Present-day cellular systems biology is producing data on an unprecedented scale. This field has generated a renewed interest in the holistic, “system” character of cell structure-and-function. Underlying the data deluge, however, there is a clear and present need for a historical foundation. The origin of the “system” view of the cell dates to the birth of the protoplasm concept. The 150-year history of the role of “protoplasm” in cell biology is traced. It is found that the “protoplasmic theory,” not the “cell theory,” was the key 19th-century construct that drove the study of the structure-and-function of living cells and set the course for the development of modern cell biology. The evolution of the “protoplasm” picture into the 20th century is examined by looking at controversial issues along the way and culminating in the current views on the role of cytological organization in cellular activities. The relevance of the “protoplasmic theory” to 21st-century cellular systems biology is considered.
- cell theory
- cell water
- cell metabolism
large-scale, high-throughput experimental methods today are producing massive amounts of data at all levels of complexity in the living cell. The generic enterprise known as “systems biology” is striving to establish order in this kaleidoscopic, information-rich empirical realm, with a newfound view of the cell as a “system.” There is, without doubt, an acute awareness of the holistic character of cellular structure-and-function in the postgenomic era. Notwithstanding the stunning successes of the modern-day analytical and computational approaches, the myopia of the data explosion in cellular systems biology has created a blinding sense of the present and a shadowy consciousness of the past. The portrayal of the cell as a “system,” in particular, has an illustrious history that bears on today's revival. The beginning of this notion, remarkable though it may seem, coincides with the birth of the protoplasm concept in the mid-19th century. Though now an archaic expression long buried in the annals of biology, the epistemological aura originally surrounding the term “protoplasm” paved the way for the pursuit of everything that we now associate with cellular structure-and-function. The story of this word, and the scientific motivation that it garnered, can enrich and inform present-day efforts to understand how the cell works.
Every scientific discipline exhibits throughout history a handful of key words that have iconic status, both in the designation of important beliefs within the field and in the conveyance of scientific knowledge in the public domain. Some of these expressions come and go, whereas others endure. In biology today, one such unique term is “cell.” It entered the scientific lexicon in the 17th century and attained notoriety in the early 19th century under the aegis of the “cell theory,” which defined the cell as the fundamental unit of all life forms. Indeed, the cell is commonly regarded as the smallest component of an organism that is “alive.” There is little (if any) mention in textbooks nowadays about one of the most powerful concepts in the history of biology, protoplasm, an expression that in times past enjoyed such romantic sobriquets as “stuff of life,” “locus of life,” “living essence,” “Urschleim,” inter alia. In fact, it was the so-called “protoplasmic theory of life,” not the “cell theory,” that yielded the original functionalist view of living cells. “Protoplasm” was the precursor expression for literally the entirety of the active components of the cell, seen as a dynamic whole. The “protoplasm theory” reached its height 150 years ago, and the perspective that it engendered lasted for only 50 years or so thereafter. Usage of the term “protoplasm” itself began to wane early in the 20th century, as the cellular makeup was subjected to the empiricism and reductionism of the molecular biology era. Yet, in no small measure it set in motion the development of the field of cell biology. With the advent of systems biology today, it seems an appropriate time to look back at the history of the inspirational role of protoplasm to appreciate the story of the first impression of the cell as a “system.”
Protoplasm: Whence It Came
The quest to understand the material basis of life, like so much of science, originates in Greek antiquity. Such ancient notions, as “proto-matter” (prote: “first, original”) - the principle that there is a fundamental substance that underlies form and function in the world around us and “hylozoism” (hyle: “matter,” zoe: “life”) - the idea that life is identifiable with (or immanent within) matter itself, have reverberated through the ages (38). The concept of “protoplasm” became the rational embodiment of these ideas in modern biology. After the onset of the Newtonian mechanical worldview, interest in the role of corpuscular “proto-matter” in the action of inanimate and animate systems mounted during the 18th century (98). Within the physical sciences such attention sharpened in the early 1800s, as evidenced markedly by William Prout's (9) provocative hypothesis that the hydrogen atom represents the “protyle” (or “prote hyle”) of the ancients. Prout, a physician, physiologist, and chemist, based his suggestion on the finding that the atomic weights for all of the (known) chemical elements appeared to be integer multiples of that of hydrogen.
Biology was not far behind physics and chemistry in its later-day search for “proto-matter.” The structural motif called the “cell” had been known since Robert Hooke's celebrated microscopic observations (and his coining of the term) in the Micrographia of 1665. As we see in present-day biology textbooks, Hooke's “cell” picture became juxtaposed with the works of Matthias Schleiden in 1838 and Theodor Schwann in 1839 to produce the “cell theory” (39). However, it is a historical fallacy to distinguish this “theory” as the primary 19th-century construct leading to the elucidation of life's ultimate physiological basis and its elementary material character. From the purely technological perspective, the advancement of the cell description may be seen as an inevitable consequence of the rapid improvements in light microscopy in the early 19th century. As to its initial impact, the cell theory signified the emergence of an ordered plan for understanding the diversity of histological information on animal and plant tissues (42). In reality, this proposal represented simply a unified anatomical view of biological forms, without an immediate concern for the substance of life per se. Originally, cells were seen simply as pores, cavities, conduits, etc., which Hooke had likened to a honeycomb. (Note that the word “cell” comes from the Latin cella meaning “inner chamber” or “small room,” and the prefix cyto- derives from the Greek for “hollow” or “receptacle.”) As described by Hall (38) (quoting from Hooke in 1665), “They were, in particular, regarded - by their discoverer among others - as avenues of communication, ‘channels, provided by the Great and Alwise Creator, for the conveyance of appropriated juyces to particular parts'.”
