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Cell membranes mainly consist of lipid bilayers with an actively regulated composition. The underlying processes are still poorly understood, in particular, how the hundreds of components are controlled. Cholesterol has been found to correlate with phospholipid saturation for reasons that remain unclear. To better understand the link between cell membrane regulation and chemical composition, we establish a computational framework based on chemical reaction networks, resulting in multiple semigrand canonical ensembles. By running computer simulations, we show that regulating the chemical potential of lipid species is sufficient to reproduce the experimentally observed increase in acyl tail saturation with added cholesterol. Our model proposes a different picture of lipid regulation in which components can be regulated passively instead of actively. In this picture, phospholipid acyl tail composition naturally adapts to added molecules such as cholesterol or proteins. A comparison between regulated membranes with commonly studied ternary model membranes shows stark differences: for instance, correlation lengths and viscosities observed are independent of lipid chemical affinity.

The human CDK-activating kinase (CAK), composed of CDK7, cyclin H, and MAT1, is involved in the control of transcription initiation and the cell cycle. Because of these activities, it has been identified as a promising target for cancer chemotherapy. A number of CDK7 inhibitors have entered clinical trials, among them ICEC0942 (also known as CT7001). Structural information can aid in improving the affinity and specificity of such drugs or drug candidates, reducing side effects in patients. Here, we have determined the structure of the human CAK in complex with ICEC0942 at 2.5 A-resolution using cryogenic electron microscopy. Our structure reveals conformational differences of ICEC0942 compared with previous X-ray crystal structures of the CDK2-bound complex, and highlights the critical ability of cryogenic electron microscopy to resolve structures of drug-bound protein complexes without the need to crystalize the protein target.

Catch bonds are cellular receptor-ligand pairs whose lifetime, counterintuitively, increases with increasing load. Although their existence was initially pure theoretical speculation, recent years have seen several experimental demonstrations of catch-bond behavior in biologically relevant and functional protein-protein bonds. Particularly notable among these established catch-bond formers is the integrin α5β1, the primary receptor for fibronectin and, as such, a crucial determinant for the characteristics of the mechanical coupling between cell and matrix. In this work, we explore the implications of single catch-bond characteristics for the behavior of a load-sharing cluster of such bonds: These clusters are shown to possess a regime of strengthening with increasing applied force, similar to the manner in which focal adhesions become selectively reinforced. Our results may shed new light on the fundamental processes that allow cells to sense and respond to the mechanical properties of their environment and in particular show how single focal adhesions may act, autonomously, as local rigidity sensors.

Eukaryotes build their complexity by means of differential gene expression, often passed from mother to daughter cell in the form of another level of genetic coding called epigenetic marking. This selective silencing of parts of the genome is presumably carried out by specific aspects of the structure of the chromatin that makes up the chromosomes. How is this achieved?The fundamental particle of chromatin structure is a self-assembled complex of basic histone proteins wrapped by approximately two turns of DNA (1xTwenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Kornberg, R.D. and Lorch, Y. Cell. 1999; 98: 285–294Abstract | Full Text | Full Text PDF | PubMed | Scopus (1124)See all References(1). This unit called the “nucleosome” is the building block in a structure that compacts DNA of lengths on the order of meters into an ∼10-μm diameter cell nucleus. This packaging also acts to repress gene expression. Promoter sites tied up in nucleosomes are not easily accessible to factors that initiate transcription. Nucleosome density and folding of the chromatin both serve to control transcription, and special “remodeling factors” are needed to change chromatin structure and expose promoter sequences (2xChromatin higher order folding: wrapping up transcription. Horn, P.J. and Peterson, C.L. Science. 