THE
DTSTRIBUTION
OF ACTIN
IN NON-MUSCLE
CELLS
The Use of’ Actin Antibody in the Localization of’ Actin within the Micrqf‘ilament Bundles qf‘ Mouse 3T3 Cells R. D. GOLDMAN,l Cold Spring
Harbor
E. LAZARIDES, Laborarmy,
R. POLLACK,
Cold Spring
Harbor,
and K. WEBER N. Y. lI724,
USA
SUMMARY Living mouse 3T3 cells display a complex array of fibrous structures which are visible with phase contrast, Nomarski and polarized light optics. When cells are fixed and stained for indirect immunofluorescence with actin antibody, the same fibers show intense fluorescence indicating that they contain actin. Electron microscopy reveals that these fibrous structures consist of submembranous bundles of microfilaments located primarily on the attached side of the cells. The results are discussed in terms of the intracellular localization of a possible submembranous contractile system involved in motile activities such as cell locomotion.
Microfilaments are found in the cortices of a wide variety of animal cells. They are frequently organized into networks or bundles beneath the plasma membrane [l-3]. Due to their diameter (40-80 A) and capacity to interact with skeletal muscle heavy meromyosin (HMM), they are considered to be actin-like [2, 41. It has also been demonstrated that an actin-like protein is present in large amounts in several types of non-muscle cells [5-91. Therefore, microfilaments are considered to contain actin which is the major morphological component of a proposed submembranous contractile system involved in many motile activities including cell locomotion, membrane ruffling, and cytokinesis [l-3, IO]. Recently, Lazarides & Weber [l I] prepared an antibody against electrophoretically puri-
fied denatured actin isolated from a clone of SV40 transformed mouse 3T3 cells (SV IOl). This antibody cross reacts with native SV 101 actin as well as chicken muscle actin. When the antibody is employed as an indirect immunofluorescent stain, it reveals the presence of an elaborate and complex array of actincontaining fibrous structures in 3T3 cells, BHK21 cells and chick embryo muscle cells observed with dark-field UV optics. Here we report that the distribution and pattern of fibrous structures seen in living 3T3 cells with phase contrast and other optical systems corresponds to the actin-containing fibers seen utilizing indirect immunofluorescence and the microfilament bundles seen with electron microscopy.
1 On leave from Department of Biological Science, Carnegie-Mellon University, Mellon Institute, Pittsburgh, Pa 15213, USA. Reprint requests should be sent to this address.
Cell cultures
22
T41804
MATERIALS
AND METHODS
Mouse 3T3 cells [I21 were grown in Dulbecco’s Modified Eagle’s medium containing IO Obcalf serum and Exptl
Cell Res 90 (1975)
334
Goldman et al.
50 pg/ml Gentamicin in Falcon tissue culture dishes maintained at 37°C in a CO, incubator. Growing stocks of cells were trypsinized (0.025 % trypsinEDTA) and replated into 35 mm culture dishes containing glass coverslips for light microscopy and without coverslips for electron microscopy.
Indirect immunofluorescence Cells growing on glass coverslips were fixed in 3.5 % formaldehyde in PBS (phosphate-buffered saline) for 20-30 min at room temperature, followed by 3 rinses in PBS. The coverslips were then immersed in cold ( -20°C) acetone for 7-10 min and then air-dried. A 1 :20 dilution of the actin antibody (see Lazarides & Weber Ill]) in PBS was added to cover the cells. The coverslips were then incubated in a moist chamber for 1 h at 37°C washed 3 times in PBS, and incubated for 1 h in a 1: 10 dilution of goat anti-rabbit globulin (Lazarides & Weber [ll]) coupled to fluorescein. The coverslips were rinsed 3 times in PBS, once in distilled water and observed immediately.
Light microscopy A Zeiss photomicroscope III equipped with darkfield UV, phase contrast, Nomarski and polarized light optics was used for observations of living, fixed, and stained cells. An FITC-filtered high pressure mercury-arc source was used for fluorescence microscopy. A Brace-Koehler rotating mica-compensator Q/20) was used for all polarized light observations. Live cells were prepared for observation as previously reported 1131 and were maintained at 37°C with a Nicholson Air Stream Incubator.
