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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9318-9326, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9318-9326.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101
Received 11 June 2003/ Returned for modification 11 August 2003/ Accepted 9 September 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent years, considerable attention has focused on the importance of macromolecular interactions in cell function (see, e.g., reference 10) and on the fact that enzymes contributing to complex processes often are bound to each other and that intermediates in the process may be channeled (see, e.g., references 6 and 16 and the review in reference 19). As a consequence of such organization, processes within cells may be able to proceed much more efficiently than those carried out by the same enzymes dispersed in solution in vitro. Thus, important questions that remain to be answered are (i) how extensive is cellular organization, (ii) what cellular components are responsible for maintaining it, and (iii) are macromolecular interactions confined to individual functional units or are they a global property of the cell?
A variety of approaches have been employed to examine the organization of macromolecules in cells. Early experiments by Kempner and Miller (15) showed that cellular contents become stratified upon centrifugation of intact Euglena cells and that the zone thought to be the cytoplasm is devoid of proteins, implying that these molecules are not free. Other experiments, employing high-voltage electron microscopy and cell extraction procedures, demonstrated the presence of an organized network in cells (22, 23) which might act as a scaffold for cell organization (20). Subsequent work revealed that some glycolytic enzymes (5) and some detergent-extractable proteins (2) are not freely diffusible in vivo, suggesting that at least some cellular components might be present in highly organized structures (reviewed in reference 26).
With the advent of new techniques to study protein-protein interactions (see, e.g., references 8, 11, 13, and 31), thousands of interactions among cellular macromolecules have been identified. However, these types of studies often lead to a high number of false-positive results, raising uncertainties about the actual extent of in vivo organization. In contrast to the aforementioned studies, another body of work (reviewed in reference 32) supports a different conclusion. The results of these studies indicate that extensive macromolecule diffusion can occur intracellularly, implying the absence of organization, but that movement is hindered by crowding and transient binding. Thus, questions about structural and functional organization, and how this might be maintained in vivo, persist.
In the present work, we have used a simple, straightforward approach that directly examines the status of endogenous macromolecules in an attempt to clarify this situation. To do this, we employed procedures that gently permeabilize a cell's plasma membrane under conditions that appear to have minimal effects on internal cellular architecture and have used such a system to examine the release from cells of various classes of macromolecules. Our data suggest that the entire mammalian cell behaves as an organized, macromolecular assembly. We show, in addition, that macromolecular organization is essential for the high efficiency of a complex cellular process, namely, protein synthesis. Finally, we demonstrate that cellular organization is largely dependent on an intact actin cytoskeleton. These observations support the conclusion that endogenous macromolecules in mammalian cells are highly organized and are not free to diffuse over large volumes. The data provide important insights into our understanding of cell structure and function.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PKB/Akt
1) Immunoglobulin G (IgG) fraction of antiserum, polyclonal affinity
isolated anti-actin, monoclonal anti-ezrin, and anti-heat shock protein
70 (hsp70) antibodies were from Sigma, and monoclonal anti-EF1
antibody was from Upstate Biotechnology. Anti-mouse and anti-rabbit IgG
(horseradish peroxidase conjugated) were purchased from Promega. All
other chemicals were reagent grade. Cell culture. Chinese hamster ovary cells (ATCC no. CRL-1781) were grown as monolayer cultures at 37°C in alpha minimal essential medium containing ribonucleosides, deoxyribonucleosides, 2 mM glutamine, and 10% fetal bovine serum in air containing 5% CO2. Cells were transferred every 2 to 3 days and were harvested for experiments 1 day after reaching confluence.
