In Search of Cellular Control: Signal Transductionin ContextDonald Ingber*

Departments of Pathology and Surgery, Children’s Hospital and Harvard Medical School,Boston, MA 02115

Abstract The field of molecular cell biology has experienced enormous advances over the last century byreducing the complexity of living cells into simpler molecular components and binding interactions that are amenable torigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanismsby what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences thatmediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventionalexperimental approaches ignore the importance of supramolecular control mechanisms that play a critical role incellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studiedin isolation because their function may vary depending on their context within the structural complexity of the livingcell. These higher-order regulatory mechanisms are based on the cell’s use of a form of solid-state biochemistry in whichmolecular components that mediate biochemical processing and signal transduction are immobilized on insolublecytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is therealization that chemistry is structure and hence, recognition of the importance of architecture and mechanics for signalintegration and biochemical control. Recent work that has unified chemical and mechanical signaling pathwaysprovides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future. J.Cell. Biochem. Suppls. 30/31:232–237, 1998. r 1998 Wiley-Liss, Inc.

Key words: cytoskeleton, mechanotransduction, integrins, cell architecture, tensegrity

Molecular cell biology is guided by the desireto understand how cells and tissues developtheir unique organic qualities, including theability to change shape, move, and grow. Oncewe understand the molecular controls that gov-ern these behaviors, we will be in a position todevelop more rational drug therapies and tocreate artificial tissues for organ repair andreplacement. The first question I would like toaddress in this essay is: are we moving alongthe correct path?

Many of the fundamental questions of controlin cell and developmental biology relate di-rectly to the mechanism of signal transduction:how an external signal produces an intracellu-lar response. This is true whether the stimulusis a soluble hormone, an insoluble extracellular

matrix (ECM) molecule, an adhesive contactwith a neighboring cell, or an external mechani-cal stress. Thus, to evaluate the correctness ofour path in this search for cellular control, wemust explore whether we truly understand howcells transduce information.

The experimental approaches used to ana-lyze cell signaling are varied. In general, theyinvolve challenging cultured cells with a con-trolled stimulus while simultaneously quanti-tating changes in cellular biochemistry or geneexpression. For example, the stimulus might bea mitogen which increases growth in a dose-dependent manner. Alternatively, it might be asecretagogue that induces protein secretion ora vasoagonist that stimulates cell contractility.Stimulus-response coupling can be similarlyanalyzed in suspended cells immediately afterthey bind to immobilized ECM components (e.g.,ECM-coated microbeads). The common themeis that the effects of all these stimuli are medi-ated by binding of a ligand to specific transmem-brane receptors on the cell surface. When theligand is bound, the receptor molecule activates

Contract grant sponsor: National Institutes of Health; Con-tract grant sponsor: NASA.*Correspondence to: Donald Ingber, Enders 1007, SurgicalResearch, Children’s Hospital, 300 Longwood Ave., Boston,MA 02115; E-mail: ingber@a1.tch.harvard.eduReceived 27 October 1998; Accepted 29 October 1998

Journal of Cellular Biochemistry Supplements 30/31:232–237 (1998)

r 1998 Wiley-Liss, Inc.

a ‘‘signaling cascade’’ inside the cell. This is aseries of chemical and molecular transforma-tions which eventually results in changes in aparticular biochemical function (e.g., gene tran-scription, translation, secretion, ion channelactivation).

Using this experimental approach, the gen-eral goal is to identify the critical transductionmolecule responsible for a particular stimulus-response coupling. This is often interpreted asthe molecule that negates the normal responsewhen deleted or inactivated and that restoresthe response when its activity is regained. How-ever, the process of signal transduction andcellular control is clearly much more compli-cated than any single signaling molecule, oreven any individual transduction pathway. Inliving tissues, cells receive many simultaneousinputs that activate numerous signaling cas-cades in parallel. For example, in a healingwound, cells simultaneously sense the bindingof soluble mitogens and insoluble ECM compo-nents as well as the pull of the surroundingtissue. Yet, one cell will proliferate in this micro-environment while its neighbors, only micronsaway, respond by remaining quiescent, differen-tiating, or even dying in different regions of thesame tissue. In fact, it is establishment of thistype of local growth differential that drivesdifferential tissue expansion and pattern forma-tion during morphogenesis in all growing tis-sues.