Electron microscopy Cells grown on plastic dishes were fixed on the dishes in 1 % glutaraldehyde in PBS for 30-60 min at room temperature. The dishes were then rinsed 3 times with PBS and 1 % osmium tetroxide in PBS was added for 3&60 min. This was followed by 3 rinses in PBS, dehydration in a series of alcohols, and embedding in Epon 812. Following polymerization of the plastic, the flat-embedded cells were removed [13] and pieces of the plastic containing cells were cut out and glued to prepolymerized blocks of Epon 812 made in BEEM capsules. Thin sections were taken parallel to the cell-substrate side of the cells on an LKB Ultrotome I. The sections were mounted on parlodioncarbon coated grids and stained with uranyl acetate [I41 and lead citrate [15]. Micrographs were taken with either a Philips 200 or 300 electron microscope.
RESULTS Fiber pattern in live cells Light microscopic observations of living 3T3 cells were restricted to cells which were spread on coverslips. Rounded up or dividing cells did not contain visible fibrous structures and were therefore not included in the Exptl
Cell Res 90 (1975)
observations. Within the spread cellsa distinct array of fibers could be seenwith phasecontrast optics (fig. 1). In polarized light these fibers are birefringent (fig. 2a, 6). Some of these fibers measureup to 2 ,um in diameter. Several patterns of these fibers could be distinguished in different cytoplasmic regions (see also [II]). In some instances the fibers ran parallel to each other for fairly long distances (fig. 3a, b). In other cases,apparent branching of fibers could be seen (fig. 4). Focal points of several radiating fibers could also be found (fig. 5). In the largest cells observed, all of these patterns could be detected. Furthermore, differential focusing with Nomarski optics revealed that the fibers were displaced toward the contact or adhesive side of the cell (fig. 6a, b). Similar fibers located towards the contact side of BHK21 cells were resolved with the Nomarski system [12] and similar observations have been made in rat embryo cells utilizing phase contrast optics [23]. Fiber pattern in fixed cells observed with light optics In order to ensure that the fibers seen within living cells were preserved in the same configuration for fluorescent and electron microscopy, 3T3 cells were examined with light microscopy following fixation. Fig. 7 (a and 6) is of the samecell observed live and then fixed for fluorescence microscopy. The array of fibers is the same in both the living and the fixed cell, although the fibers are more pronounced in the fixed cells. Glutaraldehyde fixation resulted in the same preservation of fibers. Cells followed through the osmium tetroxide postfixation and alcohol dehydration steps (see Materials and Methods) also showed that fibers were well preserved for electron microscopy. In all cases,fibers seen in fixed cells examined with polarized light retained their birefringence.
Distribution
Fig. I. A well x 650. Fig. 2. Living
spread living 3T3 cell. Note phase dense fibers throughout
of actin irl non-musclecells 335
the cytoplasm (UYYOW).Phase contrast,
3T3 cell observed with polarized light optics to demonstrate birefringent fibers at opposite compensator settings. Note change in contrast of fibers following rotation of the compensator to either side of the extinction point. x 560.
Exptl
Cell
Res 90 (1975)
336
Goldman et al.
17,q. 3. 850; F&J. 4. Fig. 5.
Regions of the cytoplasm of living 3T3 cells containing parallel arrays of fibers. (a) Phase contrast, (b) Nomarski differential interference contrast, \ I 360. Possible branching points of fibers in living 3T3 cells (arrows). Phase contrast, L I 360. Focal point of several radiating fibers in the living 3T3 cell (arrow). Phase contrast, ’ 1 360.
Indirect immunofluovescenceutilizing rather dramatic array of bright fibers similar actin-antibody to those reported previously [I l] (fig. 8). Following fixation in formaldehyde and ace- Control experiments utilizing rabbit antitone, cells were first treated with rabbit antiserum prepared prior to immunization, folactin antibody and then stained with fluores- lowed by fluorescent labelled goat anti-rabbit cein-labelled goat anti-rabbit globulin (see globulin showed no detectable fluorescent fibers when observed with UV dark-field opMaterials and Methods). When observed with dark-field UV optics, 3T3 cells displayed a tics. However, phasecontrast revealed similar Exptt
Cell Res 90 (1975)
Distribution
Fig. 6. The
same cell viewed at different contact; (b) fibers not obvious Fig. 7. The same 3T3 cell (a) observed virtually identical in both micrographs.