At its inception in the 19th century, the “cell theory” purported that all living organisms are constructed from like units, the cell, which was viewed as a “bladderlike structure with membrane, contents, and nucleus… with primary emphasis on the nucleus and the cell wall (or membrane), relegating the cellular contents to a secondary role” (31). The lack of substance in this superficial picture came under widespread attack in the mid-19th century (93). The zoologist Félix Dujardin in 1835 had already described the physicochemical properties of the living substance (which he called the “sarcode”) in rhizopods. In 1846, the botanist Hugo von Mohl demonstrated the importance of this substance in plant cells, and it is he who is usually credited with the first usage of the term “protoplasm” (from the Greek plasma: “something formed or molded”). [Actually, the physiologist Jan Purkinje had used the word in 1840 in reference to the “formative material” of animal embryos (38).] The botanist Ferdinand Cohn in 1850 unified the terminology, suggesting that “plants and animals were analogous not only because of their construction from cells, but also, at a more fundamental level, by virtue of a common substance, protoplasm, filling the cavities of those cells” (31).
Led by the prominent anatomist Max Schultze in the period 1858–1861, the conviction became entrenched that the true basis of life is to be found in the study of protoplasm, not the cell. His article (75), entitled “Über Muskelkörperchen und dass was Man eine Zelle zu nennen habe” (focusing on the properties of muscle syncytium), was a veritable milestone. As expounded by Geison (31), “The publication of this paper, more than any other single event, marked the birth of the protoplasmic theory of life. On physiological rather than structural grounds, and with special emphasis on the properties of contractility and irritability, Schultze demonstrated that a single substance, called protoplasm, was the substratum of vital activity in the tissues of all living organisms, however, simple or complex.” Even more emphatically, the historian Nordenskiöld (59) avowed that Schultze's work “laid the foundations on which cell research has since been built, and this marks a new era in the science of cytology.” Building on Schultze's seminal ideas, the noted physiologist Ernst von Brücke (10), in a widely referenced paper entitled “Die Elementarorganismen,” introduced the heuristic viewpoint that protoplasm must have a complex infrastructure (like an “elementary organism” in itself), to provide the seat of all cellular activities (see also Refs. 31, 72, and 88).
The protoplasmic theory engendered “the final repudiation of the tradition comparing living units to utricles, sacks, boxes, bubbles, or any other sort of envelope or container” (38). As the movement gained momentum, some biologists even argued that the term “cell” should be dropped, that “the advance demanded a change in nomenclature, for a cell is a box and lump of protoplasm is not” (5). The 19th-century plant physiologist Julius von Sachs (74) (obviously harkening back to Hooke) quipped that, “to call the protoplasmic unit a cell was about as appropriate as calling a live bee in a honeycomb a cell.”Thus, some 150 years ago, protoplasm had come to signify “matter possessing a certain molecular constitution permitting it to manifest life”; the cell, then, was seen as a mere housing for this matter (38).
Protoplasm: Where It Went
On 8 November 1868, Thomas H. Huxley (“Darwin's Bulldog”) gave a public lecture in Edinburgh, entitled “On the Physical Basis of Life” (published in the popular journal The Fortnightly Review in 1869), which literally made “protoplasm” a household word, and which drew the attention of scientists and nonscientists alike to the perceived role of protoplasm as the “locus of life” (93). The “cell theory” was supplanted by what came to be called the “protoplasmic theory of life” (26), with far-reaching influence. Protoplasm, as a material form and as a philosophical principle, became the symbol of the functionality of life at the most basic level. In addition to Huxley, such notables as the pioneering physiologist Claude Bernard (7) embraced the conception, calling protoplasm “life in the naked state” and declaring that, “In this amorphous, or rather monomorphous, matter life resides. Here are to be found all the essential properties of which the manifestations of the higher beings are only diversified and definite expressions, or higher modalities.” Beyond the scientific inquiry, the “protoplasmic theory” also generated the final philosophical battleground between the (ultimately victorious) advocates of mechanism and materialism and the lingering proponents of vitalism (93).
The “protoplasmic theory,” as implied by the title of Huxley's lecture, postulated that the physical basis of life (including the foundation of heredity) must be sought in the properties of this universal biological substance. As the scientific study of protoplasm unfolded, critical empirical questions emerged: How far down the scale of biological magnitude can one push the idea of “living”? What part/parts of protoplasm is/are “alive”? “Life” then (as now) eluded precise definition. The physiology of the day had compiled numerous phenomenological attributes of the living state, including irritability, sensibility, contractility, nutritive digestion/assimilation, respiration, and reproduction (21). Seeking these traits in the microcosm of protoplasm was the aim of the protoplasmic theory. Although colloidal chemistry was in its infancy at this time (76), protoplasm, early on, was known to be a gelatinous dispersion composed (aside from water) mostly of protein (or proteid) - itself regarded by some as the ultimate “proto-matter” of life. In his late 19th-century review, “On the Modern Cell Theory,” M'Kendrick (56) informs us that by the 1860s, protoplasm was defined commonly as “a diaphanous semi-liquid, viscous mass, extensible but not elastic, homogenous - that is to say, without structure, without visible organisation, having in it numerous granules, and endowed with irritability and contractility.” Nutritive matter, it was thought, somehow becomes “alive” when it enters the physical domain of parts (or all) of this protoplasmic substance.
