Download the specificity and stability of the triton

J. Cell Set. 73, 335-345 (1985)
Printed in Great Britain © The Company of Biologists Limited 1985
335
THE SPECIFICITY AND STABILITY OF THE
TRITON-EXTRACTED CYTOSKELETAL FRAMEWORK
OF GERBIL FIBROMA CELLS
MARK GILBERT* AND ALICE B. FULTONf
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, U.SA.
SUMMARY
Cellular meshworks and topography of gerbil fibroma cells can be preserved by gentle extraction
procedures using Triton X-100. We determined the stability and specificity of these cytoskeletal
frameworks by measuring extraction rate and its sensitivity to exogenous protein. Two buffers
were used, which mimicked the intracellular and extracellular ionic environments. With both
buffers, extraction was nearly complete at 5 min..This pattern of extraction was seen both in 5- and
9-day-old cultures. The same pattern of extraction was seen when three different dilutions of cells
were examined the second day after plating. Thus, extraction rate was largely independent
of minor variations in tonic composition, age in culture, or cell density. Specificity of the
cytoskeletal frameworks so produced was determined by competition with two different exogenous
proteins (bovine serum albumin or ovalbumin), which did not remove any additional material
from the cytoskeletal frameworks, even with over 10 % exogenous protein in the extraction buffer.
This pattern of extraction is not unique to gerbil fibroma cells. A similar pattern of extraction was
seen for a series of cells: mouse 3T3 cells, 3T6 cells and SVPY 3T3 cells. These experiments
indicate that the cytoskeletal framework produced by Triton extraction under appropriate conditions is stable after extraction for a period of lOmin or longer, and that the structures are
specific, in that they are not disrupted by the presence of exogenous proteins.
INTRODUCTION
The interior of most vertebrate cells contains a highly complex and finely
structured meshwork of filamentous and irregular connecting elements that extend
throughout the interior of the cell (Wolosewick & Porter, 1976, 1979). This
meshwork shows a high degree of specialization and is capable of controlled and
energy-dependent rearrangements that are important in intracellular location and
translation (Buckley, 1975; Freed & Lebowitz, 1970; Luby & Porter, 1980), cell
shape, cell movement- and endo- and exocytosis (Albrecht-Buehler & Goldman,
1976; Albrecht-Buehler & Bushnell, 1979). The interior meshwork has been the
subject of much research and is now known to contain, among other elements,
microfilaments (Buckley & Raju, 1976; Goldman, Lazarides, Pollack & Weber,
1975; Osborn & Weber, 1977), microtubules (Brinkley, Fuller & Highfield, 1975)
and intermediate filaments (Blose, Shelanski & Chacko, 1977; Hynes & Destree,
•Present address: College of Medicine, University of Iowa, Iowa City, Iowa 52242.
•)" Author for correspondence.
Key words: cytoskeleton, cytoskeletal stability, cytoskeletal specificity.
336
M. Gilbert and A. B. Fulton
1978; Lazarides, 1980). The protein chemistry of these components is beginning to
be understood. However, the visible complexity of the cell when seen intact
(Wolosewick & Porter, 1976, 1979) is greater than the complexity of the proteins
taken alone or with their cross-linking elements.
A different method of studying the complex interior meshwork has been used by
several researchers (Brown, Levinson & Spudich, 1976; Osborn & Weber, 1977;
Pudney & Singer, 1979; Webster, Henderson, Osborn & Weber, 1978; Lenk &
Penman, 1979). By exposing cells to media that are hypertonic in sucrose, have
approximately physiological salt concentration and contain a non-ionic detergent,
such as Triton X-100, it is possible to extract from the cell approximately 70 % of the
protein. The 'Triton-extracted cytoskeletal framework' prepared by this procedure
retains many of the morphological features seen in the intact cell. The distribution of
polyribosomes is unaffected (Lenk & Penman, 1979; Fulton, Wan & Penman, 1980;
Cervera, Drefuss & Penman, 1981; Dang, Yang & Pollard, 1983). Membrane
proteins remain on the cytoskeletal framework with an unchanged distribution (BenZe'ev Duerr, Soloman & Penman, 1979). Virus assembly sites are preserved (BenZe'ev et al. 1981; Lenk & Penman, 1979; van Venrooij, Silleken, van Eckelen &
Reinders, 1981). Many immunofluorescently detectable proteins are unchanged in
their distribution. Moreover, the cytoskeletal framework so prepared can often be
induced to contract in the presence of calcium and ATP.