substrate
of actin in non-muscle
cvll,~
337
focal planes with Nomarski optics. (a) fibers in focus at level of cellwhen focused up away from the substrate towards the nucleus. I 280. live; (b) fixed for fluorescence microscopy. The array of fibers is Phase contrast, 544.
fibers in both control and actin-antibody preparations. Fig. 9a and b shows the same cell with phase and fluorescence optics to demonstrate the identity of phase-densefibers and fluorescent antibody stained fibers. Parallel arrays,
branching and focal points of fibers were seenwith fluorescence microscopy (figs 8.9b). Electron
microscopJ3
Thin sections were taken parallel to the adhesive side of flat-embedded 3T3 cells. Only Exptl
Cell
Res 90 (1975)
338
Goldman et al.
Fig. 8. Well-spread 3T3 cell fixed and stained for indirect immunofluorescence utilizing actin antibody. Note the fibrous pattern in different cytoplasmic regions. UV Dark Field Optics, x 800. Fig. 9. The same cell fixed and stained with actin antibody viewed with (a) phase contrast; (b) UV dark field optics. Note relationship between phase dense fibers and fluorescent fibers. :’ 480. Exptl
Cell
Res 90 (1975)
Distribution well spread cells were selected for observation. At low magnification a fibrous pattern similar to that seen in living cells was easily distinguishable just beneath or at the same level as the plasma membrane (fig. IO). At higher magnification it became obvious that these consisted of submembranous bundles of microfilaments localized at the cell-substrate side of the cell (fig. 11). In some regions individual microfilaments appeared to be embedded in an electron-dense matrix of amorphous material (fig. 11). This distribution fits with the finding that the fibers in living cells are also displaced towards the adhesive side of the cell (see fig. 6a, b). Parallel arrays (fig. lo), possible branch points, and focal points of microfilament bundles were seen (fig. 12). Microtubules and 100 A filaments could also be seen in the cytoplasm running parallel to bundles of microfilaments (fig. 1 I), however, in many instances they did not appear to coincide with the fiber pattern seen with light microscopy (fig. 13). Furthermore, microtubules and 100 8, filaments are not localized in closely packed arrays and, therefore, it is unlikely that they could account for the fibers seen within living cells. The thickness of microfilament bundles varied enormously along their length which made it extremely difficult to obtain accurate measurements. This difficulty was due most likely to differences in the plane of the thin section as it passed through the cell. The figures presented in this paper were selected more for the ability to resolve individual microfilaments rather than to measure the thickness of bundles. However, areas of bundles up to 0.5-0.7 p are seen in figs 10-l 5. In addition, electron micrographs of thicker sections not included in this paper contained bundles which ranged up to 1.5-2.0 p thick. Therefore the maximum thickness of the fibers seen in living cells agreed reasonably
of actin in non-muscle cells
339
well with the maximum thickness of microfilament bundles seen in thin sections. Colchicine-induced breakdown of microtubules and the appearance of fibers in living and fixed cells We have found that colchicine treatment not only breaks down microtubules in BHK21 and BSC-1 cells but also prevents the dispersal of 100 A filaments through the cytoplasm [13, 241. The latter fibers accumulate in a juxtanuclear region in the presence of colchicine. However, microfilament bundles are distributed normally in colchicine-treated cells [2, 131. Therefore, we used this phenomenon as a control for the actin-antibody technique. To inhibit the assembly of microtubules as well as the normal cytoplasmic distribution of 100 A filaments, trypsinized suspensions of 3T3 cells were plated into medium containing 10 ,ug/ml colchicine. The cells spread and possessed a normal-appearing fibrous structure when viewed with phase contrast optics. These fibers stained with the actin-antibody technique (fig. 14). Electron microscopy revealed the presence of normalappearing bundles of microfilaments and no microtubules or 100 8, filaments were visible (fig. 1.5) in the same regions. Therefore, the elimination of microtubules and the prevention of the normal distribution of filaments in 3T3 cells does not change the overall pattern visible with the actin-antibody technique. These observations further emphasize the conclusion that the microfilament bundles are the actin-containing structures seen with light microscopy. DISCUSSION The evidence presented in this paper demonstrates that actin-containing fibrous structures seen within living and fixed cells consist of bundles of microfilaments. Similar fibrous Exptl
Cell Res 90 (197.5)
340
Goldman
Exptl
Cell
et al.
