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Revisiting the microtrabecular lattice
James S Clegg1
Section of Molecular and Cellular Biology, University of California, Davis, and the Bodega Marine Laboratory, Bodega Bay, CA 94923, U.S.A.
The ‘microtrabecular lattice’ (MTL) that Keith Porter described in the 1970s and 1980s is reconsidered as a proposed fundamental cytoplasmic structure of eukaryotic cells. Although considered to be an artefact by most cell biologists of his time (and probably ours), the case is made that something like the MTL may well exist, but in a much more dynamic form than images from electron microscopy imply and convey.
Key words: cell water, cytosol, microtrabecular lattice, scaffold protein, signalling proteins
Abbreviations: HVEM, high voltage electron microscopy, MTL, microtrabecular lattice
Part of a series marking the 70th birthday of the Cell Biology International Editor-in-Chief Denys Wheatley
In a special issue of Biology of the Cell, John Heuser (2003) wrote an essay entitled “Whatever happened to the microtrabecular concept?” He was referring to the proposition advanced by Keith Porter in the 1970s and 1980s that the fundamental structure of cytoplasm of eukaryotic cells is a highly branched, filamentous network that Porter called the microtrabecular lattice (MTL) that ramified throughout the cytoplasm and contained most of its formed elements and cytoplasmic proteins. The literature detailing Porter's views on the matter can be accessed through his last scientific publication (Porter, 1986) and other papers in the book in which it appeared (Welch and Clegg, 1986). These and other features of the MTL are shown in Figure 1, assembled from photomicrographs that Porter sent to me in 1987. In this case, normal rat kidney (NRK) cells were examined by high voltage electron microscopy (HVEM), revealing the MTL to the right in Figure 1. Porter once referred to the MTL as the ultimate ‘cytomatrix’, and noted that such a structure was present in every one of the many nucleated cell types he examined over his career.
Heuser points out the doubts put forward by some cell biologists about the reality of the MTL, a position he also took during the debate about the MTL. Also contained in that issue of Biology of the Cell are short articles by McNiven (2003) and Wolosewick (2003). Both having studied with Porter (John Wolosewick was probably his last graduate student), they provide a much more positive perspective about the MTL being a real structure in living cells.
Nevertheless, the MTL was generally considered to be an artefact in which freely diffusing (‘soluble’) cytoplasmic proteins aggregated with each other and onto cytoplasmic structures, notably the cytoskeleton, all this caused by procedures used to prepare the cells for microscopy. Wolosewick and Porter (1979) responded to, and attempted to counter these criticisms, in a paper whose title included the phrase ‘artifact or reality?’, but their effort apparently had little effect on prevailing opinion. It is important to realize that Porter was strictly a microscopist (and a superb one) whose evidence implied that the MTL was a rigid structure, lacking the dynamics at work in living cells. I doubt that Porter ever believed that such was the case, but I do not recall that he ever made this clear in his writings. Although a minority of scientists (including myself) supported the general idea of something like an MTL being present, based on evidence independent from microscopy (Clegg, 1984; Welch and Clegg, 1986), neither of those arguments won the day. Instead, the prevailing view then (and now) is that eukaryotic cell cytoplasm is composed of various organelles and other formed elements more or less suspended in an aqueous phase crowded with freely diffusing proteins and other macromolecules, metabolites and inorganic ions in what is usually referred to as the ‘cytosol’, but more on that deceptively simple term shortly. It has been almost 25 years since Porter's last scientific publication (Porter, 1986); in view of that, and John Heuser's question, it seemed appropriate to revisit the MTL, but this time from a different vantage point.
What is the cytosol?
The answer to this question is central, actually pivotal, to the matter in hand. Is the cytoplasmic aqueous phase, the so-called cytosol, really a crowded and chaotic solution of freely diffusing molecules — micro and macro? Is the concentration of soluble protein in the cytosol really several hundreds of milligrams per millilitre (Luby-Phelps, 2000)? One frequently sees reference to the presence of such phenomenal protein concentrations, but what is the evidence? A hint comes from the original definition of ‘cytosol’ (Clegg, 1984) being the fraction of homogenates remaining soluble in the high-speed supernatant. The protein concentration is indeed very high in such a preparation, no doubt because the cells and their contents have been smashed to pieces. I wonder how many actually believe that such a buffer-based supernatant reflects, even remotely, the composition and function of the ‘in vivo’ cytosol (which I prefer to call ‘aqueous cytoplasm’). Not only is that belief wrong, it deludes one into thinking we know something that we do not.
Much has been written about the nature of cytoplasm over the years (Welch and Clegg, 2010), but in my opinion one of the best analyses is the review by Luby-Phelps (2000). She evaluates the methods and results that have been used to describe what the aqueous cytoplasm is really like. As a result she points out that the “evidence suggests that the aqueous phase is crowded with macromolecules, yet at least 50% of cytoplasmic protein is resident in the solid phase”. In keeping with what others have proposed about the metabolic significance of this cellular compartment (Clegg, 1984; Welch and Clegg, 1986), she concluded that the “potential impact of actual intracellular conditions on the kinetics, mechanisms and regulation of metabolism make it imperative to re-examine continuum descriptions of cellular biochemistry that have been extrapolated from reductionist experiments carried out in dilute solution”. In a commentary about 10 years later, Gierasch and Gershenson (2009) championed a similar view, perhaps even more strongly, with the optimistic statement that “We have arrived at the post-reductionist era of biochemistry” noting that “…components of signal transduction pathways, metabolic networks, gene expression regulators, protein folding facilitators and so on are associated, often by weak transient interactions, into large multiprotein complexes”. Gierasch and Gershenson did not mention the MTL, but their analysis is very useful in that regard. What I am suggesting is the existence of a dynamic MTL whose structure is undergoing rapid turnover, perhaps on a scale of seconds.