Permeabilization with saponin. Cells were permeabilized as previously described (28) with the following modifications: all buffers were at room temperature and cells were not cold shocked and recovered. Cells were treated with 75 µg of saponin/ml in S buffer (130 mM sucrose, 50 mM KCl, 50 mM potassium acetate, 20 mM HEPES, pH 7.4) for 7 min at 37°C. The cell concentration was adjusted to be 1 x 107 to 2 x 107 cells per ml for each experiment. After permeabilization, cells were pelleted by centrifugation at 2,500 x g for 30 s at room temperature. In all experiments, cells were between 97 and 99% permeable (as determined by trypan blue entry). When present during the permeabilization procedure, latrunculin B (in dimethyl sulfoxide [DMSO]) and colchicine were each used at 100 µg/ml. The final concentration of DMSO was 3.75%.
Intact and permeabilized cells were subjected to ultrastructural examination to determine the effects of saponin and latrunculin treatment. After permeabilization, cell pellets were fixed overnight in 2% paraformaldehyde-2.5% glutaraldehyde in Millonic's phosphate buffer, washed in Millonic's phosphate buffer, fixed in 1% osmium tetroxide for 1 h, dehydrated, and embedded in Spurr's reagent. Thin sections were stained with uranyl acetate and lead citrate and viewed on a JEOL CX100 transmission electron microscope.
To measure the size of holes created by saponin, permeabilized and intact cells were incubated for 15 min at room temperature with fluorescent Igs (IgG, IgA, and IgM). Cells were subsequently pelleted for 30 s, resuspended in S buffer, repelleted for 30 s, and resuspended in phosphate-buffered saline (PBS)-4% formaldehyde. Cells were fixed for 30 min at room temperature, pelleted, and resuspended in SlowFade Antifade mounting medium (Molecular Probes). The suspension was transferred to a glass slide, covered with a coverslip, and sealed with nail polish. Slides were then examined on a Zeiss LSM-510 confocal laser scanning microscope.
Leakage studies. A portion of cells was counted in a hemocytometer prior to permeabilization. After the saponin treatment, cells were pelleted as indicated above and the supernatant fraction was removed and saved. Cells were resuspended in the original volume of S buffer and pelleted under the same conditions, and the supernatant fraction was saved. The final cell pellet was resuspended in the original volume of S buffer, and a portion was counted to determine cell recovery. During processing, some cells are invariably lost or lysed. The amount of leakage is presented as the percentage of a particular cell component present in the permeabilization supernatant plus the wash supernatant fractions corrected for that amount accounted for by the loss of cells. The 100% value was determined from an assay of an equivalent amount of intact cells.
Protein synthesis. Translation in intact cells was carried out in PBS medium supplemented with 25 mM glucose, 2 mM CaC12, 1 mM MgC12, and 250 µM of each of the amino acids plus a mixture of 3H-labeled leucine, lysine, phenylalanine, proline, and tyrosine. Protein synthesis in permeabilized cells was carried out in PSW buffer (130 mM sucrose, 50 mM KCl, 50 mM potassium acetate, 20 mM HEPES [pH 7.4], 5 mM ATP, 13 mM phosphocreatine, 6.1 mM MgCl2, 2.6 mM CaCl2, 5.3 mM EGTA, 5 mM glucose) supplemented with 0.1 mM GTP, 30 µg of creatine phosphokinase/ml, and 250 µM (each) amino acid plus a mixture of 3H-labeled leucine, lysine, phenylalanine, proline, and tyrosine. All protein synthesis assays were performed at 28°C. Portions were removed at the times indicated, and protein synthesis was determined as to hot-acid-precipitable radioactivity (16).
Enzyme
assays.
The activity of
glucose 6-phosphatase was determined by measuring the release of
orthophosphate (4,
30). DNA polymerase
activity was determined (using denatured calf thymus DNA as the
template) by measuring the rate of incorporation of
[32P]dATP
(21). Citrate synthase
was assayed as described by Jarreta et al.