Res 90 (1975)
Distribution
patterns or ‘tension striae’ were described years ago by Lewis & Lewis [16] in a variety of fixed and stained preparations of cultured cells. More recently these fibers have been described in living BHK21 cells and also appear to coincide with the distribution of microfilament bundles seen with the electron microscope [2, 131. In addition, Buckley & Porter [23] have seen similar fibers which they call ‘stress fibers’ in rat embryo cells. These authors also demonstrated the relationship between the fibers seen in living cells with bundles of microfilaments seen with the electron microscope. The coincidence of microfilament bundles with the actin-containing fibers seen within living cells provides convincing evidence that microfilaments contain actin. In light of the facts that large amounts of protein virtually identical to muscle actin have been isolated from a wide variety of cells [5-91, that actinlike microfilaments appear to bind heavy meromyosin in glycerinated models of nonmuscle cells [2, 4, 171 and that antibody to non-muscle cell actin cross reacts with chicken muscle actin [II], we propose that the microfilaments seen within 3T3 cells be termed mouse cell actin. This is not unreasonable if one looks at myosin, the other major muscle protein. Myosin varies significantly in its properties between non-muscle cells and even within different types of muscle cells [IQ but the term myosin is always used for this general class of contractile proteins. The localization of bundles of actin on the adhesive side of spread cells is of special interest with regard to cell locomotion in vitro. Cultured cells appear to move along a
of actin in non-mwcle
cells
341
substrate by making and breaking contacts with the substrate [19]. New contacts are apparently made at the leading edge of a migrating cell and old contacts are broken at the rear as the cell moves forward [see 19, 20, 211. It has been proposed by several investigators [2, 19, 221 that a submembranous contractile layer might account for this type of movement. It appears that the bundles of actin are the major morphologically distinct component of this layer and that this is the most likely candidate for the localization of a contractile system linked to or associated with the plasma membranes of cells. Proteins similar to myosin have also been isolated from cultured fibroblasts [8], but the intracellular localization of myosin in non-muscle cultured cells remains unknown. It is, however, possible that the myosin components are localized in these bundles within the amorphous electron dense matrix frequently seen surrounding the actin. Although we have emphasized the presence of fibrous actin within the microfilament bundles, it is likely that actin exists in other forms within cells. This is especially evident in rounded up suspended BHK21 and BSC-1 cells following trypsinization. These cells contain no submembranous bundles of microfilaments [2, 241. However, microfilament bundles assemblerapidly once the cells attach and begin spreading on a solid substrate. The formation of bundles occurs even in the absence of protein synthesis [2. 241. Thus pools of G-actin may also be present within cells which can be utilized to form the F-actin contained in the bundles of microfilaments.
Fig. 10. An electron micrograph of a thin section taken just beneath the plasma membrane of a fiat-embedded 3T3 cell. Note regions where plane of section goes outside of cell (0). Many microfilament bundles are present in this region of the cytoplasm (arrows). ” 7 750. Fig. II. Higher magnification electron micrograph of microfilament bundles (arrow). Individual microfilaments are seen as well as other electron dense material. 100 8, filaments (F) and microtubules (M) are also present. ~43ooo. Exptl
Cell Res 90 (1975)
Fig. 12. filament Fig. I3. not run Exptl
Electron bundles. Electron parallel to
micrograph x 13 200. micrograph each other.
Cell Res 90 (1975)
similar
to fig.
showing regions / I6 450.
IO showing in which
possible microtubules
branch and
points
and
microfilament
focal
points
bundles
of micro(arrays)
do
Distribution
Fig. 14. A 3T3 cell fixed and colchicine. Fluorescent fibers Fig. 15. Electron micrograph bundles are obvious, however the bundles. .34 460.
of actin in non-muscle cells
343
stained with the actin antibody technique following 12 h in medium containing are obvious in this cell. .
