Reductionism Is Dead: Long Live Reductionism! Systems Modeling Needs Reductionist Experiments.

In the era of systems biology and bigdata, reductionism is often viewed aspasse´. But it can come in handy, espe-cially when you have a theory to test.Despite decades of study, the ﬁeld ofbiochemical signal transduction is stillrife with mechanistic controversies,and one reason is that even systemsof a few interacting components canexhibit complex and nonintuitive be-havior.Manymechanismsareproposed,but most tests are confounded by thecomplexityofthe underlyingbiologicalsystems in which they are tested.Agood,andperhapsevennecessaryﬁrststeptotestinganyproposedmechanism,is to recapitulate itin a minimalbiolog-ical model that removes as many of theconfounding variables as possible, suchas a transfected cell. Mukhopadhyayet al. (1) followed exactly such anapproach to testing their hypotheticalmechanism for ultrasensitivity in T cellreceptor signaling.A longstanding question in immunereceptor signaling is why the T cellantigen receptor (TCR; Fig. 1)issocomplex, and in particular, why itscytoplasmic domains contain such alarge number of tyrosine phosphoryla-tion sites (2). In fact, multisite phos-phorylation is a common feature ofmany receptor signaling systems, butthe T cell receptor is notable for thelarge total number of sites—20—andthat these sites occur as pairs in highlyconserved regions called tyrosine-based activation motifs (ITAMs) (3).The TCR has a total of 10 ITAMs,six of which occur in the subunitcomprising the z-chain homodimer.This high degree of apparent redun-dancy has motivated numerous investi-gations, but the functional role of thehigh ITAM multiplicity of the TCR re-mains an open question (4). Severalexperimental approaches have beenemployed to examine the role of TCRITAMs in vivo during T cell develop-ment, but these studies have oftenyielded contradictory results (5,6).Takingamoremechanisticapproach,Dusheketal.(7)proposedthatmultisitephosphorylation could enable the TCRsystem to generate sharp switchlike(or ultrasensitive) responses to inputs.Recent work has shown that TCRsignaling can exhibit sharp responsethresholds to input signals, but knownmechanisms for generating such beha-vior involve components considerablydownstream of the receptor itself (8).Dushek et al. (7) proposed a novelmechanism that incorporated diffusion-limited interactions among membrane-associated kinases and phosphatasesand their multisite substrate. The TCRz-dimer subunit with its 12 tyrosinesseemed like a natural system in whichto test their model, and they proposedexperiments based on a previouslydeveloped transfectant system ( 9).Not long after, James and Vale (10)used a different transfectant system toinvestigate the mechanism of T cellactivation, demonstrating that key fea-tures such as synapse formation andrecruitment of the cytosolic kinaseZAP-70 could be recapitulated in anonimmune cell with a minimal num-ber of transfected components. Sub-sequently, Hui and Vale (11) wentfurther along a reductionist path toinvestigate T cell signaling mecha-nisms, using a fully in vitro systemcomposed of puriﬁed proteins on lipo-somes to characterize the dynamics ofearly phosphorylation events involvingthe TCR z-chain and CD45 and the ki-nases Lck, which is primarily respon-sible for z-chain phosphorylation, andCsk, which regulates Lck. Althoughboth of these studies addressed mecha-nistic questions, they did not present amathematical framework with whichto analyze and further interpret theﬁndings.In the meantime, Mukhopadhyayet al. (12) further developed theircomputational model of multisiteTCR phosphorylation building on twokey hypotheses, supported by earlierexperimental work: (1) that z-ITAMsare phosphorylated in a speciﬁcsequence and (2) that ZAP-70 exhibitsincreasing afﬁnity for each ITAM inthe sequence. Together, these wereshown to be necessary and sufﬁcient