Early consideration of the cellular composition led to the realization that protoplasm must have two primary phases: fluid and solid. As light microscopy and cytochemical staining techniques steadily improved during the late 1800s, abundant internal structure became evident. Three working models of the protoplasmic solid phase developed: reticular/fibrillar, foam/alveolar, and microsomal/granular (12, 38, 68, 76). The delineation of parts of the protoplasm as “living” relied on analysis by “vital staining.” Visibly active portions were labeled with such names (deriving from their respective Greek etymons) as “phaneroplasm” (“visible”), “kinoplasm” (“moving”), “archiplasm” (“principal”), and “ergastoplasm” (“working”). Other suggested “plasms” were “cryptoplasm” (for the “hidden” part of protoplasm), “trophoplasm” (for the “nutritive” part), and “hyaloplasm” (for the “clear” part). In 1884 the botanist Karl Nägeli modified the dualistic view of protoplasm by proposing that it consists of two fundamental portions: “hygroplasm,” the fluid region, and “stereoplasm,” the solid part - with the latter, he suggested, being the site of all vital activities (56). This particular picture of the cellular essence, as a “proteinaceous, structured, stereoplasmic” medium, would endure as a farsighted vision.
Many biologists of the late 19th century, alas, were not content simply with the assignment of the “locus of life” to the solid or fluid phase of the cell. The science of biology, like physics at the time, was heading inexorably toward an atomistic foundation. As summarized by Hall (38), protoplasmic theorists throughout the second half of the 19th century professed prolifically that, “Life is the activity of either i. individual molecules of the proper variety; or ii. an unstructured molecular complex (hyaloplasm); or iii. subvisible multimolecular structures ('metastructures') of some sort; or iv. visible intracellular structures (organelles).” Category iii represented, by far, the most popular, and speculative, view. A wave of “multimeric” protoplasmic theories ensued, as the physical basis of life came to be seen as a conglomeration of particles going by such names as “biogens,” “gemmules,” “idisomes,” “pangens,” “physiological units,” “plasomes,” inter alia (38, 93, 99). The underlying belief was that these “living molecules” represent the physical (or physiological) basis of life, as well as the principle of heredity.
With the rise of biochemistry, the metaphor for the cell as a “chemical factory” assumed a critical role in the latter part of the 19th century (71). Analogies with the division of labor in industrial factories, as well as that seen in the specialized organ systems in higher life forms, abounded in this period (55). The interface of the science of biochemistry with protoplasmic theory, though yielding much fruit over time, raised physiological concerns and questions at its inception that remain unresolved to this day. Enzymes, being the fundamental mechanistic elements that execute cellular processes, became the center of attention. Throughout the history of biochemistry, it has been taken as a matter of course that most metabolic activity of the cell results from the superposition of the action of individual enzymes dissolved in an aqueous phase, with the dynamics being governed by simple mass-action laws and random thermal motions of metabolite molecules in weak-electrolyte solution. Despite the “structural” and “organizational” views of the cell coming from protoplasmic theorists, the newfound gas-phase kinetics and the in vitro chemical laws in the late 19th century were grafted on to the analysis of cellular metabolism. This development was fostered by various observations, such as those of the botanist Wilhelm Pfeffer on the osmotic properties of cells. Pfeffer found that the cell behaves as if a semipermeable plasma membrane (the existence of which he deduced) separates two ordinary aqueous solutions, one being that inside the cell (64). The influential studies by the chemist Eduard Buchner (11) on fermentation in cell-free yeast extracts gave further credence to the view that the cell is merely a homogeneous “bag of enzymes” in aqueous solution. His findings seem to set the stage for the use of “cell-free extracts” in the development of biochemistry that was to take place during the next 50 years. One might say that the empirical concept of “soluble enzymes” was born in Buchner's work (23, 47).
Protoplasmic theorists reacted strongly against the picture of cell metabolism being procreated by the chemists in the early days. Perhaps the greatest voice was that of the aforementioned champion Claude Bernard. Enzymes, originally called “ferments” (reflecting the early interest in the nature of alcoholic fermentation), had been discovered in the mid-19th century. In 1876 the physiologist Wilhelm Kühne (who trained with Bernard) presented to the Heidelberger Naturhistorischen und Medizinischen Verein a landmark paper proposing that isolable “ferments” be called “enzymes” (meaning “in yeast” or “leavened”) (37). So was created a new term in biology, and so was created a divide that continues to be questioned today. The title of Kühne's historic article, “Über das Verhalten verschiedener organisirter und sogenannt ungeformter Fermente,” is telling: an enzyme in a test tube is one thing, an enzyme in the cell is another. Bernard (7) articulated the view that the chemically purist impression of enzyme action does not bespeak the relationship of the “ferment” to “the organization, the development, and the multiplication, that is to say the life of the cell.” He recognized that, “Today, two kinds of ferments are distinguished, according to the soluble or insoluble nature of the ferment; the one produced by the intervention of an organized or structured ferment, the other produced by nonorganized ferments, liquids, soluble products…” (Bernard's italics). Some protoplasmic theorists went so far as to propose models of cell metabolism based on the structural properties of protoplasm. There were suggestions that the catalytic agents (“ferments” or “enzymes”) form part of the integral structure of a dynamic proteinaceous network in the cell, or that they are attached to the ends of fibrillar protoplasmic tendrils (48, 51). Such early hypothetical views would prove to be rather close to reality.
Protoplasmic theory (and physiology) notwithstanding, the newly conjoined sciences of biochemistry and enzymology stayed on their independent heading as the 19th century came to a close (48). The isolation of individual enzymes and the observation of “saturation kinetics” in vitro at the turn of the century, culminating in the Michaelis-Menten and Briggs-Haldane theoretical treatments, strengthened the conviction that the mechanistic understanding of cellular function was to be found in physical chemistry. Meanwhile, espousal of the physiological idea of “living molecules” was gradually straining the limits of credibility of the protoplasmic theory in scientific circles, with charges of vitalism coming from those in the physical sciences (31, 48, 93). Early in the 20th century, with the (re)discovery of Mendel's work, the atomistic focus in biology shifted predominantly from the phenotype to the genotype (the “germ-plasm”); and the panoply of protoplasmic “particles” was swept away and replaced with a new unit: the gene. Usage of the term “protoplasm” itself began to decline, due not only to its vitalistic baggage, but, more significantly, to the increasing reductionistic focus amongst cell biologists on the intricacies of cellular (or protoplasmic) composition. The maturation of biochemistry, with the eventual elucidation of how protoplasm is metabolically built up and torn down, dispensed with the ambiguous question of where/when do nutrients become “alive;” and the empirical difference between living “protoplasm” and nonliving “metaplasm” became blurred (48, 99). And the philosophical inquiry into “What is life?” became a hunt for the Jabberwock.