2002; 297: 1824–1827Crossref | PubMed | Scopus (284)See all References(2). The structure of chromatin is highly dynamic, changing with the metabolic activity of the cell (3xVisualizing chromatin dynamics in interphase nuclei. Gasser, S.M. Science. 2002; 296: 1412–1416Crossref | PubMed | Scopus (229)See all References(3).A great deal is known about specific biochemical activities that alter chromatin structure and gene expression (4xChromatin goes global. Berger, S.L. and Felsenfeld, G. Mol. Cell. 2001; 8: 263–268Abstract | Full Text | Full Text PDF | PubMed | Scopus (38)See all References(4), but one can not help wondering whether there is some broad underlying polymer physics at work. The article by Zinchenko, Luckel, and Yoshikawa (5xTranscription of giant DNA complexed with cationic nanoparticles as a simple model of chromatin. Zinchenko, A.A., Luckel, F., and Yoshikawa, K. Biophys. J. 2007; 92: 1318–1325Abstract | Full Text | Full Text PDF | PubMed | Scopus (23)See all References(5) in this issue approaches this problem from an entirely different direction. This group has previously studied the physical chemistry of the association between cationic nanoparticles (models of the histone proteins) and DNA, mapping out rather complex phase diagrams as a function of particle size and concentration (6xCompaction of single-chain DNA by histone-inspired nanoparticles. Zinchenko, A.A., Yoshikawa, K., and Baigl, D. Phys. Rev. Lett. 2005; 95: 228101–228104Crossref | PubMed | Scopus (68)See all References(6). The way in which positively charged nanoparticles tie up DNA is not obvious, and mechanisms change dramatically with particle size.The question of what this means for gene expression is addressed in this article(5xTranscription of giant DNA complexed with cationic nanoparticles as a simple model of chromatin. Zinchenko, A.A., Luckel, F., and Yoshikawa, K. Biophys. J. 2007; 92: 1318–1325Abstract | Full Text | Full Text PDF | PubMed | Scopus (23)See all References(5). The authors used fluorescently-labeled UTP to monitor transcriptional activity in vitro. They assembled compacted DNA using the T4 genome incubated with various sizes and concentrations of cationic nanoparticle and observed the fluorescence signal generated when the fluorescent label is released into bulk solution during RNA polymerization. Interestingly, the suppression of transcription does not correlate with the previously measured compaction in a simple way. Only the smallest (10-nm) particles allowed transcription to occur at intermediate loading densities. Larger particles shut transcription down rather abruptly. These smallest particles are comparable to nuclesomes in size, although the details of the DNA wrapping must surely differ considerably between real nucleosomes and these inorganic particles. The energetics have yet to be worked out properly, but presumably the 10-nm particles represent a compromise between reasonably good electrostatic binding and the need for enough mobility to allow rearrangements that permit the passage of RNA polymerase.This work leads one to wonder about other simple physical mechanisms that might come into play. Gene expression is known to be highly localized, with clusters of related genes often located in close physical proximity within the nucleus (7xGene order and dynamic domains. Kosak, S.T. and Grioudine, M. Science. 2004; 306: 644–647Crossref | PubMed | Scopus (97)See all References(7) (which is highly structured, albeit in a dynamic way (3.xVisualizing chromatin dynamics in interphase nuclei. Gasser, S.M. Science. 2002; 296: 1412–1416Crossref | PubMed | Scopus (229)See all References, 7.xGene order and dynamic domains. Kosak, S.T. and Grioudine, M. Science. 2004; 306: 644–647Crossref | PubMed | Scopus (97)See all References)). How might this spatial order be maintained? One intriguing possibility (suggested to me by James Rothman of Columbia University) is that expressed RNA fragments tie up the DNA genome into a well determined three-dimensional structure by introducing crosslinks. Such DNA “Origami” is remarkably robust (8xFolding DNA to create nanoscale shapes and patterns. Rothmunde, P. Nature. 2006; 440: 297–302Crossref | PubMed | Scopus (2300)See all References(8). This is just one other example of what we might learn from what biochemists might consider to be absurdly simple models of the nucleus; but sometimes, simple models can be very informative.

