Imaging subcellular structures of rat mammary carcinoma cells by ...

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Journal of Cell Science 107, 2427-2437 (1994) Printed in Great Britain © The Company of Biologists Limited 1994

Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy L. I. Pietrasanta1,2, A. Schaper1 and T. M. Jovin1,* 1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, PO Box 2841, 2Instituto de Investigaciones Bioquimícas (INIBIBB), CC 857, 8000 Bahía Blanca, Argentina

37018 Göttingen, Germany

*Author for correspondence

SUMMARY Scanning force microscopy (SFM) was used for imaging subcellular structures of cultured rat mammary carcinoma cells dried in air. Identification of cellular substructures was achieved by immunofluorescence and specific fluorescence probes. Cells grown attached to a glass support exhibited submicrometer thickness in the dried state. Inside the nuclear domain the nucleoli appeared as prominent conical protrusions. Membrane extensions, microspikes and microvilli were well preserved at the cell periphery after fixation in glutaraldehyde vapor and airdrying and were distinguishable either as isolated elements or intercellular communications. The plasma membrane and soluble proteins were selectively removed with non-

ionic detergent in a buffer system. The mitochondria were concentrated primarily in the perinuclear space and exhibited a well defined filamentous shape. Their identity was confirmed by specific fluorescence staining with rhodamine 123. In the membrane-free system achieved by dry-cleaving of the sample surface, the cytoskeletal network was resolved as a complex mesh of actin-containing fiber bundles interwoven with a filigree arrangement of thinner filaments. The smallest fibrous substructures revealed by SFM with the scanning tips used to date were ∼8 to 10 nm in height and 80 nm in width.

INTRODUCTION

collapse after air-drying it could be demonstrated that a cellular fibrous pattern appeared at the subcellular level. Several methods have been elaborated to preserve intracellular structures, including removal of the cytoplasm and the plasma membrane by detergent extraction (Brown et al., 1976; Osborn and Weber, 1977a; Small and Celis, 1978; Mitsushima and Katsumoto, 1990), mechanical fracturing (Heuser and Kirschner, 1980), and dry cleaving of the specimen (Mesland et al., 1981; for a review, see Bell et al., 1989). Following exposure to the detergent Triton X-100 the fine structure of the isolated cellular scaffold, i.e. the detergent-resistant part of the cytoskeleton, was revealed by immunofluorescence (Osborn et al., 1978a). These studies showed that the organization of the isolated cytoskeleton is representative of the intact intracellular scaffold of whole cells (Webster et al., 1978; Osborn et al., 1978b). A major feature of the cytoskeleton is a sheath of actin-containing fibers (microfilaments) that form a continuous network resembling the intact cell in overall appearance (Webster et al., 1978). The remaining skeletal elements and the bulk of the cytoplasm are enclosed by this filamentous sheath (Henderson and Weber, 1979). Microfilament bundles (stress fibers) traversing the cell body are aggregates of microfilaments (Lazarides and Weber, 1974). They are preferentially expressed in adhesive cells growing and locomoting on solid substrata (Lazarides and Burridge, 1975; Weber, 1975). Occa-

The structural and morphological complexity of cells reflect to a large degree the requirements for homeostasis, signal transduction and differentiation. The advances in cell biology that have led to a better understanding of structure-function correlations have been based on novel methodological developments, including imaging techniques offering high spatial resolution and/or specificity (for a review, see e.g. Bershadsky and Vasiliev, 1988). The cytoplasm of higher eucaryotic organisms contains a complex set of cytoskeletal fibers comprising three major classes of filaments: microfilaments, microtubules and intermediate filaments. Apart from determining cell shape and motility, the filament network is important for the interaction with the extracellular matrix via transmembrane receptors, e.g. in adhesive cell-matrix interactions of confluent cells (Wehland et al., 1979), and cell-cell contact (Geiger et al., 1987; for a review, see Carraway and Carothers-Carraway, 1989). The intracellular matrix has been probed by electron microscopy (EM) of whole fixed cells (Metuzals, 1969; Kilarski and Koprowski, 1976) and of thin sections (Goldman and Knipe, 1972), and by immunofluorescence microscopy (Lazarides and Weber, 1974; Weber et al., 1975). Efforts have been made to view substructures of fixed and air-dried cells (Brown et al., 1976; Small and Celis, 1978). Despite cell