20th-Century Cell Biology: What Is Protoplasm Really Like?
During the 20th century, a wide array of purification methods (including selective protein precipitation, ion-exchange chromatography, gel filtration, electrophoresis, and ultracentrifugation) and analytical procedures (including high-voltage electron microscopy, increasingly sophisticated light microscopy, confocal microscopy, fluorescence techniques, and in situ probe analysis) were brought to bear on the examination of cells and their components. Such investigations gradually revealed an extensive arrangement of organelles, membranous structures, and cytoskeletal elements interlaced throughout the cell. We have only to open the pages of any cell biology textbook today to appreciate the intricate and highly integrated structural tapestry of the cellular interior. Among the myriad of experimental approaches and published studies constituting the colorful history of the discovery of cellular ultrastructure, one might identify many singular works that stand out as key advances in our understanding of how the whole cell is put together. It is beyond the scope of the present article to review the entirety of this original literature. Suffice it to say that, what we find over time is an emerging jigsaw puzzle, whose interlocking pieces began to generate in the holistic eye a sense of synergy far beyond the properties of the components.
Today's visualization of the makeup of protoplasm, at face value, would appear to be a reification of the dualistic view that had arisen in the late 19th century: there are two distinct phases of the cell, “solid” and “liquid.” By the turn of the century, the expression “cytoplasm” was becoming a popular descriptor of the cellular makeup. This word was coined by Albrecht von Kölliker in the 1860s to designate the fraction of the cell outside the nucleus, but it gradually replaced “protoplasm” in the course of the 20th century (100). Today, the discernibly solid parts (aside from organelles) have acquired such labels as “cytomatrix,” “cytoskeleton,” etc., whereas the purely liquid portion became known as the “cytosol;” the latter term introduced in the 1960s by Henry A. Lardy, as an operational expression referring to the in vitro supernatant remaining after all the solid material in cell extracts is pelleted by high-speed ultracentrifugation (17, 18).
The physiological oversimplicity of such a biphasic “structuralist” representation of the cell was called into question early in the 20th century. The leading cell biologist Edmund B. Wilson (99), in a summary lecture on the “Structure of Protoplasm” (delivered at the Marine Biological Laboratory, Woods Hole, MA) at the end of the 19th century, concluded that, “If we except certain highly specialized structures, the hope of finding in visible protoplasmic structure any approach to an understanding of its physiological activity is growing more, instead of less, remote, and is giving way to a conviction that the way of progress lies rather in an appeal to the ultramicroscopical protoplasmic organization and to the chemical processes through which this is expressed.” Wilson's monumental text The Cell in Development and Heredity (100), which went through three editions (1896, 1900, and 1925, respectively, ultimately growing to 1,000+ pages in length), reconciled the various views of protoplasmic structure from the late 19th century and was highly influential in setting the stage for analysis in the early 20th century. He recognized the importance of the dynamic interplay between the structured and unstructured “phases” of the cell, prophesying in the third edition of his book that, “There is reason to conclude that of all the cell constituents the ‘structureless hyaloplasm' is the most constant and active, and may perhaps be regarded as forming the fundamental basis of the protoplasmic system from which, directly or indirectly, all other elements take their origin.” If protoplasm is truly a “system,” then there can be only one physiological “phase” underlying the visual microscopic representation.
In the early 20th century, the exposition of the “submicroscopic structure” of protoplasm attracted considerable attention, as seen perhaps most distinctly in Albert Frey-Wyssling's research and writings (28, 29). As it became apparent that protoplasmic structures are dynamic and labile, the interrelationship of these entities with the fine-grained “ground substance” of the hyaloplasm was a major concern very early. The reality of micrographic pictures of cells, subject to fixatives (or “preservatives,” as they were sometimes called), was subject to debate. Gwendolen Foulke Andrews (who trained under Wilson), in a turn-of-the-century exposé on the methods for studying cells micrographically, professed that, “I have convinced myself that ‘preservatives' fix for us little of the true structure of the living substance, and can, at best, keep for us grosser relations, of a mixed sort in point of time; hiding an infinite complexity of form, and destroying perforce those infinitely delicate relations whose fleeting harmonies make up life phenomena” (3). This argument persists today.
Among those contributing in a major way to the pursuit of the intricacies of cell structure in the 20th century was Keith R. Porter, whose extensive observations and critical ideas, beginning in the 1940s, have earned him a prominent place in the history of cell biology. Porter was greatly influenced by the likes of Wilson and Frey-Wyssling, as he followed the quest to describe and understand “submicroscopic protoplasm.” Although best known for his description of formed elements like the endoplasmic reticulum, Porter believed that the finer-level cytomatrix organization would turn out to be of exceptional importance. For example, Porter (67) wrote that “The matrix therefore occupies, in present day concepts of the cytoplasm, the same position as that held by the ground substance or hyaloplasm of the earlier light microscope image. It is the ‘structureless' medium in which are suspended all the resolvable elements of the cytoplasm…” He expounded further something that would predict his own quest in the decades to come: “There is no reason to believe that with improved methods of microscopy and specimen preparation this part of the cytoplasm will not in time be shown to contain organizations of macromolecules.” In the early 1970s Porter played a key role in the installation of a high-voltage electron microscope (HVEM) at the University of Colorado. Training this powerful instrument on fixed, whole tissue culture cells, he set out on this mission, soon to describe what he termed the Microtrabecular Lattice (MTL). Using HVEM images, Porter described the presence of an extensive network of narrow cylinders (∼7 nm in diameter and of variable length), the “microtrabeculae,” that ramified throughout the cell, seemingly connecting all the formed elements. These and other features of the MTL can be seen in Fig. 1 (assembled from photomicrographs that Porter sent to J. S. Clegg in 1987). It is easy to understand why Porter referred to such a unit structure as the ultimate “cytomatrix,” the most elementary solid phase of the cytoplasm.