All of these features suggest that the extraction procedure using Triton preserves
faithfully the specific relationships between proteins within the cell and provides an
experimental approach to the integrated behaviour of the complex meshworks
within the cell. Direct evidence of the specificity of the structures produced by this
procedure has not previously been available. There has been concern (Dang et al.
1983) that during the extraction procedure proteins might adventitiously become
associated with the meshwork and thus over-represent the complexity or the mass of
the structural components within the cell. The experiments presented below
represent an evaluation of the stability and specificity of the structures produced by
this procedure.
MATERIALS AND METHODS
CCL146 cells (a kind gift from Dr James Feramisco) were maintained in culture on Dulbecco's
medium + 1 0 % horse serum. From 2-9 days after subculturing, cells were washed once with
phosphate-buffered saline (PBS) and once with the extraction buffer without Triton. Extraction
was then done with either of two buffers, made up daily, by covering the cells with the buffers
containing 0-25 % Triton. After the appropriate time, samples were removed with Pasteur
pipettes. The cytoskeletons that remained on the dish were then prepared for Biorad protein assay
by treatment with 100/xl of DNase at 2mg/ml for lOmin; 10/xl of 1-Oin-NaOH were added and
the samples were allowed to incubate for an hour at room temperature. Then 380/xl of the
extraction buffer and 10/xl of 1 - 2 M - H C L were added and the samples were vigorously scraped
with a rubber policeman. The samples were removed from the dishes and the protein content of
the soluble and cytoskeletal fractions were determined using the Biorad assay. The protein content
of samples was corrected for the presence of DNase; the protein standards contained an amount of
Triton equal to that of the samples to correct for interference by Triton.
Cytoskeletal specificity and stability
337
Measurement of protein solubility in the presence of competing exogenous protein
Cell culture was as above. Before extraction, cells were labelled with [35S]methionine at
10^iCi/ml for lOmin in Tnethionine-deficient MEM. They grew in complete unlabelled medium
for an additional 2-3 hours. The extraction procedure was performed as above, with the
appropriate amounts of protein added. To remove cytoskeletons from the culture dishes, the
cytoskeletons were solubilized using 0-5 % sodium dodecyl sulphate (SDS), 0-1 M-NaCl, lOmMTris, 1 mM-EDTA, and allowed to stand at room temperature for 30min. Samples were precipitated with trichloroacetic acid and radioactivity was measured in a scintillation counter.
To measure extraction of lipids (Lenk, Ransom, Kaufmann & Penman, 1977), cells were
labelled with 0'8/xCi/ml of tritiated choline overnight before extraction. The tritium radiation in
the soluble and cytoskeletal fractions was counted in the tritium spectrum.
Extraction buffers
The final concentrations of the components of internal buffer were as follows: lOmM-Hepes,
1 mM-EGTA, 300mM-sucrose, 0-15 mg/ml phenylmethylsulphonyl fluoride (PMSF; from an
absolute ethanol stock kept at - 2 0 ° C ) , 2 % or 0-25% Triton, lOmM-NaCl, 15mM-MgSO4,
10mM-Na2HPO4, 1 mM-K2SO4, 60mM-K 2 HPO 4 , 10mM-H2SO4 (pH7-4). The external buffer
contained the following components at their final concentrations: 10mM-Hepes, 1 mM-EGTA,
300mM-sucrose, 0-15 mg/ml PMSF, 2 % or 0-25 % Triton, 120mM-NaCl, 2 5 mM-MgSO4, 5 nwNa 2 HPO 4 , 20mM-sodium acetate, ISmM-NaOH (pH7-4).