(14). The activities of
lactate dehydrogenase, glucose 6-phosphate dehydrogenase, and
glyceraldehyde 3-phosphate dehydrogenase were determined as described
in the Worthington Biochemical Corp. manual. Aminoacyl-tRNA synthetase
assays were performed at 37°C in reaction mixtures containing
the following ingredients: 250 mM Tris-HCl (pH 7.5), 5 mM
MgC12, 0.2 mM EDTA, 0.2 mg of bovine serum albumin/ml, 1.5
mg of rabbit liver tRNA/ml, 0.1 mM 3H- or
14C-labeled amino acid (
20 to 100 cpm/pmol), and
sufficient cell extract or supernatant fraction to measure synthetase
activity. Reactions were stopped by the addition of 10%
trichloroacetic acid containing 0.5% Casamino Acids (Difco).
Aminoacyl-tRNA precipitates were collected and counted as described
previously (1). Release of
protein and RNA from prelabeled cells was performed as previously
described
(18).
Western
blotting.
After
electrophoresis on 8% polyacrylamide gels, proteins were
transferred to a polyvinylidene difluoride membrane in Tris-glycine
buffer (0.375 M Tris, 0.192 M glycine, 20% methanol). Blocking
of the membrane was performed with a 3% solution of nonfat milk
in PBS buffer for 2 h at room temperature with shaking. The
membrane was then treated with the antibodies mentioned above in PBS
buffer supplemented with 3% nonfat milk (2 to 5 µg of
antibody/10 ml of buffer) overnight at 4°C with shaking.
Visualization of the protein bands followed the ECL Western blotting
protocol using anti-mouse IgG (for anti-ezrin, anti-hsp70, and
anti-EF1 antibodies) or anti-rabbit IgG (for anti-actin and
anti-AKT antibodies) conjugated with horseradish peroxidase as the
secondary antibody.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Structural characterization of permeabilized CHO cells. To carefully evaluate the effects of saponin treatment on cell structure, permeabilized CHO cells were examined by light and electron microscopy and compared to untreated cells. The extent of cell permeabilization was monitored by trypan blue dye entry and amounted to 97 to 99% in all experiments presented. Ultrastructural examination revealed no gross differences between intact and permeabilized cells (Fig. 1A and B, respectively). Moreover, in both cases, the cell populations were homogeneous. Less than 1% of the cells were lysed, and many microvilli were present on the cell surface in each case. In contrast, latrunculin-treated, permeabilized cells were quite heterogeneous. About 15% of these cells were lysed and about one-third were more electron dense (Fig. 1C). In addition, few microvilli were present. Analysis at higher magnification showed no damage to either the cytoplasm or the internal membranes of the permeabilized cells (data not shown). This is consistent with the known differential sensitivity of the plasma membrane to saponin (33). Mitochondria in the permeabilized cells appeared to be slightly distended but were otherwise intact. The endoplasmic reticulum was not disrupted. Several indistinct areas of the plasma membrane were seen. However, it has not been determined whether these areas represent holes in the membrane or just regions of the membrane oblique to the plane of the thin sections. The relative intactness of the permeabilized cells prepared by the methods described here is in contrast to that found with other permeabilization procedures (3, 5), which appeared more destructive.
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Release of
macromolecules.
To determine
the degree of organization of proteins in CHO cells, the cells were
prelabeled with [3H]amino acids and then subjected
to the usual saponin permeabilization procedure followed by
centrifugation to separate those proteins that were released from those
that remained with the cell. As shown in Fig.
4A, only 12% of total cellular
protein was released from the permeabilized cells, indicating that the
vast majority of proteins were in some way sequestered. This situation
changed dramatically upon the addition of the microfilament-disrupting
agent, latrunculin B. Under these conditions, total protein leakage
increased to 40%. It should be noted that proteins do not leak
from intact cells (Fig.
4A), and this was not
altered by the addition of latrunculin B (data not shown). These data
suggest that a large percentage of cellular proteins are sequestered by
direct or indirect association with the actin cytoskeleton. In
contrast, the addition of colchicine, a microtubule-disrupting agent,
led to only a small increase of protein leakage. Thus, microtubules
appear to play a less important role in macromolecule
organization.
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Similar studies were
carried out using cells prelabeled with
[3H]uridine to assess leakage of RNA (Fig.