DTSTRIBUTION
OF ACTIN
IN NON-MUSCLE
CELLS
The Use of’ Actin Antibody in the Localization of’ Actin within the Micrqf‘ilament Bundles qf‘ Mouse 3T3 Cells R. D. GOLDMAN,l Cold Spring
Harbor
E. LAZARIDES, Laborarmy,
R. POLLACK,
Cold Spring
Harbor,
and K. WEBER N. Y. lI724,
USA
SUMMARY Living mouse 3T3 cells display a complex array of fibrous structures which are visible with phase contrast, Nomarski and polarized light optics. When cells are fixed and stained for indirect immunofluorescence with actin antibody, the same fibers show intense fluorescence indicating that they contain actin. Electron microscopy reveals that these fibrous structures consist of submembranous bundles of microfilaments located primarily on the attached side of the cells. The results are discussed in terms of the intracellular localization of a possible submembranous contractile system involved in motile activities such as cell locomotion.
Microfilaments are found in the cortices of a wide variety of animal cells. They are frequently organized into networks or bundles beneath the plasma membrane [l-3]. Due to their diameter (40-80 A) and capacity to interact with skeletal muscle heavy meromyosin (HMM), they are considered to be actin-like [2, 41. It has also been demonstrated that an actin-like protein is present in large amounts in several types of non-muscle cells [5-91. Therefore, microfilaments are considered to contain actin which is the major morphological component of a proposed submembranous contractile system involved in many motile activities including cell locomotion, membrane ruffling, and cytokinesis [l-3, IO]. Recently, Lazarides & Weber [l I] prepared an antibody against electrophoretically puri-
fied denatured actin isolated from a clone of SV40 transformed mouse 3T3 cells (SV IOl). This antibody cross reacts with native SV 101 actin as well as chicken muscle actin. When the antibody is employed as an indirect immunofluorescent stain, it reveals the presence of an elaborate and complex array of actincontaining fibrous structures in 3T3 cells, BHK21 cells and chick embryo muscle cells observed with dark-field UV optics. Here we report that the distribution and pattern of fibrous structures seen in living 3T3 cells with phase contrast and other optical systems corresponds to the actin-containing fibers seen utilizing indirect immunofluorescence and the microfilament bundles seen with electron microscopy.
1 On leave from Department of Biological Science, Carnegie-Mellon University, Mellon Institute, Pittsburgh, Pa 15213, USA. Reprint requests should be sent to this address.
Cell cultures
22
T41804
MATERIALS
AND METHODS
Mouse 3T3 cells [I21 were grown in Dulbecco’s Modified Eagle’s medium containing IO Obcalf serum and Exptl
Cell Res 90 (1975)
334
Goldman et al.
50 pg/ml Gentamicin in Falcon tissue culture dishes maintained at 37°C in a CO, incubator. Growing stocks of cells were trypsinized (0.025 % trypsinEDTA) and replated into 35 mm culture dishes containing glass coverslips for light microscopy and without coverslips for electron microscopy.
Indirect immunofluorescence Cells growing on glass coverslips were fixed in 3.5 % formaldehyde in PBS (phosphate-buffered saline) for 20-30 min at room temperature, followed by 3 rinses in PBS. The coverslips were then immersed in cold ( -20°C) acetone for 7-10 min and then air-dried. A 1 :20 dilution of the actin antibody (see Lazarides & Weber Ill]) in PBS was added to cover the cells. The coverslips were then incubated in a moist chamber for 1 h at 37°C washed 3 times in PBS, and incubated for 1 h in a 1: 10 dilution of goat anti-rabbit globulin (Lazarides & Weber [ll]) coupled to fluorescein. The coverslips were rinsed 3 times in PBS, once in distilled water and observed immediately.
Light microscopy A Zeiss photomicroscope III equipped with darkfield UV, phase contrast, Nomarski and polarized light optics was used for observations of living, fixed, and stained cells. An FITC-filtered high pressure mercury-arc source was used for fluorescence microscopy. A Brace-Koehler rotating mica-compensator Q/20) was used for all polarized light observations. Live cells were prepared for observation as previously reported 1131 and were maintained at 37°C with a Nicholson Air Stream Incubator.