Key words: atomic force microscopy, cytoskeleton, mitochondria

2428 L. I. Pietrasanta, A. Schaper and T. M. Jovin sionally, these bundles terminate abruptly at the cell margin at sites of firm attachment to the underlying substratum. The actin fiber sheath is interwoven with elements of the polymorphic intermediate filament system and to a lesser extent with tubulin-containing microtubules. From EM measurements the filaments are classified into three distinct sizes according to diameter: microfilaments (6 nm), intermediate filaments (10 nm), and microtubules (25 nm). In this study we have combined scanning force microscopy (SFM) and immunofluorescence microscopy for imaging structural features of a mammalian cell line. The SFM offers the potential for atomic/molecular resolution of cellular and molecular structures in air and in liquid even with living cells under physiological conditions (for a recent review, see Hoh and Hansma, 1992). From its inception, the primary goals of SFM have included the imaging of biological cells (prokaryotic, eucaryotic) and subcellular structures in air, and particularly in the living-functional state in liquid (Butt et al., 1990; Gould et al., 1990). SFM has been applied to studies of the cell membrane, cell organelles, and the membrane transparent cytoskeleton of fixed and living cells (Hörber et al., 1992; Radmacher et al., 1992; Chang et al., 1993; Kasas et al., 1993; Parpura et al., 1993; Putman et al., 1993). Recently, the cell surface of living cells was imaged by SFM, thereby providing some insight into the dynamics of cell motility (Häberle et al., 1992; Henderson et al., 1992; Radmacher et al., 1992). In these investigations, the fine structure of the intracellular cytoskeleton was necessarily unavailable to the scanning tip. In this SFM study we present the extracellular and the exposed intracellular topography of a culture rat mammary carcinoma (RMCD) cell (Rathke et al., 1975). The complex nature of the cytoskeleton (Henderson and Weber, 1979) is revealed in suprising detail. We show that the SFM is a valuable tool for visualizing nanometer structures of the isolated cytoskeleton of the detergent-treated cell, and that it can be used for the non-destructive inspection of filigree biological microassemblies without contrast enhancement (as required for EM) or a labeling procedure (as in immunofluorescence microscopy). MATERIALS AND METHODS Cells Rat mammary carcinoma cells (RMCD; Rathke et al., 1975) (a gift from Dr M. Osborn) were harvested on glass coverslips in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum at 37°C in a CO2 incubator. Chemical treatment for dissection of the RMCD cell Procedure 1 Specimens were prepared as previously described (Henderson and Weber, 1979; Heuser and Kirschner, 1980). In short, cells were washed for 1 minute in phosphate-buffered saline (PBS) at 37°C and incubated for 1 minute at 37°C in stabilization buffer (S-buffer): 0.1 M piperazine N,N′-bis(2-ethane sulphonic acid) (PIPES), pH 6.9, 0.5 mM MgCl2, 0.1 mM EDTA. They were then washed with PBS and fixed in the vapor phase of 25% glutaraldehyde (Webster et al., 1978; Henderson and Weber, 1979) for 15 minutes. After washing with PBS and distilled water the samples were dehydrated in ethanol, air dried, and imaged with the SFM.