Measurement of aldolase activity
Aldolase activity was measured using the Boyer modification of the hydrazine assay (Jagan-
nathan et al. 1956) as described in Worthington Enzymes Manual.
RESULTS
All of the experiments followed the same basic experimental design. Cells were
grown in monolayer in plastic dishes and exposed for varying lengths of time to
buffers of different compositions. The amount of protein removed from the cells by
these procedures was then determined.
Fig. 1 shows the time-course of extraction for cells of different ages and in the
presence of two different buffers. The two buffers used, called internal and external,
mimic as closely as is reasonably feasible the ionic composition of the intracellular
and extracellular fluids (Loewy & Siekevitz, 1963). Since these two media differ
markedly in their Hofmeister properties (Bull, 1971), it might be anticipated that
they would have an effect on whether proteins associated with the cytoskeletal
framework are extractable or not. However, as is clear in the two graphs, no
difference was found betwen these two buffers during the extractions. Secondly, it is
clear that for cells of either age, the early extraction of protein proceeds rapidly and
was nearly complete at 5 min; not much more protein was removed from the cells at
15min. In both cases, approximately 70% of the protein in the cell was readily
removed by the detergent. The resulting cytoskeletal framework remains stable for
at least a further 10 min under our conditions.
In Fig. 1, the cells were present at different densities due to different lengths of
time in culture, and this presents an additional variable. We controlled for length of
time in culture by using three different initial plating densities; these results are
M. Gilbert and A. B. Fulton
1(X) r
5 day culturc
-
Internal
0
0
/k-
-
e,
13
3
External
I
I
2
5
1
Expasure to Triton (min)
9 day culture
Internal
Exposure to Triton (min)
Fig. 1
Cytoskeletal specificity and stability
339
shown in Fig. 2. In this case, cells were either sparse on the dish, subconfluent, or
completely confluent. All cultures had been subcultured 2 days earlier. Again, the
time-course shows that most of the protein that can be extracted has been extracted
at 5 min and not much more is removed during the following lOmin. In addition, it
is clear that there are only minor differences in the extractability of protein with cell
density. The shape of these extraction curves is reminiscent of those seen by Lasek &
Morris (1984), when they examined the extraction rate of specific components of the
axoplasmic matrix. However, the cytoskeletal frameworks described here are too
complex to determine the specificity of the cytoskeletal framework by kinetic
analysis alone, although that could be done for specific components.
To determine whether major components were non-specifically adhering to the
cytoskeletal frameworks, a different approach was taken. We sought to remove from
the cytoskeletal frameworks any adventitiously bound proteins by competition with
100 i-
Q
50
o
1/6
1/12
1/4
Culture dilution
Fig. 2. Rate of protein extraction as a function of cell density. Two days before
extraction, cells were subcultured at three different dilutions. At the time of extraction
the culture subcultured at 1:4 dilution was confluent, the 1 :6 culture was subconfluent,
and the 1:12 culture was sparse. Extracted protein was measured by the Biorad assay.
(D—D) 15 min; ( A — A ) 5 min; (O—O) 2min.
Fig. 1. Rate of extraction of soluble proteins. Five- and 9-day-old cultures were exposed
to either of two buffers containing 0-5 % Triton; the protein extracted was measured by
the Biorad assay. The 5-day cultures were subconfluent and the 9-day cultures were
confluent. No substantial differences are seen either with age of culture or with
extraction buffer used. Extraction is largely complete at 5 min.
M. Gilbert and A. B. Fulton
340
100
2 % Triton
c
'i
50
20
10
% Exogenous protein
100 r
0-25 % Triton
% Exogenous protein
Fig. 3
Cytoskeletal specificity and stability
341
various amounts of exogenous protein. Fig. 3 shows the results for two proteins,
bovine serum albumin and ovalbumin. It is clear from Fig. 3 that the extent of
extraction seen at 5 min does not vary even in the presence of as much as 20 %
protein in the extraction medium. The only differences from the previous results are
the simultaneous presence of high protein concentrations and low detergent concentrations; under these conditions the lipid also failed to be extracted. In no case
were we able to extract more protein from the cytoskeletal frameworks by increasing
either the concentration of the detergent or of competing exogenous protein.
