidin diluted in buffer from a stock solution of 3.3 f