Procedure 2 For visualization of the mitochondria the cells were washed according to procedure 1 and fixed in solution with 1.5% glutaraldehyde in Sbuffer at room temperature for 20 minutes. They were treated with 0.2% Triton X-100 in S-buffer and with 4 M glycerol at 37°C for 3 minutes prior to dehydration and air drying. Procedure 3 For imaging the cytoskeleton, cells were washed according to procedure 1, treated with Triton X-100 according to procedure 2, and fixed in the vapor phase of glutaraldehyde (procedure 1). For SFM imaging of the cytoskeleton the air-dried sample surface (plasma membrane) was removed by application of sticky tape (Tesa; Baiersdorf, Hamburg, FRG; Mesland et al., 1981). Scanning force microscopy A NanoScope III-contact SFM (Digital Instruments (DI), Santa Barbara, CA) was used for SFM imaging under ambient conditions. In contact-SFM a very sharp stylus mounted at the tip of a cantilever is in continuous contact with the surface of a sample mounted on a piezoelectric transducer/actuator (PZT) and is scanned in a raster fashion over a selected area. The force sensor is integrated into an optical readout system for detecting bending motions due to changes in surface topography. These motions are registered with Å accuracy via the reflection to a split photodiode of a laser beam focused on the rear of the cantilever. In the constant force (topographic) mode an electronic feedback control compensates for changes of the topography by modifying the z-extension of the PZT, i.e. by adjusting the height of the sample. Microfabricated Si tips integrated into triangular cantilevers (Ultralevers, Park Scientific, Sunnyvale, CA) with force constants between 0.03 and 0.06 N/m were used. Scanning was with a J-tube PZT with 135×135 µm (x, y) and 5.4 µm (z) scan range. The scan rate was 8 lines/second and only the standard planefit correction was applied to the data. Image size was 512×512 pixels, so that the acquisition time for an image was ∼1 minute. Image processing was with the NanoScope software combined with manipulation with NIH-Image (NIH, Bethesda), Photoshop (Adobe Systems), and Canvas (Deneba Systems). In the analysis of cross-sections we report the full width at half maximum height (FWHM) as a first-order compensation for the systematic distortions introduced by the conical tip geometry. These effects increase progressively for steeper, higher structures and constitute an inherent limitation of the SFM technique. We propose that the FWHM provides a good compromise in the interpretation of the data obtained from the point and face detection modes of the conical tip at edge structures (Fritzsche et al., 1994; see also Discussion). Fluorescence microscopy of mitochondria, actin and tubulin For labeling of mitochondria, rhodamine 123 (Calbiochem, San Diego, CA; Johnson et al., 1980) was dissolved in dimethylformamide to a concentration of 1 mg/ml and diluted 100-fold into the cell culture medium immediately prior to use. Living cells were incubated with the dye for 30 minutes at 37°C, washed three times with medium, and maintained in medium supplemented with 10% fetal calf serum. For visualization of actin in the fluorescence microscope the detergent-treated and fixed cells (procedure 3) were labeled with FITC-conjugated phalloidin (Sigma, St Louis, MO). The samples were incubated prior to labeling with NaBH4 in S-buffer for 4 minutes in order to block aldehydes and thereby reduce the background fluorescence signal and the non-specific antibody binding of the fixed samples (Weber et al., 1978). For visualization of tubulin an undiluted hybridoma supernatant of antibodies (Osborn and Weber, 1977b) (a gift from Dr M. Osborn) was applied to detergent-treated and fixed cells (procedure 3) for 30 minutes at room temperature after blocking aldehydes with borohy-

Scanning force microscopy of cells 2429 dride in S-buffer as described above. The sample was washed with PBS for 3 minutes, incubated with rhodamine-labeled goat anti-mouse IgG (Rh-GAMIG; Jackson Immunoresearch Laboratories, West Grove, PA), and diluted 1:200 in PBS for 30 minutes at room temperature. Excess antibody was removed by washing the samples with PBS for 2 minutes. Fixation of the sample was with 1.5% glutaraldehyde in PBS for 10 minutes at room temperature. The sample was rinsed in PBS and mounted on slides with Mowiol 4-88 (Hoechst, Frankfurt, FRG). Fluorescence imaging was with a Zeiss Axioplan epifluorescence microscope equipped with a Photometrics CH220 camera system (Photometrics, Tucson, AZ) incorporating a thermoelectrically cooled Kodak KAF 1400 CCD sensor.