This pattern of extraction, i.e. largely complete at five min and unaffected by
exogeneous competing protein, is also seen when a single protein, aldolase, is
examined. Aldolase was chosen as one of several enzymes reported to associate to
some degree with cytoskeletal structures (Morton, Clarke & Masters, 1977). The
aldolase activity found in the soluble fraction after 5 min extraction is 72 % of the
total activity; at 10min the soluble fraction has only increased to 80%. In separate
experiments, the aldolase activity extracted after 5 min was 67% in the absence of
exogenous protein, and 68% in the presence of 0-1 % bovine serum albumin (BSA)
in the buffer.
This pattern of extraction is not unique to gerbil fibroma cells. Fig. 4 compares
the pattern of extraction for 3T3 cells, 3T6 cells and the virally transformed
derivative of 3T3, SVPY-3T3. Although there are minor differences in the rate and
extent of extraction for the three cell lines, they all show the same pattern of rapid
extraction during the first 3—5 min, with little more protein removed after that.
DISCUSSION
The results described above show that the Triton release of protein proceeds
rapidly during the first 5 min of extraction and is largely complete at that time, with
only a small amount of protein then being removed. Our studies of other cells have
shown that although the precise time at which extraction is complete may vary for
cell types, the general pattern of extraction is similar. In addition, for most cell
types, after the second day in culture there is no further variation seen in the pattern
of extraction; nor does cell density largely affect the pattern of extraction.
The ionic compositions of the intracellular medium and the extracellular fluid of
most cells are usually different (Loewy & Siekevitz, 1963), such that the internal
Fig. 3. Protein extraction in the presence of competing exogenous protein. Cells were
labelled for 20 min with radioactive methionine, and then allowed to grow in the presence
of unlabelled methionine for 2-3 h. Extracted protein was measured by radioactivity of
[3SS]methionine in trichoroacetic acid-precipitable counts. Two different proteins were
used as exogenous competing proteins: bovine serum albumin (D—D) and ovalbumin
(O—O). Extraction of lipids was monitored by extraction of tritiated choline added the
previous day. These values are indicated by C on each graph. In the presence of 2%
Triton, no increase in extraction was seen with increasing competing protein. In the
presence of 0-25 % Triton, there was a decrease in extraction, of both protein and
choline, with increasing competing exogenous protein.
342
M. Gilbert and A. B. Fulton
10
Exposure to Triton (min)
Fig. 4. Comparison of rates of extraction for three related cell types. The cells were
labelled with radioactive methionine for 15 min and allowed to grow for 3 h in the
presence of unlabelled methionine. Extracted protein was measured as radioactivity in
trichloroactic acid^precipitable counts.
milieu has Hofmeister properties that decrease the solubility of protein (Bull, 1971).
However, our data show that extraction is largely insensitive to such variations. In
other systems, low or high salt concentrations can considerably change the pattern of
extraction (Anderton, 1981), but we restricted these studies to the physiological
range of ionic strength.
Specificity is a subtle concept and is usually measured by either of two parameters: the strength of interaction or the uniqueness of the interaction. To measure
the strength of the interactions would require an equilibrium analysis of extraction
for the entire cytoskeleton. However, the cytoskeletal framework is complex and
contains over 70 protein components easily detected by two-dimensional gel electrophoresis. Thus, such analyses would be impossible unless done by computer-aided
systems such as those used by Garrels or Anderson.
The second criterion, however, is easily applied. The idea that any protein can
satisfy non-specific binding sites is frequently made use of when purifying rare
proteins. The use of bovine serum albumin to cover up sticky sites on an apparatus
has a long tradition (Ramadoss, Steczko, Uhlig & Axelrod, 1983). When either BSA
or ovalbumen was used up to 200mg/ml, no difference was seen in the pattern or
Cytoskeletal specificity and stability
343
extent of extraction. The only variation seen in extractability of the cytoskeletal
framework was in the presence of high concentrations of protein and low concentrations of detergent; under these conditions lipid also failed to be extracted. Since
the critical micelle concentration of Triton X-100 is 19 mg/100ml (Helenius,
McCaslin, Fries & Tanford, 1979), equal amounts of free detergent monomer are
available at the two detergent concentrations in the absence of exogenous protein.
