Examination of microextensions of NIH3T3 fibroblast by scanning electron microscopy
We used NIH3T3 cells because their morphology and localization of cytoskeletal proteins including actin, tubulin, vinculin, and moesin have been extensively investigated using various fixatives. When removed from stock cultures with trypsin and inoculated into coverslip-containing dishes, NIH3T3 cells readily attached to the serum-coated substrate, began to spread and flattened within one hour (data not shown). Consistent with the report by Rajaraman et al.[20
] on human diploid WI-38 cells and Albrecht-Buehler and Goldman [21
] on 3T3 fibroblasts, microextensions appeared within 20 min after seeding. Initially during spreading, the cells lost microvilli from their upper cell surface and formed microextensions with a diameter of about 0.2 μm and a maximal length of about 10 μm. Subsequently, the cells continued to spread and to randomly elongate microextensions up to 40 μm. However, closer examination of the fixed cells with a 60° tilt angle showed numerous additional protrusions extending from the cell body with a diameter of only about 0.05 μm (Figure ). These structures were not observed in cells prior to 12 hours after inoculation. At 48 hour, about 5% (7/130) of the cells had extended such structures that usually were < 0.5 μm in length, and rarely exceeded 1 μm. Because of these unique features, we will use the term ultramicroextensions. Three days after reaching confluency (at about one week in culture), about half of the surface of the cells was covered with ultramicroextensions (Figure ). When cells were inoculated at high density, ultramicroextensions appeared earlier, but ultramicroextensions were rarely seen even after incubation for prolonged periods in sparse cultures or when the cells did not make contact (data not shown). Ultramicroextensions originated either from microextensions of neighboring cells or from tips of other microextensions in a broom-like fashion (Figure ). Ultramicroextensions also originated from the cell surface similar to microvilli (Figure ), or as branches from other microextensions and ultramicroextensions (Figure ). Identical results were obtained when the cells were grown on poly-L-lysine-coated coverslips (data not shown).
Figure 1 Scanning electron micrographs of NIH3T3 fibroblasts with a tilt angle 60°. (A-C) Exponentially growing NIH3T3 cells were seeded onto serum-coated glass coverslips at relatively low density (1 × 103 cells/cm2) thereby avoiding cells to (more ...)
Exposure of cytoskeleton in microextensions and ultramicroextensions by DOTMAC extraction
Fixation with formaldehyde followed by extraction with Triton X-100 is a fairly standard method for immunofluorescence microscopy of cells, but microextensions were fragmented and sometimes even lost during this treatment as shown in Figure . an alternative method, we extracted cells also with DOTMAC and discovered that fixation and extraction with 1.0 % PFA/0.5 % DOTMAC in PBS(+) on ice for 5 min, followed by fixation with 1.0% PFA in PBS on ice for 20 min (DOTMAC/PFA method), preserved not only micropodia (white arrow, Figure and ), but also microvilli (black arrow, Figure and ), and ultramicroextensions (white arrowhead, Figure ). This treatment extracted lipids (data not shown) and exposed cytoplasmic filaments giving origin to ultramicroextensions (Figure ). At the cell edge the ultramicroextensions are derived from a deeper cytoskeletal meshwork (white arrowhead, Figure ) rather than from thick cytoskeletal filaments.
Figure 2 (A) Scanning electron micrographs of NIH3T3 fibroblast extracted with Triton X-100 or DOTMAC at a tilt angle 60°. (A-C) NIH3T3 cells on glass coverslips were rinsed with PBS(+) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. (more ...)
We have performed ultrastructural analysis by SEM of cells fixed and/or extracted by the methods listed in Table 1. When the cells were treated with coagulant fixatives, the cytoskeleton was apparently exposed, but the three dimensional structures at the edge of the cells were not preserved as well as those prepared by the DOTMAC/PFA method (data not shown). When the cells were extracted with Triton X-100, NP-40 or saponin after relatively weak fixation with formaldehyde, the cytoskeleton apparently was lost, fragmented, or scraped off depending on the concentration of detergents and the period of incubation (Table 2). Too high a concentration of glutaraldehyde or incubation for extended period of time in formaldehyde, on the other hand, did not allow to expose the cytoskeleton (data not shown).
Comparison of different fixation/extraction methods for preservation and fluorescence labeling of microextensions
We also compared the different fixation and extraction procedures listed in Table 1 with respect to fluorescence intensity and labeling of actin stress fibers and microtubules, and with respect to morphological preservation of cells and microextensions (Table 2). The latter was also assessed from SEM micrographs before and after preparation for fluorescence microscopy and these results are summarized in Table 2 as well.
Staining of actin and tubulin filaments differed depending on the probe and on the fixative. Among the coagulant fixatives, acetone produced the best results with respect to preservation of microextensions, staining of actin with either phalloidin or with monoclonal β-actin antibodies, and staining of microtubules with a monoclonal anti-β-tubulin reagent. After methanol or ethanol fixation, some cells were partially detached and changed their shape, but this did not occur with other coagulant fixatives. Acetone also provided good preservation of microextensions even after preparing the cells for immunolabeling, whereas some of the microextensions were lost, when cells were fixed with other coagulant fixatives (Table 2).