But how does each cell ‘‘know’’ what to dowhen confronted by a specific stimulus? Theanswer is that the same stimulus can producean entirely different response depending on thecellular context. A simple analogy would be alight switch at the top of a flight of stairs: iteither can turn the lights on or off, dependingon position of the switch on the floor below. In asimilar manner, raising the concentration ofgrowth factor in culture medium results in adose-dependent increase in growth when cellsare plated on a highly adhesive ECM substratewhereas varying the density of the immobilizedECM component can produce similar dose-dependent control of cell growth under condi-tions in which growth factor levels are optimal[Ingber and Folkman, 1989; Ingber, 1990; Ing-ber et al., 1990]. Furthermore, cells can bestimulated by optimal growth factors and anoptimally adhesive ECM, yet downstream sig-naling events and cellular behavior can varydramatically (growth vs differentiation vs apop-

tosis), depending on the degree to which the cellmechanically stretches or retracts [Ingber andFolkman, 1989; Singhvi et al., 1994; Chen etal., 1997; Huang et al., 1998]. In other words,the cellular response is dependent on both thechemical and the mechanical context in whichsignal transduction proceeds. Thus, the keyquestion is not which signaling molecule is acti-vated, as currently dominates existing ap-proaches. Rather, it is how all these differentsignaling pathways are integrated inside thecell.

This new challenge in the field of signal trans-duction is not unlike the multibody problem inphysics and it similarly requires a new frame ofreference. We tend to teach physics in terms oftwo body problems because they are readilyamenable to analysis, however, the reality isthat the two body problem is the rare exception,rather than the rule, in the real world. Simi-larly, we prefer to describe biological mecha-nisms in terms of what we can measure, with-out considering the structural complexity inwhich these pathways must function in theliving cell. This was a sound approach when wehad no way to deal with cellular complexity.However, this is rapidly changing and it isclearly our challenge for the future.

Further insight into the mechanism of signalintegration has come from analysis of the pro-cess by which ECM and mechanical stressesregulate cellular form and function. Binding ofECM molecules to their own cell surface recep-tors, known as integrins, can activate many ofthe same intracellular signaling pathways thatare stimulated when growth factors bind totheir receptors [Clark and Brugge, 1995]. Giventhat most normal (anchorage-dependent) cellssimultaneously require both adhesion to ECMand soluble mitogens for their own growth andsurvival, much may be gained by understand-ing how these two signals are integrated insidethe cell.

Work in the integrin signaling field has shownthat part of the mechanism of signal integra-tion and cellular control is based on the spatialorganization of signal transducing moleculesinside the cell.Although most analyses of signal-ing proteins are carried out in solution, many ofthese molecules normally function when immo-bilized on insoluble cytoskeletal scaffolds. Forexample, when cells bind to microbeads coatedwith ECM molecules that induce integrin recep-tor clustering, a specialized cytoskeletal struc-

In Search of Cellular Control 233

ture, known as the focal adhesion complex(FAC), forms at the site of integrin binding[Plopper and Ingber, 1993] and physically con-nects the internal actin cytoskeleton to inte-grins and thus to ECM on the outside the cell[Wang et al., 1993]. Importantly, many key sig-naling molecules are simultaneously recruitedto the FAC [Plopper et al., 1995; Miyamoto etal., 1995a,b]. These signaling components in-clude protein kinases (FAK kinase, c-src, MEK,ERK, JNK, Raf), inositol lipid kinases, small Gproteins, ion transporters (Na1/H1 antiporter)and a subset of high-affinity growth factor recep-tors, to name a few.

Elements of different signaling cascades maydirectly interact, and hence integrate, whenthey are brought into close proximity withinthe FAC. One example of signal integrationbased on this form of ‘‘solid-state’’ signaling isthe finding that integrin clustering activatessynthesis of the inositol lipid substrate, phos-phatidylinositol-bis-phosphate (PIP2), whereasits breakdown (and the associated release ofthe downstream signaling molecules, diacylglyc-erol and inositol-tris-phosphate) is controlledby growth factor clustering-dependent activa-tion of the membrane-associated enzyme, phos-pholipase C-g [McNamee et al., 1993; McNa-mee and Ingber, 1996]. In fact, this is a beautifulexample of how growth factors and ECM workhand-in-hand to control cell form and function.Cell binding to ECM activates synthesis of thissignaling substrate, however, in the absence ofa soluble stimulus, signaling proceeds no fur-ther. Similarly, growth factor binding may acti-vate phospholipase C, yet if no PIP2 substrateis available, there would be no release of down-stream signaling molecules. However, when thecell both adheres to ECM and binds to growthfactors, full activation of downstream signalingcascades occurs and cell behavior is altered.

The important point is that the inositol lipidkinases that mediate the effects of ECM andthe phospholipase C that is activated by growthfactors appear to colocalize within the sameinsoluble cytoskeletal microdomain (i.e., FAC)as integrins and growth factor receptors [McNa-mee et al., 1996; Plopper et al., 1995]. It is thespatial positioning of these signaling compo-nents that facilitates signal integration. ECMand growth factors also interact to control othersignaling mechanisms (e.g., Na1/H1 exchange,expression of growth response genes), but inthis case the integrin and growth factor recep-

tor signaling cascades are distinct and additive[Ingber et al., 1990; Schwartz et al., 1991; Dikeand Ingber, 1996]. Yet, again it is the proximitybetween these different signaling componentsand their colocalization with the FAC that likelyoptimizes signal processing and integration inthese cells.