Porter championed the reality of this structure for the remainder of his career. However, few cell biologists have accepted the existence of the MTL, believing that the elaborate edifice Porter described was an artifact of fixation or critical point drying resulting in the deposition of previously “soluble” proteins on other cell components, as well as the formation of artificial protein-protein aggregates. John Heuser (41), who was a participant in this debate, but not supporting the MTL, has written a recent history of the subject, entitled “Whatever happened to the ‘microtrabecular' concept?” in a special issue of Biology of the Cell. There the reader can find a variety of photomicrographs of the MTL, as well as Heuser's interesting historical account and micrographs of his own work. Also contained in this issue of Biology of the Cell are short articles by Mark McNiven (54) and John Wolosewick (101). Both having studied with Porter, they provide an additional perspective about the MTL. We might note that Wolosewick and Porter (102) coauthored a paper in 1979 that responded to criticisms of the MTL, with hope of establishing the MTL as a “real” structure. A recent opinion paper by Kim and Coulombe (46) compiles abundant evidence that the spatial organization and regulation of the protein-synthesizing system of eukaryotic cells are determined by the cytoskeleton, notably actin microfilaments. Those authors posited that, “The cytoskeleton has evolved as a scaffold that supports diverse biochemical pathways.” Perhaps Keith Porter would have said: “I told you that in the mid-1970s.”
The relationship between the “solid” and “liquid” phases of the cell at the “submicroscopic” level is most surely a dynamic one, though an exact and encompassing model remains to be determined. Porter's interpretation may have been wrong in detail but right in principle. Because of the continuing controversies underlying empirical fixation procedures, static micrographs alone cannot give us a complete picture of the structure-and-function of protoplasm. Porter was a realist as well as dedicated to describing the MTL. In a 1987 letter to one of us (accompanying the photographs from which Fig. 1 in this paper was assembled; J. S. Clegg, personal communication), he wrote: “Do you really think that we or anyone else can make sense of such a mess.”
A key objection to such “submicroscopic” structural models as the MTL concerns the question of “soluble proteins.” What fraction of the cytoplasmic protein constituency is, in fact, “soluble”? What is the actual concentration of “free” proteins in the cell? The standard view today is that the aqueous phase of cytoplasm (the “cytosol”) contains a very large concentration of “dissolved” proteins of various kinds, as well as a variety of other macro- and micromolecules. Yet, this description flies in the face of a body of evidence, some of it quite old (84), indicating that the majority of macromolecules (particularly protein) is normally associated, in some manner, with cytomembrane and cytomatrix structures. Such support for an organized rendering of the “submicroscopic” realm has come from the advancement of biochemistry and enzymology.
While the sundry subcellular structures were being discovered during the progression of 20th-century cell biology, the major metabolic pathways were gradually revealed by the application of analytical chemistry, as well as by the isolation and study of individual enzymes using a “grind-and-find” approach. The “bag of enzymes” representation of the cell was modified in the 1940s and 1950s, as electron microscopy and cell-fractionation techniques began to show that certain enzyme activities are associated with specific organelles that could be separated and identified. Since then, a variety of more refined microscopy methods, in situ analyses, and extraction procedures has provided increasing evidence that the surfaces of intracellular membranes and cytomatrix structures are the location for a multitude (perhaps the majority) of intermediary metabolic reactions, as well as such larger-scale operations as signal transduction, intracellular “trafficking,” protein synthesis, and DNA/RNA processing (for reviews see Refs. 2, 20, 46, 61, 84, and 86; see also Molecular INTeraction [MINT] database, http://mint.bio.uniroma2.it/mint/Welcome.do). For example, enzymes of the glycolytic pathway were once considered to be classic examples of “soluble proteins,” but we now know that many of them (possibly all) are not freely diffusing in three dimensions in eukaryotic cells (see Refs. 18, 19, 60, plus various chapters in Ref. 86). In reality, the actual aqueous portion of the cytoplasm may not be so “crowded” after all (17, 80).
Renewed attention of late has been given to the role of localized, heterogeneous microenvironments in defining the true kinetic and thermodynamic nature of biochemical events in living cells (e.g., Ref. 32). The importance of surface activity in the understanding of subcellular processes, in fact, was realized long before the nature of cellular infrastructure was clearly established. A noteworthy discussion of this view and its implications came in 1930 from the biochemist Rudolph Peters (62) in a prophetic paper entitled “Surface Structure in the Integration of Cell Activity” (later refined in Ref. 63). Modern-day calculations of protein “concentration” in association with cytomembranes and cytomatrix elements indicate high, crystal-like densities in (on) the particulate structures of the cell (80, 82, 91). Studies with artificially immobilized enzyme systems, commencing around 1970, have proved crucial in establishing the importance of the microenvironment in enzyme action. The ground-breaking works of Ephraim Katchalski (43), Klaus Mosbach (57, 85), and Daniel Thomas (87) are particularly noteworthy (for reviews see Refs. 78, 90, 92, and 97).