RESULTS Overall morphology of the dried RMCD cell The RMCD cell exhibits a fibroblastic morphology and appears flat after air drying. The cell body can extend over several hundred µm2 (Rathke et al., 1975). These characteristic features were revealed in the SFM. Fig. 1A shows a surface area of cultured RMCD cells in a confluent state mounted on a coverslip and air dried (procedure 1). Typically, a large cytoplasmic region surrounded the oval nucleus about 50-100 nm above the glass surface, which appeared between adjacent cells with a roughness on the nanometer range. In Fig. 1B a selected surface area depicts the cytoplasm of four cells in contact with part of their plasma membranes overlapping in a narrow region. Such structures appear in the late confluent state of cell growth in which contact inhibition is in effect. The height of the cytoplasmic region in Fig. 1B was ∼60 nm; i.e. the dried cell appeared very flat in this part of the cell body. The cell surface was perceptibly modulated by intracellular fibers with corrugations 20-30 nm in height (Fig. 1C). This fibrous pattern is typical for bundles of actin filaments traversing the cytoplasm towards the cell border (see below). The oval and planar nuclear regions are shown at a higher resolution in Fig. 1D. Typically, the dried nuclear region extended up to 20 µm in length, 10 µm in width and ∼300 nm in height. The surface of the nucleus appeared smoothly contoured in the SFM (see Discussion below). The most prominent structures inside the nucleus were the nucleoli, appearing as a single large conical protrusion or, occasionally, as a cluster of smaller individual units (Fig. 1D). From the SFM micrograph in Fig. 1D the typical dimensions of these nuclear protrusions were ∼2 µm in width and 900 nm in height. The maximum peak height of the specimen, typically at one of the nucleoli, did not exceed 1.5 µm, rendering the RMCD cell a very favorable specimen for SFM investigations. Membrane extensions and organelles: microspikes, microvilli and mitochondria Thin membranous extensions, microspikes, were visible at the borders of the RMCD cells (Fig. 2A). Microspikes were commonly observed in the case of confluent cells, indicative of fibroblastic growth and locomotion. Firm anchorage of microspikes (Fig. 4E) enables the cell to retract the cell body (Wehland et al., 1979). According to our SFM measurements, these extensions can extend more than several micrometers from the cell body, typically ∼300 nm in width, 2-30 µm in length, and ∼50 nm in height (Fig. 2D). The latter value is appreciably greater than expected for two stacked membrane

bilayers, and may reflect the presence of the actin-containing core (Wehland et al., 1979; cf. Fig. 4E). Similar thin membranous extensions, constituting part of the extracellular matrix and involved in cell-cell contact, were discerned (Figs 2B, 4E). They were 30 nm in height and 250 nm in width (Fig. 2E). Microvilli were distributed over the whole cell surface and displayed an overall morphology similar to that of microspikes (Fig. 2C). These fingerlike extensions of the cell membrane are mechanically supported by small bundles of actin filaments projecting outwards from the cortex to form the stiff core (Follet and Goldman, 1970; Wehland and Weber, 1980). Our SFM measurements showed structures 58±10 nm in height, 270±45 nm in width, and 1.55±0.37 µm in length (Fig. 2F). Part of the soluble cytoplasma of the unfixed RMCD cell was selectively removed by exposure to nonionic detergent at moderate conditions and postfixation (procedure 2). From inspection of the samples by phase-contrast light microscopy it was obvious that the nuclei resisted the extraction step, but that the cytoplasmatic cell fraction was significantly depleted (data not shown). A region around a nucleus in which part of the dorsal cell envelope was removed was imaged with the SFM (Fig. 3A). The arrows point to a presumed demarcation of the partially removed cellular membrane exhibiting a step of ∼50 nm. In the membrane-free region, the mitochondria were clearly distinguishable from the microvilli (see also Fig. 2C). The characteristic elongated cylindrical shape of the mitochondria is best perceived in a perspective view (Fig. 3B). The dimensions were several µm in length, 290±40 nm in width, and 70±15 nm in height (mean values from data collected from 10 structures). A rhodamine-123-stained fluorescence micrograph of the mitochondria (Fig. 3C) confirmed that they were primarily clustered in the perinuclear region and exhibited a wormlike morphology. Morphology of the exposed cytoskeleton of the RMCD cell After removing lipids and soluble proteins with Triton X-100, the detergent-resistant part of the RMCD cell was subjected to fixation and dry-cleaving of the sample surface with adhesive tape (procedure 3). A gallery of SFM and fluorescence images obtained under these conditions is shown in Fig. 4. Drycleaving removed the dorsal part of the RMCD cell, thereby creating a fresh stable surface in the SFM. A well-defined cleavage plane free from surface contamination is displayed in Fig. 4A. Neither rupture nor detachment of the subcellular structures from the glass surface was observed in this and other such experiments. Close to the nucleus the area of the cell body appeared amorphous with several holes, presumably due to the lipid extraction (Fig. 4A). The most prominent structures of the flat cell body were the thick parallel fibers constituting the microfilaments extending out from the cell center and primarily oriented along the long axis of the cell. Cross-sectional analysis in the SFM showed fibers 200-500 nm in width and 70-150 nm in height. The microfilaments terminated abruptly at or near the cell margin (Fig. 4D), often coinciding with the loci of focal contacts (see below). The fluorescence images of RMCD cells labeled with fluorescein-phalloidin (Fig. 4E) displayed the corresponding bundles of actin filaments or, more generally, stress fibers incorporating the microfilamentassociated proteins, myosin, tropomyosin, α-actinin and filamin (Weber and Groeschel-Steward, 1974; Lazarides and