Phospholipid vesicles referred to as li-posomesallowreconstitutionofproteinfunction in the absence of other pro-teins.Liposomeshavebeenwidelyusedin exploring viral fusion proteins and,more recently, proteins that mediateintracellular fusion. An evolutionarilyconserved family of proteins calledSNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptors) is critically important inmembrane and protein trafficking ineukaryotic cells including docking andfusion of synaptic vesicle carryingv-SNARE VAMP with plasma mem-brane carrying t-SNAREs SNAP-25and syntaxin (1). The specific role ofSNAREs in intracellular fusion hasbeen explored by many groups and inmany experimental systems. Howeverthe important question whether pairingof v- and t-SNAREs directly mediatesfusion or acts upstream of it remainsunder debate (1–5). One of the mostimportant arguments that SNAREsmight act as fusogens came fromreports on fusion between SNARE-bearing proteoliposomes (6). Two arti-cles in this and previous issues providenew insights into fusogenic activity ofreconstituted SNAREs (7,8).Do SNAREs represent minimal fu-sion machinery? According to one ofthe conventional definitions, a fusionprotein is a protein that is critical forfusioninabiologicallyrelevantcontextand is sufficient to promote merger ofboth outer and inner leaflets of themembranes in model systems. SNAREssatisfy this definition, as evidenced byfusion reported for SNAREs reconsti-tuted in lipid bilayers (6,9–11) andexpressed at cell surface (12). A stron-gerdefinitionwouldadditionallyrequirethis reconstituted fusion to be com-parable with the biologically relevantone in rates of both lipid and contentmixing under conditions that mimicphysiological conditions; in particular,in dependency onthe lipid andmediumcompositions; and in the numbers ofthe proteins required for fusion. Thesurface density of fusion proteins de-terminesnotonlytheratesoffusionbutalso the observed fusion phenotypes,with fewer proteins required for themerger of outer leaflets (hemifusion)than for complete fusion (10,13).In many earlier articles, SNARE–mediated liposome fusion was studiedatveryhighsurfacedensitiesofSNAREswith protein/lipid ratios of v-SNAREVAMP as high as 1:20–50 (6,10) cor-responding to 300–750 proteins pervesicle. This exceeds v-SNARE densi-ties characteristic for synaptic vesicles,widely quoted as 30–100 v-SNAREsper vesicle (3) but might be as low as10–15v-SNAREs/vesicle(protein/lipid;1:1000) as estimated in Dennisonet al. (8). Two new articles explore thefusogenic activity of proteoliposomeswith SNARE (synaptobrevin) surfacedensityintherangefrom1:950to1:120.Both groups formed proteoliposomesbyadirectreconstitutionmethod,wheredetergent-solubilized SNAREs wereadded to preformed liposomes ratherthan by a comicellization techniquebased on mixing of proteins and lipidsindetergent.Whereasmostoftheearlierwork on SNARE proteoliposomes wasdone on comicellization proteolipo-somes (6,11) but see Lu et al. (10),proteoliposomes prepared by direct re-constitution are much more homoge-neousinsizeandproteincontent(7)andthus are better suited for analysis of theeffects of the surface density of recon-stituted proteins. In contrast to studiesusing proteoliposomes with high sur-face densities of SNAREs (6,10), bothChen et al. and Dennison et al. reportthat at lower protein/lipid ratios of upto 1:120–250, fusion is very slow andinefficient (7,8), and does not exceed4% even after 75 min (8). At theseproteindensities,fusionisnotobservedeven when efficient docking of proteo-liposomes is induced by subfusogenicconcentrations of polyethylenglycol(PEG) (8), indicating that in artificialsystems, a high density of SNAREs isneeded not only to increase dockingprobability but for the actual fusionreaction as well. Interestingly, for thesame average protein density, proteoli-posomes formed by comicellization aremorefusogenic.Thismightbeexplainedby the heterogeneity of these liposomescontaining a fraction of smaller andapparently more fusogenic vesicles thatare enriched in SNAREs (7).At high enough density, SNAREsmediate robust lipid mixing betweenproteoliposomes (7,10), and, even atlower densities, reconstituted SNAREspromote fusion driven by PEG (8). Inbothcases,SNARE-promotedfusionbe-tween liposomes proceeds by the samefusion-through-hemifusion pathway asdiverse biological fusion reactions in-cluding intracellular fusion (13). Basedon the analysis of the effect ofSNARE proteins on the kinetics ofPEG-induced fusion, Dennison et al.(8) conclude that SNAREs promoteformation of a local hemifusion con-nection, referred to as a stalk interme-diate (13). Finding that similar to manyother fusion reactions (13), includingSNARE-dependent fusion betweenyeast vacuoles (5), SNARE-dependentliposome fusion observed at high pro-tein densities is inhibited by lysophos-phatidylcholine (7) further strengthensthis conclusion.Although work on SNARE proteoli-posomes, including the new studies,has already brought important insightsinto the mechanisms of the interactions