Importantly, mechanotransduction is directlyoverlayed on top of this solid-state signalingmechanism. Mechanical stresses are not trans-mitted equally across the plasma membrane atall points on the cell surface. Instead, integrinsand other transmembrane adhesion receptorsthat link extracellular attachment scaffolds(ECM, other cells) to the internal cytoskeletonappear to provide preferred paths for transmem-brane mechanical signal transfer [Wang et al.,1993; Yoshida et al., 1996; Maniotis et al., 1997;Potard et al., 1997]. The finding that mechani-cal signals also converge on the FAC and influ-ence the activity of many of the signaling mol-ecules that are immobilized on the cytoskeletalbackbone of this complex, raised the possibilitythat signals from ECM, growth factors, andmechanical distortion may be integrated di-rectly within the FAC at the site of integrinbinding [Ingber, 1991, 1997; Chicurel et al.,1998a]. In fact, recent studies show that cellstructure, biochemistry, and signal transduc-tion differ depending on the level of mechanicalforces balanced across the FAC [Wang etal., 1993; Chen and Grinnell, 1995; Maniotis etal., 1997; Choquet et al., 1997; Chicurel et al.,1998a,b].

In contrast to signaling by soluble agonistsand insoluble ECM components, external me-chanical signals are always imposed on a pre-existing force balance [Ingber, 1991]. All livingcells continually generate mechanical tensionwithin their contractile cytoskeletal microfila-ments and they transmit these forces to all theparts of the cell. Thus, this form of signal trans-duction involves modulation of biochemicalevents by changing the level of stress in thecell. For this reason, the response of the cell toan external stress may vary depending on cellextension and the initial prestress (internaltension) in the cytoskeleton [Wang and Ingber,1994], much like how the quality of a musicaltone varies when one tunes a guitar string.

The FAC and other membrane adhesion com-plexes are not the only sites of signal integra-tion. Because cells use a tension-based sytem ofarchitecture, known as tensegrity, to structure

234 Ingber

themselves [Ingber, 1993, 1998], a local stressapplied to integrins may promote long-rangestructural rearrangements throughout the celland nucleus [Wang et al., 1993; Maniotis et al.,1997]. Coordinated restructuring of the wholecell may help orchestrate the entire cellularresponse to both chemical and mechanical sig-nals. This dependence on cell architecture mayhelp explain how altering the balance of me-chanical forces in a cell and cell shape caninduce it to produce entirely different func-tional outputs (growth vs differentiation vsapoptosis) given the same set of inputs (e.g.,growth factors and ECM) [Chen et al., 1997].

How could this work? How could a mechani-cal distortion alter intracellular biochemistry?The answer comes from molecular biophysics.If the cytoskeletal framework of the cell isabruptly deformed by applying mechanicalstresses, and if the framework does not break,at least a subset of the molecules that comprisethese scaffolds must physically distort. Chang-ing molecular shape or mechanics alters thermo-dynamic and kinetic parameters and thus di-rectly influences biochemistry [Ingber, 1997].An interesting historical point is that the pref-ace of certain biochemistry textbooks publishedin the early part of this century explained thatpressure and volume were to be ignored be-cause it was assumed that all biochemical reac-tions under study were occuring in a test tube(free in solution). However, the preface alsowarned the reader to be aware that real livingsystems would never be understood if viewed asbased on a structureless chemistry and thus,that we must return to address this question inthe future when techniques become available todeal with structural complexity. Unfortunately,this preface was deleted from subsequent edi-tions, so many readers of these basic textbooksfail to realize the fundamental importance ofphysical parameters (local changes in pressureand volume and related stress tensors) on bio-chemical reactions. This link between mechan-ics and chemistry helps to explain how all liv-ing cells respond to mechanical stresses andhow they alter their form and function to bestaccommodate these stresses [Ingber, 1991,1997].