4A). Very little RNA
leaked from permeabilized cells or from permeabilized cells treated
with colchicine. In contrast, upon the addition of latrunculin B, RNA
leakage increased to 15% (the presence of DMSO to which
the latrunculin was added had no effect on leakage). Analysis by
polyacrylamide gel electrophoresis (PAGE) of the RNA that is released
under these conditions revealed that it was primarily tRNA (data not
shown). Thus, these data indicate that tRNA also is normally
sequestered in cells and that its retention is dependent on an intact
actin cytoskeleton. The data do not allow any conclusions regarding
ribosomes. They also may be sequestered or they may be too large to
pass through the holes generated by
saponin.
Release of specific
proteins.
We next addressed
the issue of whether the total protein release presented in Fig.
4 was representative of
all proteins or whether proteins of different pathways or cellular
locations might behave differently. Accordingly, proteins from
different cellular compartments and several different cytoplasmic
proteins were each examined for release in the absence or presence of
agents that affect the cytoskeleton. We turned our attention first to
actin and to the actin-binding proteins EFl and ezrin. As
shown in Fig.
5, the results of Western blot analysis indicated that only relatively
small amounts of these proteins were released from permeabilized cells.
Likewise, very little actin was released from intact cells or
permeabilized cells treated with DMSO (data not shown). In contrast,
upon treatment with latrunculin B, which leads to actin filament
depolymerization, or gelsolin, which severs the filaments, there was a
dramatic increase in the release of the three proteins. In contrast,
the presence of colchicine, which disrupts microtubules, had
essentially no effect on release of any of these proteins (Fig.
5). Essentially identical
results were obtained with AKT (protein kinase B) and hsp70
(Fig. 5). Given the low
level of actin release from permeabilized cells, it is likely that even
the populations of monomeric actin that are known to be present at
discrete sites in cells complexed with other proteins
(7) remain bound in some
manner.
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Kinetics of
cytoplasmic protein leakage.
To examine in more detail the two
putative populations of cytoplasmic proteins, we followed the time
course of their release from permeabilized cells (Fig.
6A). The leakage of total protein was also measured for comparison. Over the
course of 60 min, the amount of total protein released increased from
10 to 25%. This increase of 15%
represents the maximum amount of additional protein leakage, because
some of it may have been due to cell lysis during the extended
incubation. Nevertheless, these data indicate that protein release
remained relatively low, even during 1 h of incubation.
Leucyl-tRNA synthetase was released in concert with that of total
protein, in keeping with the single time point measurement (Table
1). Lactate dehydrogenase,
on the other hand, was released at a much more rapid rate during the
60-min time period, reaching a level of 75% at 60 min
and confirming the existence of at least two populations of cytoplasmic
proteins. The data suggest that lactate dehydrogenase, and presumably
other proteins of this group, is transiently sequestered but can leak
from permeabilized cells during the period in which it is not
associated with cellular structures. Moreover, it appears (as
determined by total protein release) that the majority of cellular
proteins are more stably sequestered.
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70%; however, the additional
presence of colchicine did not change the rate or total amount (within
5%) of protein leakage (compare Fig.
6C to
6B). These data suggest
that sequestration of proteins is largely due to the actin
cytoskeleton. Under these conditions, 90% of
leucyl-tRNA synthetase and 95% of lactate dehydrogenase
were released in 60 min and, again, the presence of colchicine had no
additional effect. The high level of release of the two cytoplasmic
enzymes suggests that cytoplasmic proteins are able to leak essentially
to completion once the actin cytoskeleton has been disrupted. In
contrast, since 30% of total protein remained with the
cell it is likely that organellar proteins were not released, even
under these extreme
conditions. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data also suggest that
the cytoskeleton, particularly the actin microfilament network, plays
an important role in maintaining this organization. On the basis of an
examination of protein synthesis (the results of which are presented
here and elsewhere) (18,
29), both supramolecular
organization and the actin cytoskeleton are necessary for maintaining
the high efficiency of this process in vivo compared to the same
reactions in vitro. Thus, components of the translation apparatus such
as tRNA, aminoacyl-tRNA synthetases, and EFl are normally
tightly sequestered in permeabilized cells. However, upon disruption of
microfilaments there is a dramatic reduction of protein synthesis
accompanied by release of these translation components from the cells.