The evolution of our perception of the microworld of enzyme action in vivo, like the generation of our present-day holistic understanding of cell ultrastructure noted above, has been a gradual process that may be likened to a puzzle work, with the elucidation of many interlocking pieces and many contributing scientists. The original literature here, as well, is far too extensive for a review within the scope of the present article. Though, we should like to single out the personage of the biochemist Paul Srere for mention. Few scientists have had as much impact on the discovery landscape of enzyme organization in the living cell, not only in terms of his own pioneering studies, but also in regard to the number of researchers and the breadth of ideas that he influenced. Over the course of the last 30 years or so, Srere led the way in planning a host of scientific meetings on this topic, including an ongoing biennial Gordon Research Conference founded in 1987, whose title (and scope) has morphed over the years from “Organization of Metabolic Sequences,” to “Macromolecular Organization and Cell Function,” to (the current title) “Cellular Systems Biology.”
Despite the mounting evidence for metabolic organization and microenvironmental effects in vivo, the vision of soluble metabolic pathways has remained, chiefly as a time-honored deduction from high-speed centrifugation experiments on cell and organelle extracts; and it is still a widespread and firmly rooted way of thinking in cell biology and biochemistry. Thus “soluble metabolism” and “dilute aqueous phase properties” have become the conventional wisdom, forming the basis for experimental design, interpretation of data, and construction of models. But we must not lose sight of the fact that these beliefs are built more on assumption and extrapolation of 19th-century physical chemistry than on hard evidence. Not to belittle the ensuing reductionism of 20th-century cell biology and biochemistry, as it has yielded an ever-flowing fountain of information on cellular structure-and-function, from the level of genes and proteins, to metabolic pathways, to organelles, to the cell itself. After all, most purified enzymes do “work” in a test tube, under the dilute aqueous conditions that we define in vitro. Information on the in vitro properties of isolated enzymes has been of much utility, for example, in piecing together such theoretical constructs as “Metabolic Control Analysis,” which has provided a window (at least qualitatively) into the global distribution of metabolic control in vivo (24). Aside from basic knowledge, abundant applications in areas of biomedicine and biotechnology have sprung from the long history of painstaking study on individual enzymes in vitro. Yet, there remains the “Humpty-Dumpty” quandary: how do we fit the pieces together into a functional whole? (32, 45). Disparities abound, as we attempt to reconcile the observed features of isolated enzymes with the observed behavior of metabolism in the intact cell. For example, it is often found that the observed kinetic properties of individual enzymes in vitro simply do not account quantitatively for metabolic fluxes in vivo (90, 97). The algebra of holism dictates that 1+1>2. Enzyme action is, in reality, part-and-parcel of an intricate structural edifice in living cells. Albeit complicated physically (and mathematically) to grasp, this is the portrait of “protoplasm” for the 21st century.
Today, a suited pictorial representation of the global unity of structure-and-function in cells, as in all organismal levels of biology, in fact, comes from the realm of fractals (1, 89). It is becoming apparent that localized fractal dynamics provides the proper theoretical principles for comprehending the character of enzyme kinetics, mass transport, and thermodynamic flow-force relationships in the cellular microenvironments (4, 52, 92). The particular fractal image that seems to us most apropos of cellular protoplasm, as regards its present form as well as its emergent evolution, is the “Sierpiński sponge” (Fig. 2), which is a confined geometric object whose progressive “fractalization” results in the surface area increasing to (the theoretical limit of) infinity as the volume shrinks to zero (53). Had Robert Hooke's 17th-century microscope been more powerful (we might muse), perhaps he would have concluded, as we do today, that his honeycomb “cells” are actually filled with vibrant, hydrated sponges?!
With the spotlight on the functional significance of microenvironmental organization in vivo, speculation has arisen as to whether there is, in fact, some kind of unitary, corpuscular basis for the physiological activity of cellular processes. For example, in 1985 Srere proposed the term “metabolon” for complexes of metabolically sequential enzymes that exist in organized states in vivo (83). Whereas such aggregates vary in their size and structure, the intent of the expression is to draw attention to the “particle” aspect of cellular metabolism. Unbeknownst to Srere (G. R. Welch, personal communication), the word “metabolon” (from the Greek for “change”) had existed fleetingly in the scientific vernacular in the early 20th century, having been introduced by the physicist Ernest Rutherford in 1903 to designate sequences of atomic radioactive-decay species (73). Ironically, Rutherford was looking to biology for metaphorical concepts at the time (13). The symbolism of the ancient term “protyle” lingered into the 20th century, both in physics and biology. In 1920 Rutherford named the proton, the newly discovered subatomic particle that forms the nucleus of the hydrogen atom, in honor of Prout's original conception of “protyle” (9). Heretofore, the atom was envisioned as an amorphous blob (a plasma) of positive charge, within which freely moving electrons are interspersed - the so-called “plum pudding” model that enjoyed brief popularity at the beginning of the 20th century.
In an article entitled “The Particle Size of Biological Units: A Review” published in 1932, the physical chemist John Ferguson (27) surveyed the (then) current findings on the sizes of entities that seemed to be minimally related to the manifestation of “life.” With the use of data obtained via the methods of ultramicroscopy, gel-membrane ultrafiltration, and (the relatively new area of) ultracentrifugation, he considered a variety of protein molecules, viruses, and “genes” (whose size, as well as composition, was a matter of conjecture at the time). Having drawn no unifying conclusion, Ferguson concluded that, “The conception of a self-perpetuating catalyst of molecular (protein?) size is an alluring prospect to the materialist in search of the ‘Protyle' of life. Speculation is fruitless, however, until such entities are definitely established by physicochemical data on the one hand, while the biologist from his (sic) viewpoint recognizes their ‘vital' powers of reproduction, assimilation, and adaptation.” With the influence of 19th-century thought in evidence, Ferguson (27) closed by opining that, “Life still is a great mystery, and in the creed of the biologist its essence is that peculiar ‘integrative force' which still defies analysis. For the biologist, biology, and not materialism, must yet remain the ‘working hypothesis'.”