To uncover the secrets that lie hidden within the intricate structure of muscle, Ishiwata's group have developed a hybrid system that lies halfway between muscle-fiber and single-molecule studies. They hold a single actin filament at one end by attaching it specifically to a plastic microbead that is held and manipulated using optical tweezers. They then take a muscle myofibril from which the native actin filaments have been specifically removed (by treatment with gelsolin) and position it so that the actin filament can be threaded into the frayed end of the myosin filament lattice. Since the preparation is immersed in an MgATP-containing solution, the actin is pulled in toward the M-line by the myosin heads. This motion is monitored using a videocamera system. Movement of the actin filament can be arrested and even reversed by adjusting the power of the laser tweezer. By stopping the motion at various predetermined positions, the researchers were able to measure the relationship between force and filament overlap. Their results seem to match the classical results obtained from intact muscle fiber preparations: The average force is directly proportional to overlap between actin and myosin filaments and hence is determined by the number of myosin heads available to bind.The results so far seem consistent with our classical understanding of the muscle contractile mechanism but the experiment remains tantalizing as the time response of the recording equipment is currently insufficient to analyze the stochastic noise produced by the relatively small number (∼100) of myosins. In the future, experiments made with increased time resolution should give important additional information and permit more advanced statistical analysis (10xThe principles of the stochastic interpretation of ion-channel mechanisms. Colquhoun, D. and Hawkes, A.G. : 397–482CrossrefSee all References(10). One hopes that such studies will shed light on some of the mechanistic questions raised earlier. We also eagerly await studies of the diverse muscle types that were used early on in our quest to “solve muscle”, for instance, insect flight and scallop muscle fibers. The possibility also exists to mix and match different thin-filament regulatory systems with different myosin thick-filament lattices. The new ability to look at small ensembles of myosins in their native lattice arrangement and also vary the type of actin or myosin used means we can let the frog see the rabbit.

Recent advances in atomic force microscopy allowed globular and membrane proteins to be mechanically unfolded on a single-molecule level. Presented is an extension to the existing force spectroscopy experiments. While unfolding single bacteriorhodopsins from native purple membranes, small oscillation amplitudes (6-9 nm) were supplied to the vertical displacement of the cantilever at a frequency of 3 kHz. The phase and amplitude response of the cantilever-protein system was converted to reveal the elastic (conservative) and viscous (dissipative) contributions to the unfolding process. The elastic response (stiffness) of the extended parts of the protein were in the range of a few tens pN/nm and could be well described by the derivative of the wormlike chain model. Discrete events in the viscous response coincided with the unfolding of single secondary structure elements and were in the range of 1 microNs/m. In addition, these force modulation spectroscopy experiments revealed novel mechanical unfolding intermediates of bacteriorhodopsin. We found that kinks result in a loss of unfolding cooperativity in transmembrane helices. Reconstructing force-distance spectra by the integration of amplitude-distance spectra verified their position, offering a novel approach to detect intermediates during the forced unfolding of single proteins.