The fact that protein synthesis in these permeabilized cells remains at
such a high level, despite the complexity of the process, also suggests
that this system has retained much of the original structural integrity
of intact cells.
Permeabilized cells have been used by other investigators to examine cell architecture (5, 22). Compared to other permeabilizing agents, however, saponin, the permeabilizing agent used here, has the advantage when carefully titrated of causing minimal damage to internal membranes and leading to a very uniform population (>97%) of permeabilized cells (17). Although earlier work was limited largely to some glycolytic enzymes, those studies also supported the conclusion that extensive enzyme organization was a probable feature of animal cells (5, 22). In the present work, the analysis has been extended to total protein, to enzymes of multiple organelles, to structural proteins, to cytoplasmic enzymes of several metabolic pathways, and to RNA. Thus, these studies have revealed that organization extends to many components of the cell. Moreover, it has led to the finding that at least two populations of cytoplasmic proteins exist, those that are stably associated with cellular structure and those that appear to associate transiently, which results in their partial release from permeabilized cells. Differential release of glycolytic enzymes and the idea of stable and transient association with cell structure have been suggested earlier (12). Most significantly, these studies have also revealed the importance of the cytoskeleton in maintaining the organization of cytoplasmic macromolecules. While many macromolecules are known to bind directly to cytoskeletal elements, it remains to be determined how much of the organization is due to direct binding and how much is due to secondary binding to already bound molecules.
The extensive organization of cellular macromolecules that we propose does not mean that they are unable to move in cells. It is well known, for example, that upon microinjection many macromolecules can diffuse to specific cellular sites. However, our data suggest that diffusion is not the primary means by which endogenous macromolecules are transported. Rather, we would propose that macromolecules and macromolecular complexes normally are actively transported by molecular motors along the cytoskeleton which might be considered the "railroad tracks" of the cell (for reviews, see references 9 and 25). The increasing number and diversity of known molecular motors underscores the importance of their function in the cell and the large number and types of cargo that need to be transported.
How then does one reconcile our observations that endogenous macromolecules do not freely diffuse throughout cells with the large body of work (32) indicating that such molecules are able to move? There are several possibilities. First, measurements of macromolecule diffusion in cells generally use techniques (such as fluorescence recovery after photobleaching) that examine movement over relatively short distances whereas, in our experiments, long-range movement is necessary for molecules to be released from cells. One would expect that even if macromolecules were part of an organized structure, they would dissociate and rebind allowing for movements within small volumes. The extent of movement would depend on the rate of rebinding compared to the rate of diffusion. In fact, this is what we propose for the more rapid rate of leakage of the glycolytic enzymes. The second consideration is that measurements of diffusion generally employ microinjected or modified macromolecules that may exceed the available binding sites or may bind more weakly, either of which could result in movement that does not occur with the endogenous molecules. Further work will be needed to clarify these differences.
The data and conclusions presented here supporting the idea that the entire cell is a macromolecular assembly fits well with observations in other closely related areas. Thus, the increasing evidence that intermediates in many metabolic pathways are channeled demands associations among components of the pathway, at least transiently. Extensive organization among cellular components would also be in keeping with the tenets of metabolic control analysis and the behavior of metabolic networks such that perturbations in one part of a system can have profound effects on components elsewhere in the system. Such functional interrelationships are best understood in terms of structural organization. Finally, the thousands of interactions among cellular proteins that are now being reported in a variety of systems are exactly what would be expected from cells organized as we propose. It is becoming clear that if we wish to understand how cells function in vivo, we will have to use more integrative approaches and study the biochemistry of organized systems (10, 20).
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ACKNOWLEDGMENTS |
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This work was supported by grant GM16317 from the National Institutes of Health.
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FOOTNOTES |
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