While evaluating evidence for and against a crowded cytosol (Clegg, 1984), it seemed likely that such a view was heavily influenced by extrapolation from cell fractionation rather than direct experimental evidence. On the other hand, a substantial amount of information existed, derived from different kinds of studies, that was at least consistent with an MTL-like cytoplasm. I attempted to argue that the MTL was not a static entity as suggested from microscopy, but a dynamic structure in which various proteins were associating with, or dissociating from, the actin cytoskeleton and other cytoplasmic structures on a rapid timescale. This is not the place to resurrect that analysis (Clegg, 1984, 1992; Welch and Clegg, 1986). What have we learned about the cytosol since then? We are told that several classes of proteins assigned to this compartment are involved with the critical function of signalling, for at least part of their lifetimes. But what is their location in the cytoplasm?
Scaffolds, adaptors and signalling proteins
Zeke et al. (2009) reviewed the substantial literature dealing with the so-called scaffold or adaptor proteins that are abundant in cytoplasm and play an important role in cellular signalling circuits (e.g. Ramirez and Albrecht, 2010; Chapman and Asthagiri, 2009). This area of study continues to be at the forefront of research in biochemistry and cell biology. Although this literature is too massive to be covered in this brief paper, it is my impression that the majority of the components of signalling pathways are, indeed, believed to be ‘soluble’, i.e. located in the cytosol. What if they also spend part of their lives as components of a dynamic MTL?
Another question of importance concerns the actual concentrations (not the relative ones) of cytoplasmic proteins involved in these interactions — are we talking about a small fraction or an abundant one? Although that information seems to be scarce, it appears that the total concentration of these proteins is not trivial, and that their interactions are certainly dynamic. Even less clear to me is their cytoplasmic location because, as far as I have been able to determine, the study of these proteins is usually carried out using cell extracts.
Porter considered the actin cytoskeleton to be a critical component of the MTL, a view held by others at that time (see Clegg, 1984). Kim and Coulombe (2010) make the case that the spatial organization and regulation of the protein-synthesizing system of eukaryotic cells are determined by the cytoskeleton, notably actin microfilaments. Those authors proposed that “The cytoskeleton has evolved as a scaffold that supports diverse biochemical pathways”. Indeed, that idea has been around for decades (for a few of many examples over the years, see Masters, 1981; Knull and Walsh, 1992; Penman, 1995).
I realize that I am stretching things a bit in this minireview, but given some leeway in that direction, I would argue that such a dynamic MTL is possible, and that it would have enormous importance, including things like the signalling pathways and much of intermediary metabolism. It would be nice if the opening sentence in the review by Gierasch and Gershenson (2009) is actually true: “We have arrived at the post-reductionist era of biochemistry”. Those are welcome words for the future of cell biology as well.
Porter's description of the MTL implied to some that it was a static structure. But viewed in the more recent and emerging picture of a dynamic cytoplasm, perhaps the idea of a MTL should be re-examined, not constrained by having to comply with the HVEM images. It is widely accepted that a great many protein–protein interactions occur in cytoplasm, the question is: how many proteins are involved, what are their locations and turnovers, ‘bound and unbound?’ (associated and unassociated). The MTL of HVEM images (Figure 1) surely does not exist in living cells, but I think it could well be a reflection of something that does, being a snapshot of a dynamic entity involving interactions of the kind already mentioned. I am not suggesting that all, or even most, of the scaffold and adaptor proteins are associated with formed cytoplasmic structures; what I am suggesting is that, at any given time, a significant number of them find themselves to be part of a dynamic MTL. Porter's image of cellular structure was holistic, dominated by the cytoplasmic matrix he worked so hard to describe. Reductionism has taken us a long way in cell biology and biochemistry, and that approach will likely continue to be productive. But I believe very few would take the position that ‘grind and find’ methodology will be sufficient to understand the structure and function of cells. Gierasch and Gershenson (2009) mention the difficulty of “putting Humpty Dumpty back together again”, referring to the formidable task of reconstructing cells from the bits and pieces studied in vitro. They also write that “It is virtually impossible to reassemble an in vivo environment…” I agree, although that is too bad, because in the present climate the best evidence for a MTL of the kind I suggest here would be to build one in vitro.
Many thanks to Denys Wheatley, whom I have been privileged to know and collaborate with for over 30 years. He has frequently made me rethink my positions on scientific matters, often on major questions in cell biology that today do not get aired because too many scientists are now primarily concerned with specific experimental details and not with general concepts in this discipline, and their wider implications and applications.
Clegg, JS (1992) Cellular ultrastructure and metabolic organization. Curr Topics Cell Reg 33, 1-14
Knull, HR and Walsh, JL (1992) Association of glycolytic enzymes with the cytoskeleton. Curr Topics Cell Reg 33, 15-46
McNiven, MA (2003) The solid state cell. Biol Cell 94, 555-556
Ramirez, F and Albrecht, M (2010) Finding scaffold proteins in interactomes. Trends Cell Biol 29, 2-4
Zeke, A, Lukács, M, Lim, WA and Reményi, A (2009) Scaffolds: interaction platforms for cellular signaling circuits. Trends Cell Biol 19, 354-374
Received 12 July 2010; accepted 26 August 2010
Published online 28 September 2010, doi:10.1042/CBI20100511
© The Author(s) Journal compilation © 2010 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)