Electron microscopy Cells grown on plastic dishes were fixed on the dishes in 1 % glutaraldehyde in PBS for 30-60 min at room temperature. The dishes were then rinsed 3 times with PBS and 1 % osmium tetroxide in PBS was added for 3&60 min. This was followed by 3 rinses in PBS, dehydration in a series of alcohols, and embedding in Epon 812. Following polymerization of the plastic, the flat-embedded cells were removed [13] and pieces of the plastic containing cells were cut out and glued to prepolymerized blocks of Epon 812 made in BEEM capsules. Thin sections were taken parallel to the cell-substrate side of the cells on an LKB Ultrotome I. The sections were mounted on parlodioncarbon coated grids and stained with uranyl acetate [I41 and lead citrate [15]. Micrographs were taken with either a Philips 200 or 300 electron microscope.
RESULTS Fiber pattern in live cells Light microscopic observations of living 3T3 cells were restricted to cells which were spread on coverslips. Rounded up or dividing cells did not contain visible fibrous structures and were therefore not included in the Exptl
Cell Res 90 (1975)
observations. Within the spread cellsa distinct array of fibers could be seenwith phasecontrast optics (fig. 1). In polarized light these fibers are birefringent (fig. 2a, 6). Some of these fibers measureup to 2 ,um in diameter. Several patterns of these fibers could be distinguished in different cytoplasmic regions (see also [II]). In some instances the fibers ran parallel to each other for fairly long distances (fig. 3a, b). In other cases,apparent branching of fibers could be seen (fig. 4). Focal points of several radiating fibers could also be found (fig. 5). In the largest cells observed, all of these patterns could be detected. Furthermore, differential focusing with Nomarski optics revealed that the fibers were displaced toward the contact or adhesive side of the cell (fig. 6a, b). Similar fibers located towards the contact side of BHK21 cells were resolved with the Nomarski system [12] and similar observations have been made in rat embryo cells utilizing phase contrast optics [23]. Fiber pattern in fixed cells observed with light optics In order to ensure that the fibers seen within living cells were preserved in the same configuration for fluorescent and electron microscopy, 3T3 cells were examined with light microscopy following fixation. Fig. 7 (a and 6) is of the samecell observed live and then fixed for fluorescence microscopy. The array of fibers is the same in both the living and the fixed cell, although the fibers are more pronounced in the fixed cells. Glutaraldehyde fixation resulted in the same preservation of fibers. Cells followed through the osmium tetroxide postfixation and alcohol dehydration steps (see Materials and Methods) also showed that fibers were well preserved for electron microscopy. In all cases,fibers seen in fixed cells examined with polarized light retained their birefringence.
Distribution
Fig. I. A well x 650. Fig. 2. Living
spread living 3T3 cell. Note phase dense fibers throughout
of actin irl non-musclecells 335
the cytoplasm (UYYOW).Phase contrast,
3T3 cell observed with polarized light optics to demonstrate birefringent fibers at opposite compensator settings. Note change in contrast of fibers following rotation of the compensator to either side of the extinction point. x 560.
Exptl
Cell
Res 90 (1975)
336
Goldman et al.
17,q. 3. 850; F&J. 4. Fig. 5.
Regions of the cytoplasm of living 3T3 cells containing parallel arrays of fibers. (a) Phase contrast, (b) Nomarski differential interference contrast, \ I 360. Possible branching points of fibers in living 3T3 cells (arrows). Phase contrast, L I 360. Focal point of several radiating fibers in the living 3T3 cell (arrow). Phase contrast, ’ 1 360.
Indirect immunofluovescenceutilizing rather dramatic array of bright fibers similar actin-antibody to those reported previously [I l] (fig. 8). Following fixation in formaldehyde and ace- Control experiments utilizing rabbit antitone, cells were first treated with rabbit antiserum prepared prior to immunization, folactin antibody and then stained with fluores- lowed by fluorescent labelled goat anti-rabbit cein-labelled goat anti-rabbit globulin (see globulin showed no detectable fluorescent fibers when observed with UV dark-field opMaterials and Methods). When observed with dark-field UV optics, 3T3 cells displayed a tics. However, phasecontrast revealed similar Exptt
Cell Res 90 (1975)
Distribution
Fig. 6. The
same cell viewed at different contact; (b) fibers not obvious Fig. 7. The same 3T3 cell (a) observed virtually identical in both micrographs.