Some 50 years after Ferguson's review treatise, there appeared a more penetrating study bearing upon the question of “biological units” in living cells. In 1980 the cell biologist Peter Sitte (80) presented an in-depth examination of subcellular structures, motivated by the awareness of the high density of protein molecules in (on) these structures and the realization of the importance of surface activity in localized metabolic processes. He pointed out a remarkable homology in the surface-area-to-volume ratio (the “specific surface”) for all membranous cytological substructures, corresponding to an upper limit of 150 μm−1 and a “sphere of influence” of about 100 nm in diameter (see Fig. 3). Taking an integrated (what he viewed as a distinctly “systems”) view of the cell, Sitte (79) suggested that this homology reflects a common evolution in the structure-and-function of the microenvironmental domains that define cellular activities. Theoretical analysis has shown that, by coincidence (!), the observed size of the homologous spatial “units” noted by Sitte corresponds to the dimensional limit for efficient reaction-diffusion coupling in metabolic processes, based on typical values of enzyme catalytic-turnover constants and diffusion coefficients (91). Accordingly, we are led to modify the fractal design in Fig. 2 to include idealized spheres denoting confined regions, ranging from organelles to protein complexes (“metabolons”), responsible for the execution of localized metabolic processes in situ (see Fig. 4). Thus it would seem that we have arrived at a “plum pudding” picture, of sorts, for the structure-and-function of protoplasm - 100 years after this transitory icon existed in the annals of physics. We have also arrived at the realization that the secret of protoplasm is to be found not in substance but in process. As recapped fittingly by Sitte (80), “2500 years ago the Greek philosopher Heraclitus assumed all things to be in a state of steady motion and change. A living cell would have given him plenty of satisfaction.”
Water: The Overlooked Component of Protoplasm
Water has been recognized since very distant times as profoundly important in both living and nonliving systems, as seen for example in the ancient Hindu scriptures: “About 3,000 years ago the Upanishad Thinker said ‘it is water that assumes the form of this Earth, midregion, this heaven, these mountains, these gods and men, cattle and birds, herbs and trees, and animals together with worms, flies and ants. Water is indeed all these forms: meditate on water'” (15). Legions of thinkers have since “meditated” on water, generating a rich history on this deceptively simple molecule, with its own romantic side (6). In fact, water was the original “protomatter” (or “protyle”) in ancient Greek philosophy, having been proposed by Thales of Miletus as the fundamental material basis of the world and everything in it. Water is so intrinsic to the living state that it is easily taken for granted and, alas, ignored. Interesting then, that early descriptions of protoplasm overlooked water, except to recognize its presence and obvious importance. Understandable perhaps, because those who studied protoplasm in those days had no way to actually describe the physical properties and detailed roles of water. When did the physical study of water in biological systems begin? Difficult to say, but an early insight was provided by the remarkable scientist Thomas Graham (36), who found that some of the water in complex systems, like gelatinous starch, does not dissolve sugar. Those studying protoplasm at the time did not pick up on this empirical fact and draw the inference that some of the water in colloidal substances like protoplasm could differ from ordinary water, with potentially significant consequences. Graham did not use the term “bound water” to describe that fraction of water, but one can posit that he had, indeed, come across the idea.
The term “bound water” has had a focal position in biology since the inventive work of Ross A. Gortner. He and his students showed that a substantial amount of the water in certain plant systems does not freeze at temperatures where bulk water should do so (58). That fraction of water was referred to as “bound,” and research on biological (protoplasmic) water would be influenced down to the present time. (Of course, we now know that such water is not bound in a static sense but is in dynamic exchange with the surrounding aqueous phase.) Gortner's work attracted the attention of those studying the properties of colloids, a hot topic of that era. A meeting of major importance was held at the University of Cambridge 29 Sept-1 Oct 1930 on “Colloid Science Applied to Biology,” and the matter of cell and tissue water was a significant part. Although Gortner did not attend (apparently due to ill health), his paper (33) was communicated at the meeting and published (along with the extensive discussion that followed it) in the Transactions of the Faraday Society (vol. 26) that same year. Although Gortner's notion of “bound water” attracted considerable attention at the 1930 meeting, it was not completely adopted. The very marked difference in opinion about protoplasmic water, leading to a polarization of views, was summed up by two participants. The distinguished physiologist A. V. Hill wrote, “… in living tissue, at least in muscle, practically the whole of the water is free…” (p. 72). Obviously, Hill was not a proponent of bound water. In contrast, W. Ramsden proposed that, “… as regards the water inside a biological cell, I should expect the whole of it to be ‘modified' water…” (p. 697). However, the arguments of Hill prevailed, and his work was cited for the next 40 years as evidence that almost all cell water is that of an ordinary bulk solution.
Gortner was a key actor on the stage of protoplasmic water during in the early 20th century. He wrote that, “It is my belief that many of the reactions characteristic of living processes have to do more with the water relationships of the organism than any other single factor” (33). After the Cambridge meeting, Gortner published two major reviews in the (then) young Annual Reviews of Biochemistry (34, 35), one of which focused on the role of water in protoplasm. But his words apparently had little impact. For example, William Seifriz, a prominent student of protoplasm at the time, wrote a review with the title The Properties of Protoplasm (77), in which Gortner's papers were not mentioned, and the concept of bound water not considered at all.