2430 L. I. Pietrasanta, A. Schaper and T. M. Jovin

Fig. 1. Overall morphology by SFM of cultured RMCD cells grown on coverslips and air dried. The x,y dimensions in each panel are denoted by the horizontal bar and the height is color-coded according to the horizontal bar placed below the figure. (A) Top view. Bars, 30 µm (x,y) and 1.5 µm (z). Cells were fixed with glutaraldehyde and air dried (procedure 1). The nuclei (nuc) appear as bright oval protrusions and the cell body is flattened on the glass surface (S). Fingerlike membrane extensions, microvilli (mv), are visible around the nucleus. In other cell regions the membrane region (mem) appears flatter. (B) Membrane region of four cells in contact. Top view. The membrane protrusions in this very flat region of the cell membrane are modulated by the cortical cytoskeleton and by the partial overlap of membrane regions of adjacent cells. Bars, 10 µm (x,y) and 1 µm (z). (C) Zoom and perspective view (tilt angle 60°) of the region indicated with the arrow in B. The small arrows point to the filamentous pattern of the membrane surface modulations. Bars, 3 µm (x,y) and 0.6 µm (z). (D) Zoom and perspective view (tilt angle 60°) of the nuclear region. The width of the nucleus is about 10 µm, and the length is about 20 µm. A cluster of 3 conical nucleoli appears inside the nucleus. Bars, 4 µm (x,y) and 1.8 µm (z).

Burridge, 1975; Wang et al., 1975), and of central importance in cell motility and locomotion. Fig. 4B presents an additional example of the polymorphic cellular architecture defined by the

stress fibers and resolved by SFM. The pattern was reminiscent of and presumably bore a causal relationship to the membrane modulations in Fig. 1C. Regions of three and two

Scanning force microscopy of cells 2431

Fig. 2. SFM of membranous extensions of the RMCD cell. (A) Micrograph of microspikes. Top view. These membrane extensions (mem) are similar to microvilli but are in contact with the solid support (S) and involved in cell adhesion and cell growth. Bars, 2 µm (x,y) and 0.5 µm (z). (B) Cell-cell contact by thin membrane extensions. Top view. Bars, 3 µm (x,y) and 0.4 µm (z). (C) Top view of a region of the cell membrane in the nuclear halo (see Fig. 1A). The whole cell surface is covered by fingerlike protrusions, the microvilli. Bars, 2 µm (x,y) and 0.5 µm (z). (D, E, F) Cross-sectional scans of selected cellular substructures along the broken line in A, B and C, respectively. Distances between markers are labeled in color.

cells in contact are shown in Fig. 4C and D, respectively. The cytoskeletons of adjacent cells did not intermingle and cell-cell contacts were effected by thin filaments about 20 nm in height and 150 nm in width (this figure and Fig. 1B).