Fixation with chemical crosslinkers, followed by extraction with detergents or organic solvents, are standard procedures for preparing cells for immunofluorescence. First, we investigated effects on the preservation of microextensions by variations in detergent concentration and time of incubation with formaldehyde. As summarized in Table 2, with shorter time periods of fixation and with higher detergent concentrations more cells lost microextensions. For example, when cells were fixed with 3.7% FA for 20 min and extracted with 0.2% TX100 for 5 min (Table 1), stress fibers were stained well with phalloidin, but microextensions were lost (Figure and ). The SEM observation before and after preparation for fluorescence microscopy (washing and incubation with probes) indicated that microextensions were lost during these steps of the procedure. On the other hand, microextensions of cells prefixed with glutaraldehyde or DSP prior to extraction were very well preserved indeed (Figure and ). However, the cytoskeleton of glutaraldehyde-fixed cells could not be exposed by the detergent and some structures apparently were lost in DSP-Tsb-fixed cells when analysed by SEM (data not shown). Quick extraction with 0.2% Triton X-100 in MTSB (30 seconds, 2 times), followed by fixation with 1% glutaraldehyde also preserved microextensions well indicating that fragmentation or removal of microextensions by extraction with 0.2% Triton X-100 does not occur within 1 min. The DOTMAC/PFA method provided excellent morphological preservation and immunolabeling with high fluorescence intensity of actin stress fibers and microextensions (Figure and ). Although phalloidin staining and immunolabeling of tubulin were poor in cells prepared by the DOTMAC/PFA method (Figure and ), this method provided the widest spectrum of reactivity with the probes investigated here (Figure and Table 1). The monoclonal antibody reagent specific for β-actin worked only in TCA or DOTMAC/PFA-treated cells (Figure and ).
Figure 3 Scanning electron and confocal laser scanning micrographs of NIH3T3 cells fixed with various procedures. (A, E, I, M, and Q) SEM image of NIH3T3 cells shown with no tilt angle. Cells were double labeled with TRITC-phalloidin (B, F, J, N, and R) and anti-β-tubulin (more ...)
Next, we compared the staining patterns of two well-characterized cytoskeletal proteins, vinculin and moesin, in cells prepared by DOTMAC/PFA and other conventional fixation methods (Figure ). As expected. vinculin localized at focal contacts in cells prepared by either PFA/Triton X-100 or by DOTMAC/PFA (Figure , and ). Moesin staining, on the other hand, was primarily at the edge of cells and in microextensions, when cells were fixed with TCA or DOTMAC/PFA (Figure and ). DOTMAC/PFA was superior to TCA and the standard Triton X-100 permeabilization procedure to preserve microextensions and for moesin staining (Figure , and ).
Figure 4 Comparison of fluorescence labeling of actin, vinculin, moesin and threonine558-phospho-moesin in NIH3T3 cells. Cells were rinsed with PBS(+) and fixed with TX100 (3.7% FA/0.2% TX100, see Table 1, A-D), TCA (see Table 1, E-H), or DOTMAC/PFA (see Table (more ...)
TCA fixation was reported recently to be useful for staining of phosphorylated moesin [12
]. Therefore, we tested our anti-phospho-moesin antibodies with this method and, as shown in Figure , the fluorescence intensity was too low to distinguish characteristic structures probably because the amount of phosphorylated moesin is low in unstimulated NIH3T3 cells (cf. Figure ). Figure demonstrates another example of NIH3T3 cells stained with actin monoclonal and moesin polyclonal antibodies after DOTMAC/PFA fixation. Many branched microextensions were double-stained with these antibodies, but occasionally, part of a microextension or an entire microextension apparently lacked actin [17
Figure 7 Determination of detergent-resistant components of NIH3T3 cells before (C) and after treatment with kinase, staurosporine (ST), or phosphatase inhibitors, calyculin A (CA) or pervanadate (PV). The subconfluent cells were rinsed with PBS(+), and detached (more ...)
Figure 5 Double staining of actin and moesin in NIH3T3 cells fixed with DOTMAC/PFA shown at high magnification. Note that branched micropodia were well preserved by the DOTMAC/PFA fixative and stained with both β-actin monoclonal and moesin polyclonal (more ...)
Evaluation of crosslinked polypeptides after fixation and extraction
Our results suggested that some of procedures listed in Table 1 were inadequate for preserving fine structural details. Therefore, we re-evaluated the extent of crosslinking of polypeptides by gel electrophoresis and western blotting. The polypeptide banding patterns of extracts from unfixed and fixed cells with SDS sample buffer are shown in Figure . Incubation in 0.5% Triton X-100 in PBS detached cells from the dish (Figure , lane 2), but addition of 1 mM magnesium and 1 mM calcium ions in PBS prevented detachment to some extent (Figure , lane 3). Microtubule stabilizing buffer also prevented detachment (Figure , lane 5). On the other hand, most polypeptides were retained on the dish after DOTMAC treatment (Figure , lane 6). Although most of β-actin was retained after 0.5% detergent extraction, moesin was not unless 1% paraformaldehyde was added (Figure , lanes 4 and 7). Under these conditions, moesin and β-actin migrated predominantly as monomeric polypeptides at 45 and 78 kDa positions on the SDS-PAGE gels and only very small amounts of crosslinked moesin and β-actin were detected by western blotting at higher molecular weight positions (data not shown).