substrate
of actin in non-muscle
cvll,~
337
focal planes with Nomarski optics. (a) fibers in focus at level of cellwhen focused up away from the substrate towards the nucleus. I 280. live; (b) fixed for fluorescence microscopy. The array of fibers is Phase contrast, 544.
fibers in both control and actin-antibody preparations. Fig. 9a and b shows the same cell with phase and fluorescence optics to demonstrate the identity of phase-densefibers and fluorescent antibody stained fibers. Parallel arrays,
branching and focal points of fibers were seenwith fluorescence microscopy (figs 8.9b). Electron
microscopJ3
Thin sections were taken parallel to the adhesive side of flat-embedded 3T3 cells. Only Exptl
Cell
Res 90 (1975)
338
Goldman et al.
Fig. 8. Well-spread 3T3 cell fixed and stained for indirect immunofluorescence utilizing actin antibody. Note the fibrous pattern in different cytoplasmic regions. UV Dark Field Optics, x 800. Fig. 9. The same cell fixed and stained with actin antibody viewed with (a) phase contrast; (b) UV dark field optics. Note relationship between phase dense fibers and fluorescent fibers. :’ 480. Exptl
Cell
Res 90 (1975)
Distribution well spread cells were selected for observation. At low magnification a fibrous pattern similar to that seen in living cells was easily distinguishable just beneath or at the same level as the plasma membrane (fig. IO). At higher magnification it became obvious that these consisted of submembranous bundles of microfilaments localized at the cell-substrate side of the cell (fig. 11). In some regions individual microfilaments appeared to be embedded in an electron-dense matrix of amorphous material (fig. 11). This distribution fits with the finding that the fibers in living cells are also displaced towards the adhesive side of the cell (see fig. 6a, b). Parallel arrays (fig. lo), possible branch points, and focal points of microfilament bundles were seen (fig. 12). Microtubules and 100 A filaments could also be seen in the cytoplasm running parallel to bundles of microfilaments (fig. 1 I), however, in many instances they did not appear to coincide with the fiber pattern seen with light microscopy (fig. 13). Furthermore, microtubules and 100 8, filaments are not localized in closely packed arrays and, therefore, it is unlikely that they could account for the fibers seen within living cells. The thickness of microfilament bundles varied enormously along their length which made it extremely difficult to obtain accurate measurements. This difficulty was due most likely to differences in the plane of the thin section as it passed through the cell. The figures presented in this paper were selected more for the ability to resolve individual microfilaments rather than to measure the thickness of bundles. However, areas of bundles up to 0.5-0.7 p are seen in figs 10-l 5. In addition, electron micrographs of thicker sections not included in this paper contained bundles which ranged up to 1.5-2.0 p thick. Therefore the maximum thickness of the fibers seen in living cells agreed reasonably
of actin in non-muscle cells
339
well with the maximum thickness of microfilament bundles seen in thin sections. Colchicine-induced breakdown of microtubules and the appearance of fibers in living and fixed cells We have found that colchicine treatment not only breaks down microtubules in BHK21 and BSC-1 cells but also prevents the dispersal of 100 A filaments through the cytoplasm [13, 241. The latter fibers accumulate in a juxtanuclear region in the presence of colchicine. However, microfilament bundles are distributed normally in colchicine-treated cells [2, 131. Therefore, we used this phenomenon as a control for the actin-antibody technique. To inhibit the assembly of microtubules as well as the normal cytoplasmic distribution of 100 A filaments, trypsinized suspensions of 3T3 cells were plated into medium containing 10 ,ug/ml colchicine. The cells spread and possessed a normal-appearing fibrous structure when viewed with phase contrast optics. These fibers stained with the actin-antibody technique (fig. 14). Electron microscopy revealed the presence of normalappearing bundles of microfilaments and no microtubules or 100 8, filaments were visible (fig. 1.5) in the same regions. Therefore, the elimination of microtubules and the prevention of the normal distribution of filaments in 3T3 cells does not change the overall pattern visible with the actin-antibody technique. These observations further emphasize the conclusion that the microfilament bundles are the actin-containing structures seen with light microscopy. DISCUSSION The evidence presented in this paper demonstrates that actin-containing fibrous structures seen within living and fixed cells consist of bundles of microfilaments. Similar fibrous Exptl
Cell Res 90 (197.5)
340
Goldman
Exptl
Cell
et al.