The substructure of focal contacts The morphology of focal contacts is seen at higher magnification in Fig. 5A. Stress fibers were visible that traversed the cytoskeleton and ended abruptly at the cell margin, often at the

2432 L. I. Pietrasanta, A. Schaper and T. M. Jovin Fig. 3. RMCD cell after mild treatment with Triton X-100. (A) SFM micrograph of a region close to the nucleus in which part of the cell membrane is disrupted. Top view. The short arrows are distributed along the fracture line. The mitochondria are exposed in the membrane-free region and show a snakelike morphology. Bars, 5 µm (x,y) and 1.7 µm (z). (B) Zoom and perspective view (pitch 45° and tilt angle 0°) of the membrane-free region in (A). Bars, 4 µm (x,y) and 0.6 µm (z). (C) Fluorescence image of rhodamine-123-labeled mitochondria of RMCD cells, exhibiting a clustering of the mitochondria in the perinuclear region. Pseudo-colored. Bar, 5 µm.

focal contacts demarcating the points of firm cell-substratum adhesion (Izzard and Lochner, 1976). In Fig. 5B, a stress fiber (short arrow) appears to terminate inside the cellular domain by dispersing into an array of microfilaments. In other regions filaments (most probably microfilaments) were oriented perpendicular to the larger stress fibers (Fig. 5B, arrow). At higher magnification (Fig. 5C) the cell margin was clearly defined not only by actin bundles but also by a matrix of thinner filaments, some of which were coated with granules (g), presumably reflecting associated proteins. Careful inspection of these and other SFM images failed to reveal structures that could be assigned to residual membrane components from the ventral side of the cell. SFM of the cytoskeleton reveals a thin filigree web on the ventral cell interior A three-dimensional web of fine interconnecting filaments was discerned below the bundles of actin filaments on the ventral cell side (Fig. 5D,E,F). This structure appeared throughout the cell and cross-connections were numerous. Most of the smaller fibers were about 20 nm in height and 160 nm in width. We also found some elements 45 nm in height and 200 nm in width, and the smallest fiber dimensions resolved by SFM were about 6-9 nm in height and 75-80 nm in width (areas indicated by arrows in Fig. 5E). The thinner filaments of the cytoskeleton were composed of actin, tubulin and vimentin (see Dis-

Fig. 3

Fig. 4. SFM images of the detergent-resistant part of the cytoskeleton of the RMCD cell with direct identification of structural elements by fluorescence microscopy. (A) Top view. The complex structure of the resistant cytoskeleton of confluent cells is revealed. The nuclear region remains attached to the surface and appears amorphous whereas the cell margin is well structured. Large bundles of actin filaments (‘stress fibers’) traverse the cell, oriented mostly along the long axis of the cell. Bars, 15 µm (x,y) and 1 µm (z). (B) Top view of an internal structure close to the cell margin of an RMCD cell. The cell margin is located beyond the left image side. The stress fibers radiate from a backbone scaffold. In the upper left corner a cell-cell contact region is visible. A stress fiber (arrow, cell 2) is congruent with the cell margin and cell 1 is connected by thin filaments. Bars, 5 µm (x,y) and 0.6 µm (z). (C) Contact area of three adjacent cells. The cytoskeletons of adjacent cells are not interdigitated, but bridges are formed by thin and short filaments. Bars, 6 µm (x,y) and 0.5 µm (z). (D) Cytoskeleton along an extended region of cell-cell contact. Zoom into the central region in (A). Bars, 10 µm (x,y) and 0.5 µm (z). (E) Fluorescence pattern of actin filaments visualized by fluorescein-phalloidin staining. Stress fibers (sf, see arrows) traverse the cell body; the cell margin on the lower part of the cell is terminated by actin fibers (sf, see B). Cell-cell contacts (cc) and microspikes (ms) are seen. Pseudo-colored. Bar, 10 µm. (F) Microtubule system of an RMCD cell visualized by immunofluorescence microscopy. Pseudo-colored. Bar, 10 µm.