Figure 6 SDS-polyacrylamide gel electrophoresis of extracted polypeptides from NIH3T3 cells after fixation and/or permeabilization. Subconfluent cultures of cells were rinsed with PBS(+), fixed and/or extracted as indicated. After rinsing with PBS, the material (more ...)
Crosslinking of polypeptides was also evaluated after treatment with several other fixatives that preserved microextensions well and that were found to be useful previously for moesin staining (Figure ). Only a small amount of polypeptides was extracted when cells were fixed with glutaraldehyde or DSP-Tsb (Figure , lane 2, 3 and 6) and the cells were morphologically well preserved by these fixatives as observed by SEM, indicating that most proteins were irreversibly crosslinked. On the other hand, when cells were fixed with PLP or LP and treated with 1% saponin in 3% BSA for permeabilization, most of the polypeptides were either not crosslinked by these relatively mild fixatives or crosslinks were reversed by the addition of SDS sample buffer (Figure , lanes 4 and 5). Similar results were obtained when cells were fixed with 3.7% formalin or 4% PFA, followed by permeabilization with 0.1% Triton X-100. Threonine558-phosphomoesin was not dephosphorylated in these fixatives, as determined by western blotting with specific phosphomoesin antibodies (Figure ). We examined the effect of incubation time and temperature during fixation with 4% PFA in PBS(+), since conditions for irreversible crosslinking of polypeptides in cultured cells with formaldehyde are unknown. As shown in Figure , fixation under the widely used conditions at room temperature (25°C) for 20 min, is not sufficient to irreversibly crosslink actin, moesin and many other polypeptides. At 37°C, the required time for incubation can be shortened, but most of the actin molecules were not irreversibly crosslinked even after 30 min. Incubation with sodium borohydride after fixation in PFA had no effect either (data not shown) suggesting that reversible bridges may have formed that could not be reduced.
Biochemical analysis of DOTMAC-insoluble materials of NIH3T3 cells
NIH3T3 cells suspended in PBS were treated with 1 μM staurosporine, 1 μM calyculin A or 100 μM pervanadate and fractionated with 0.5% Triton X-100 or DOTMAC. Similar to results in platelets [19
], the polypeptide patterns on SDS-PAGE gels differed for DOTMAC- and Triton X-100-extracted cells and more polypeptides were recovered in the DOTMAC- as compared to the Triton X-100-insoluble fraction (Figure ). Many signaling molecules are known to be sequestered into the Triton X-100-insoluble fraction during activation and/or aggregation of human platelets, and cytoskeletal association has been inferred from this result. While this may be true for some proteins, the postulated cytoskeletal association of threonine558-phosphorylated moesin was maintained only when platelets were extracted with DOTMAC, but not with Triton X-100 [19
]. By contrast, phosphorylated moesin was found in the insoluble material of NIH3T3 cells after extraction with both types of detergent (Figure ). When extracted with 0.5% detergent, a larger amount of insoluble phosphomoesin was detected with Triton X-100 as compared to DOTMAC. Different concentrations of detergents (0.1%, 1% and 2%) were tested and correlated with the amount of moesin released from NIH3T3 cells, but the insoluble fraction of moesin was always higher with Triton X-100 than with DOTMAC (data not shown).
We also analysed for tyrosine-phosphorylated proteins in the insoluble fraction of cells treated with pervanadate (PV), a potent inhibitor of tyrosine phosphatases, extracted with 0.5% detergent. A larger fraction of tyrosine-phosphorylated proteins were recovered in the DOTMAC-insoluble pellet (unpublished observation)
DOTMAC induces bundling of actin filaments and microtubules
The excellent preservation of the cytoskeleton in microextensions of cells fixed with PFA/DOTMAC could be due to stabilization of the cytoskeleton in general or of actin filaments selectively. We, therefore, explored whether DOTMAC has a direct effect in vitro on actin filaments prepared from purified actin. As shown in Figure , actin filaments bundled in the presence of DOTMAC (Figure ) suggesting that this might contribute to the greater stability of the actin cytoskeleton that we have observed in cells extracted with this detergent. This effect is relatively specific since DOTMAC disrupted microtubules and they appeared as amorphous filaments (Figure ).
Figure 8 Negative staining electron micrographs of rabbit skeletal muscle F-actin (A and B) and bovine microtubules (C and D) in the absence (A and C) and presence (B and D) of DOTMAC. Note that DOTMAC induces bundling of actin filaments and microtubules. Bars: (more ...)