Res 90 (1975)
Distribution
patterns or ‘tension striae’ were described years ago by Lewis & Lewis [16] in a variety of fixed and stained preparations of cultured cells. More recently these fibers have been described in living BHK21 cells and also appear to coincide with the distribution of microfilament bundles seen with the electron microscope [2, 131. In addition, Buckley & Porter [23] have seen similar fibers which they call ‘stress fibers’ in rat embryo cells. These authors also demonstrated the relationship between the fibers seen in living cells with bundles of microfilaments seen with the electron microscope. The coincidence of microfilament bundles with the actin-containing fibers seen within living cells provides convincing evidence that microfilaments contain actin. In light of the facts that large amounts of protein virtually identical to muscle actin have been isolated from a wide variety of cells [5-91, that actinlike microfilaments appear to bind heavy meromyosin in glycerinated models of nonmuscle cells [2, 4, 171 and that antibody to non-muscle cell actin cross reacts with chicken muscle actin [II], we propose that the microfilaments seen within 3T3 cells be termed mouse cell actin. This is not unreasonable if one looks at myosin, the other major muscle protein. Myosin varies significantly in its properties between non-muscle cells and even within different types of muscle cells [IQ but the term myosin is always used for this general class of contractile proteins. The localization of bundles of actin on the adhesive side of spread cells is of special interest with regard to cell locomotion in vitro. Cultured cells appear to move along a
of actin in non-mwcle
cells
341
substrate by making and breaking contacts with the substrate [19]. New contacts are apparently made at the leading edge of a migrating cell and old contacts are broken at the rear as the cell moves forward [see 19, 20, 211. It has been proposed by several investigators [2, 19, 221 that a submembranous contractile layer might account for this type of movement. It appears that the bundles of actin are the major morphologically distinct component of this layer and that this is the most likely candidate for the localization of a contractile system linked to or associated with the plasma membranes of cells. Proteins similar to myosin have also been isolated from cultured fibroblasts [8], but the intracellular localization of myosin in non-muscle cultured cells remains unknown. It is, however, possible that the myosin components are localized in these bundles within the amorphous electron dense matrix frequently seen surrounding the actin. Although we have emphasized the presence of fibrous actin within the microfilament bundles, it is likely that actin exists in other forms within cells. This is especially evident in rounded up suspended BHK21 and BSC-1 cells following trypsinization. These cells contain no submembranous bundles of microfilaments [2, 241. However, microfilament bundles assemblerapidly once the cells attach and begin spreading on a solid substrate. The formation of bundles occurs even in the absence of protein synthesis [2. 241. Thus pools of G-actin may also be present within cells which can be utilized to form the F-actin contained in the bundles of microfilaments.
Fig. 10. An electron micrograph of a thin section taken just beneath the plasma membrane of a fiat-embedded 3T3 cell. Note regions where plane of section goes outside of cell (0). Many microfilament bundles are present in this region of the cytoplasm (arrows). ” 7 750. Fig. II. Higher magnification electron micrograph of microfilament bundles (arrow). Individual microfilaments are seen as well as other electron dense material. 100 8, filaments (F) and microtubules (M) are also present. ~43ooo. Exptl
Cell Res 90 (1975)
Fig. 12. filament Fig. I3. not run Exptl
Electron bundles. Electron parallel to
micrograph x 13 200. micrograph each other.
Cell Res 90 (1975)
similar
to fig.
showing regions / I6 450.
IO showing in which
possible microtubules
branch and
points
and
microfilament
focal
points
bundles
of micro(arrays)
do
Distribution
Fig. 14. A 3T3 cell fixed and colchicine. Fluorescent fibers Fig. 15. Electron micrograph bundles are obvious, however the bundles. .34 460.
of actin in non-muscle cells
343
stained with the actin antibody technique following 12 h in medium containing are obvious in this cell. .