Scanning force microscopy of cells 2433

2434 L. I. Pietrasanta, A. Schaper and T. M. Jovin

Scanning force microscopy of cells 2435 Fig. 5. Fine structure of the isolated cytoskeleton of the RMCD cell by SFM. (A) Top view of a cell margin reveals stress fibers (sf), focal contacts (fc), and smaller filaments (f). Bars, 3 µm (x,y) and 0.4 µm (z). (B) Cytoskeleton at a cell margin. A short arrow (ap) points to a surface region where the terminus of a stress fiber falls apart into microfilaments and creates what is presumably an adhesion plaque. In another area microfilaments (mf) are bundled to create a focal contact. Top view. Bars, 4 µm (x,y) and 0.35 µm (z). (C) Zoom of the cell margin in B. Top view. As well as the more aligned filaments, a filigree network is apparent. A filament 14 nm in height and 130 nm in width, probably a microtubule (mt), is seen (see text). The smaller fibers have some granules (g) on their surface. Bars, 2 µm (x,y) and 0.3 µm (z). (D,E,F) Filament distribution in selected regions of the cells. Examples of smaller-sized filaments that are branched and/or crossed are indicated with arrows. Top view. (D) Bars, 3 µm (x,y) and 0.3 µm (z); (E) top view. Bars, 3 µm (x,y) and 0.3 µm (z); (F) f1 is ~25 nm in height and 140 nm in width; f2 is ~15 nm in height and 140 nm in width; and f3 is ~9 nm in height and 100 nm in width. Top view. Bars, 2 µm (x,y) and 0.2 µm (z).

cussion below). In the immunofluorescence image of Fig. 4F the microtubule system was seen to have survived the detergent treatment. Microtubules were characteristically present as long unbranched but tangled fibers with a uniform diameter (Weber et al., 1978). They could not be detected specifically in the SFM images, although some filaments 14 nm in height were tentatively identified as such (Fig. 5C, mt). DISCUSSION In this paper we have investigated the subcellular structure of an air-dried mammalian cell by SFM. Details of the threedimensional architecture of the RMCD cell were obtained at different levels as a function of the biochemical dissection procedure applied. The study of such air-dried specimens represents an initial step towards the investigation of subcellular structures by SFM, specifically the cytoskeleton, in a normally hydrated state. For this project preparation methods had to be specially adapted for the SFM. As is well known from electron microscopy, the main problem was to prevent or minimize drying artefacts. The most intriguing example of such an artefact is provided by the morphology of the dried nucleus in Fig. 1C. The spherical nucleus is collapsed and the nuclear envelope is strongly modulated by the nucleoli. More sophisticated drying techniques, e.g. critical point drying, might prevent this effect, but with the disadvantage (for SFM) of preserving or producing very high surface features with step heights in the micrometer range, frequently beyond the zscanning range of the piezoelectric drive system. Cell surface structures of submicrometer dimensions, e.g. microvilli and microspikes, are clearly distinguishable by SFM (Fig. 2). Typical dimensions of the microvilli are about 100 nm in diameter and up to 5 µm in length. These structures, often of unkown function and appearing on almost all higher eucaryotic cells containing an actin-based scaffold, are dynamic and fragile. The dimensions for the microvilli derived from our measurements could be indicative of some flattening during air drying but the shape seems to be preserved, presumably due to the actin core. We have also tried to image critical-point-dried specimens of RMCD cells by SFM in order to visualize the microvilli originally extending from the cell body. This