Well, then, how does binding of a receptor‘‘activate’’ intracellular signaling cascades? Theconventional answer is that the transmem-brane protein autophosphorylates or that it in-teracts with a cytoplasmic signaling protein

when it binds to its external ligand. However,the reality is that chemistry is structure. Bind-ing of a growth factor to a small region of theextracellular portion of a large transmembranereceptor protein results in higher-order struc-tural transformations that propagate through-out the length of the molecule and across theplasma membrane. This is possible because theprotein itself is composed of discrete stiff andflexible regions (e.g., a-helices, b-strands, hingeregions) that interact via tensile hydrogen bond-ing to pull themselves into a stable three-dimensional form. This dependence of the struc-tural stability of individual molecules oninternal prestress, continuous tension, and lo-cal compression is characteristic of tensegrityarchitecture, which also guides the organiza-tion of living cells and tissues [Ingber, 1998].Because of tensegrity, a local stress induced bygrowth factor binding to its receptor results inglobal structural rearrangements throughoutthe membrane protein molecule. These changesin molecular shape and mechanics (e.g., stiff-ness, vibration mode) may result in exposure ofpreviously sequestered catalytic regions withinthe cytoplasmic portion of the receptor mol-ecule or changes in its binding affinity for differ-ent cytoplasmic components. In this manner, astructural remodeling cascade is triggered in-side the cell and biochemical changes result.

Another example of how chemistry is struc-ture can be seen in signaling based on proteintyrosine phosphorylation, perhaps the classicparadigm of signal transduction. The conven-tional approach is to view this as an on/offsignal and to focus on the protein sequencesthat mediate these events. However, from astructural or biophysical perspective, the keysignaling event is a change in molecular me-chanics. The addition of a phosphorylation moi-ety to a tyrosine group changes molecular flex-ibility and shape (e.g., from globular to linear);in fact, this specific type of modification is usedto create adhesive substrates for tissue engi-neering with controlled flexibility [Urry, 1992].I would suggest that similar cascades of molecu-lar restructuring events occur when tyrosinephosphorylation signaling pathways are acti-vated in living cells. Furthermore, it is likelythat it is through these coordinated changes inmolecular mechanics and resulting alterationsin the flexibility and architecture of larger ten-sionally coupled supramolecular scaffolds thatmany different signaling pathways can be simul-

In Search of Cellular Control 235

taneously orchestrated, even though they existin different locations inside the FAC.

It is important to note that this form of mecha-nochemical regulation is not limited to the FAC[Chicurel et al., 1998a]. Similar integration mayoccur at lateral cell-cell junctions which alsoexhibit high levels of cytoskeletal-associatedsignaling molecules [Yamada and Geiger, 1997],as well as efficient transmembrane mechanicalforce transfer [Yoshida et al., 1996; Potard etal., 1997]. Furthermore, solid-state biochemis-try also plays a key role in other types of meta-bolic processing events (i.e., other than signaltransduction) and in different locations in thecell. For example, many of the biochemical reac-tions involved in DNA synthesis, RNA process-ing, protein synthesis, and glycolysis also ap-pear to proceed in a solid-state along insolublecytoskeletal scaffolds within both the cyto-plasm and the nucleus [Ingber, 1993b]. In thecase of protein synthesis, intermediates in theprocess, aminoacyl-tRNAs, are channeled di-rectly from the aminoacyl-tRNA synthetases tothe elongation factor to the ribosomes withoutdissociating into the cellular fluid [Stapulionisand Deutscher, 1995]. Solid-state biochemistryand enzymatic channeling may help explainthe high level of efficiency of biochemical reac-tions that is observed in living cells but oftencannot be mimicked in a test tube. It also pro-vides a mechanism by which cell shape modula-tion and associated cytoskeletal distortion canresult in changes in cellular biochemistry andgene expression [Ingber, 1997]. Most impor-tantly, it emphasizes how we will never be ableto fully understand cellular control if we onlyanalyze individual molecules in isolation.

In summary, the reductionist experimentalapproach that dominates current thinking inthe field of molecular cell biology clearly hashad a major positive impact in terms of increas-ing our knowledge base. It also has greatlyfacilitated drug discovery. However, the ques-tion remains: is this the correct paradigm forthe future? As briefly discussed above, it isunlikely that this solution chemistry approachcan provide all the answers we are seeking.Thus, the new challenge is to develop methods,imaging techniques and experimental ap-proaches to study critical biochemical processeswithin the complexity of the living cell and tocontrollably vary cell structure and mechanics.A look to the past shows that many of the majorbreakthroughs were facilitated by the develop-

ment of new theories (and methods to test them)that changed the frame of reference that previ-ously dominated the field. The recent conver-gence of molecular cell biology with bioengineer-ing has attracted new types of investigators tothis field and has led to the generation of en-tirely new experimental and analytical ap-proaches [Ingber, 1993b]. As these fields mergewith computer science and informatics, newadvances will likely be accelerated.

Understanding how biochemistry proceedswithin the context of the structural complexityof living cells may lead to development of en-tirely new approaches in medicine, engineer-ing, and materials science that could makefunctional genomics appear primitive by com-parison. This will be a brave new world inwhich science and technology will be driven bybiomimetics rather than genomics, by struc-ture and mechanics rather than chemistryalone, and by fabrication strategies that mimicbiological mechanisms of self-assembly andstructural remodeling. The challenge for thenext millenium is to choose our first steps wisely.

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