Presumably, the inhibition of lipid extraction at high protein/detergent ratios
reflects adsorption of detergent onto protein. Other cytoskeletal studies have seen no
differences between 0-1% and 1% Triton X-100 (Bell, 1981). None of the conditions examined — variations in salt composition, elevation in detergent concentration, or the presence of competing' non-specific proteins — produced a depleted
cytoskeletal framework. This strongly suggests that the structures produced by such
an extraction procedure represent a reasonably stable and physiologically significant
complex within the cell.
The cytoskeletal frameworks produced by this procedure probably represent a
lower limit of the proteins present in this structure within the cell at any one
moment. From the discussion above, it is clear that a protein that associates with and
dissociates from the cytoskeleton more frequently than every 2min would most
probably be removed during our extraction procedures. Therefore, the structures
studied in these Triton-extracted cytoskeletal frameworks probably comprise proteins that exchange less than once every 10-15 min. In addition, the concentration of
protein within most cells would tend to increase the extent of association between
proteins (Minton, 1981; Fulton, 1982).
These conclusions, that a suitable extraction with a non-ionic detergent can
separate the cell into two useful fractions and that the cytoskeletal frameworks so
produced faithfully reflect the spatial properties of the living cell, will be readily
accepted by most. Indeed, to some, the accumulated body of studies cited earlier
presents sufficient evidence for these conclusions, so that the present study will only
confirm what has already been shown or implied earlier.
However, concern about possible artifacts due to this procedure has been expressed, both in discussions and, less frequently, in print. One recent study (Dang
et al. 1983) raised two important, but quite distinct, questions: to what ought the
term 'cytoskeleton' refer, and what does resistance to Triton extraction imply?
The cytoskeleton was first used metaphorically, to imply that structures exist
within the cell which like bones, resist compression and confer shape upon the cell.
The strict application of this metaphor has been undercut recently. Both microfilaments and microtubules have been shown, in different cells, to be capable of
extension, retraction and load bearing; intermediate filaments have been effectively
removed from cells without altering cell shape or motility. These observations,
together with the fact that most animal cells round up if removed from an
extracellular matrix, suggest that the early metaphorical sense of cytoskeleton,
although stimulating, probably cannot be applied in a simple and uniform way.
The cytoskeleton, in the usage of Dang et al. (1983), is "the cellular fibre system
(keratin, actin/stress fibres and microtubules)". Unfortunately, this prescriptive
344
M. Gilbert and A. B. Fulton
definition is open to two complementary difficulties. If it is taken to exclude proteins
that associate with these fibres, clearly proteins such as tropomyosin and microtubule-associated proteins (MAPs) are not 'cytoskeletal'. If, on the other hand, such
associated proteins are included the definition ceases to be strict, since the range of
such proteins cannot be delimited a priori.
These are real difficulties in framing an exhaustive definition for the cytoskeleton.
They were among the reasons that an operational definition was chosen for the
cytoskeletal framework (Lenk & Penman, 1979).
What, then, does resistance to extraction by Triton indicate? First, it does not
indicate insolubility in Triton. This is addressed directly in several studies (Fulton
& Wan, 1983; Quinlan & Knipe, 1983) and implicity whenever shear is used to
disrupt cytoskeletons and release proteins into solution. Second, resistance to Triton
extraction is not a general property of membrane proteins. Only the plasmalemma
retains enough protein to be visible ultrastructurally (Lenk & Penman, 1979); even
some of its proteins are extracted (Ben-Ze'ev et al. 1979). What such Tritonresistant proteins represent is the class of proteins that in vivo were engaged in a
three-dimensionally inter-connected structure, a structure that includes the structural fibres and their multiple interactions in the cell.
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(Received 25 July 1984 - Accepted 28 August 1984)