Among those who took up the study of cell and tissue water in the 1950s and 1960s, the names of Afanasii Troshin, Walter Drost-Hansen, and Gilbert Ling stand out. They each had differing ideas about the nature of cell water, but all three agreed that it was very unlikely to be the same as ordinary bulk water. But, like Gortner, they failed to convince most of the scientific community. Then, in 1969, two papers appeared that would change the balance. Freeman Cope (22) and Carlton Hazelwood et al. (40) published evidence suggesting that much (even all) of cellular water has properties markedly different from “ordinary” bulk water. They interpreted their findings as evidence for Gilbert Ling's “Association-Induction Hypothesis” (50), which considers all of the water in cells to be in the form of polarized multilayers, much different from the ordinary bulk fluid. The vibrant controversy that raged over the next 20 years can be referred to as the “water wars.” It is beyond the scope of the present article to review the abundant literature and the excitement of that period. But we should note that the battlefield has now grown rather quiet; and, although the issues have not been resolved, the consensus view seems to be not much different from that put forth by Hill in 1930 - which is to say, that almost all of cellular (protoplasmic) water is not unusual. Notwithstanding, the discussion continues (16, 30, 65, 66, 103). It seems odd that the most vital and widespread of biological molecules remains to have its physical properties and participation in the cellular milieu (that is, protoplasm) defined in detail. Perhaps we should heed the advice of the ancients and meditate on protoplasmic water and then return to its study.
Protoplasm: Is It Relevant Today?
Amidst the flood of data that has inundated the study of the living cell in the postgenomic 21st century, there is a grand synthesis proceeding under the rubric of systems biology. This burgeoning epistemic framework, while only a decade old, has rapidly assumed a defining role in the organization and integration of empirical information, as well as in driving knowledge discovery. A large and rapidly expanding taxonomic terminology has arisen within the systems biology vocabulary, using the suffix “-ome” to denote component sets for the specific biological entities that are the subject of data collection, annotation, curation, and interpretation at each level of organization in the living system. Instead of the perception of the cell as a family of “-plasms” in 19th-century protoplasmic theory, we see an assortment of “-omes” in 21st-century cellular systems biology.
Surprisingly, there is no established definition of “systems biology”, as is obvious from a simple search on the World Wide Web. Powell et al. (70) put it simply that, “What is often meant by ‘systems biology' today is the attempt to make sense of the vast amounts of data that have been accumulated by the genome sequencing projects and other data-gathering exercises.” As suggested by Westerhoff and Alberghina (96), the definition of systems biology is “heterogeneous” and specified “by examples,” including many topics “all focusing on the mechanics behind the emergence of functionality.” Absent a standard definition, it seems universally accepted that this field seeks to study the relationships and interactions among the parts of biological systems and the integration of such information into a picture of the functioning organism (25). Despite frequent reference to such pioneering systems theories as that of Ludwig von Bertalanffy, systems biology in its current guise focuses heavily on static informational arrays and component mappings. The incorporation of a dynamical representation would complete the picture, and this stands as an aspirational goal at the moment. As expressed by Boguski (8), “The future may lie in a new vision of annotation that supersedes static, ‘repository biology' with a dynamic ‘virtual cell' in which most properties and behaviors can be quantitatively modeled and dynamically represented in all of their interconnected complexity” (for example, see Ref. 81). Such reasoning, of course, lay at the foundation of the protoplasmic theory in the 19th century. Cellular systems biology would appear, at least superficially, to be the emperor in new clothes.
The challenge today, daunting though it may be, is to create a vision of the cell in silico by piecing together the “-omes,” in conjunction with the vast amount of facts on the organizational character of the life of the cell. The branch of cellular systems biology known as “interactomics,” in particular, is generating inroads here (Ref. 94; see also Molecular INTeraction [MINT] database, http://mint.bio.uniroma2.it/mint/Welcome.do). Yet, the data-driven advancement of knowledge continues to spawn an expansive, hierarchically reductionistic collection of “-omes,” and the path to the integration of the disparate “-omic” information into a description of the cell as a functioning whole is not altogether obvious. As argued cogently by Kell and Oliver (44), the true course of study demands that “data” and “ideas” be complementary and iterative partners along the way. The idea of “protoplasm,” at its height, served a supremely important role in organizing thought and focusing attention on the cellular makeup as the fundament of life. The protoplasm principle died when the inquiry “What is life?” died amidst a haze of varied “-plasms” and “living particles.” Today, the overarching idea of the cell as a “system,” though certainly an appropriate identifier, seems self-evident to the point of being nondescript. It remains to be seen if this idea will avoid becoming too suffused with the assortment of “-omes” to achieve the goal of an integrated view of cell structure-and-function.
Stripped of its vitalistic and esoteric trappings, protoplasmic theory in its day (like cellular systems biology today) is really about cell physiology (95). The aforementioned cell biologist E. B. Wilson, a leading visionary (and transitional figure) in the history of cell biology, remarked presciently in the opening pages of the first edition of his text The Cell in Development and Heredity (100), “No attempt is here made to identify a living ‘protoplasm' as such or to distinguish between ‘living' and ‘non-living' cell-components. Life is treated as a property of the cell-system as a whole, the components of that system differing only in the degree and manner of their activity… Logically, no doubt, this is correct; and from a purely physiological point of view is perhaps the only possible mode of treatment.” “Physiology,” itself, is one of those deeply rooted biological subjects whose central role waned [relegated to the status of “a quaint and vaguely anachronistic” discipline (14)] in the course of the 20th century, as life's processes were dissected with increasing precision by the developing sciences of biochemistry, biophysics, genetics, and molecular biology. Along with this analytical reductionism, though, came a loss in our appreciation of the holistic function of the life form. While physiology today may have lost its scientific independence, it nevertheless represents “a point of view, an attitude toward the study of ‘functions' (not merely ‘mechanisms' or ‘processes') that gives meaning to biological systems” (49). The study of living cells, we would suggest, does not need a term, such as “system,” to give it direction; rather it requires a physiological way of thinking. It is in this sense that the word (nay, the idea) “protoplasm” deserves its rightful place in the history of biology.
No conflicts of interest are declared by the author(s).
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