approach failed due to major surface instabilities during scanning in the SFM (data not shown). However, from the success in visualizing mitochondria (Fig. 3), we conclude that appropriate conditions for specimen preparation can be found with which structures demarcated by membranes can be preserved. Since positive identification of subcellular structures appearing in the SFM was most readily achieved parallel with fluorescence microscopy we could establish that the apparent topography in the SFM was related to intrinsic cell morphology. The cell envelope, i.e. the outer surface of the plasma membrane, appears granular in the SFM after air drying. We must emphasize the crucial role played by tip geometry in the SFM images. A fine granularity smaller than the diameter of the scanning tip (see below) and with subnanometer features could produce a patchwork of mirror images of the tip, such that the latter is effectively scanned by the surface. The dry-cleaving of the detergent-treated cell gives access to the cytoskeletal components most accessible to the surface of the cell (Fig. 4). It appears that sticky tape removes an upper layer along a well preserved sheath of stress fibers and on a plane about 100 nm above the glass surface. In the original work of Mesland et al. (1981), critical-point-dried cultured hepatocytes were used for preserving internal cellular structures. Our results indicate that the success of the dry-cleaving depends on the proper choice of the cellular system as well as on the drying conditions. The cytoskeleton can be regarded as an assemblage of different filamentous structures embedded in an intracellular matrix of proteins and lipids. Two layers of microfilaments exist parallel to the upper and lower plasma membranes (Wehland et al., 1979). Dry-cleaving with the sticky tape removes the dorsal part of the cell, and therefore the membrane-associated dorsal microfilament layer is also presumably lost at this step. The stress fibers, however, persist, since they are rigid, aligned and firmly attached to the glass surface. The stability of the three-dimensional web of fine filaments below the stress fibers is probably due to abundant biochemical crosslinks, e.g. actin with filamin. Our SFM measurements do not provide evidence for such crosslinking, since there is no way to differentiate between this phenomenon and fiber collapse during air drying (Fig. 5). Intermediate filaments are the most stable components of the cytoskeleton, since they are retained under essentially all extraction conditions. From our SFM determinations of filament dimensions it is not possible to discriminate within the population of the smaller cytoskeletal elements, the lateral dimensions of which are systematically overestimated in comparison to their apparent height (Fig. 5). Our results are in agreement with SFM data obtained with purified microfilaments from Delain et al. (1992), who reported an apparent width of ∼50 nm for a single microfilament in contradiction to the 7 nm obtained by EM. These difficulties can be attributed to the inherent limitations of surface reconstruction from profilometry by SFM. The lateral dimensions of an object in SFM are broadened due to the nonlinear convolution with the tip structure (Vesenka et al., 1992). As a first approximation we can assume that the estimation of the lateral dimensions of biological objects reflects an arithmetic broadening corresponding to the additive contribution of the mean tip diameter (~10 nm, Park Scientific Instr.). Such an approximation is valid only for very flat structures a few nm in height. The strong positive correlation between the

2436 L. I. Pietrasanta, A. Schaper and T. M. Jovin apparent height and width of cytoskeletal elements is striking and probably indicative of the geometric factors alluded to. We believe that future advances in tip fabrication technology as well as more elaborated deconvolution procedures in image reconstruction will increase the lateral resolution of the SFM by at least one order of magnitude. Another possible source of error in the fiber dimensions derived from SFM is the process of drying. From EM, it is known that microtubules shrink from their normal 20-22 nm diameter to 13-15 nm after sample drying (Buckley and Raju, 1976; Temmink and Spiele, 1978; Webster et al., 1978). Thus, in principle, it could be difficult to differentiate between intermediate filaments and microtubules or microfilaments on dried specimens, particularly in the presence of the granular structures associated with these filaments (Fig. 5). The granules are probably part of the residual intracellular ground substance but the existence of such a microtrabecular system (Wolosewick and Porter, 1979; Porter and Tucker, 1981) is currently a matter of discussion (Heuser and Kirschner, 1980; Lea and Temkin, 1992). An interesting extension of our present SFM investigation would be the combination of specific fluorescence labeling of filaments and the use of colloidal gold coupled to antibodies. Since the latter can be prepared with different sizes of gold reporter particles, positive identification should be possible in the SFM even from the topographic patterning with mixtures of labeled antibodies. The SFM offers the opportunity for studying cells under physiological conditions, a potential that has attracted particular attention (Henderson et al., 1992; Hörber et al., 1992; Radmacher et al., 1992). The nanometer resolution of the scannning probe technique offers new perspectives in the study of the hydrated cytoskeleton and its dynamics. In conclusion, we have shown that the dried cellular structures of a confluent mammalian cell are accessible to SFM and that images with good spatial resolution can be obtained after detergent extraction of the soluble part of the cell and drycleavage. The detergent-resistant part of the intracellular filament network imaged by SFM exhibits the various fiber elements expected from the known features of the cytoskeleton. We are indebted to Drs D. Arndt-Jovin and M. Osborn for helpful discussions and critical reading of the manuscript. This work was partly supported by the German Research Council (DFG; grants Jo105/9 and Jo105/7).

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