Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

Directed cell migration on fibronectin gradients: effect of gradient slope

The migration of human microvascular endothelial cells (hMEC) was measured on a range of fibronectin gradient slopes. hMEC drift speed increased with increasing gradient slope with no concurrent change in cellular persistence time or random cell speed. The frequency of discrete cellular motion in the gradient direction increased with gradient slope. Morphological polarization of cells on the gradients is also characterized and correlated with cellular drift speed. These experiments present the first demonstration of cellular response to changing haptotactic gradient slope using an in vitro system for the quantitative study of cell migration.     

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-d-erythro-sphingosine (C₁₆Cer), N-palmitoyl-d-erythro-sphingosylphosphorylcholine (C₁₆SM) and cholesterol. The amounts of DOPC, sum of C₁₆Cer and C₁₆SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C₁₆Cer/(C₁₆Cer + C₁₆SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C₁₆Cer (X = 0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X = 0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X ≥ 0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

Morphogenesis as a macroscopic self-organizing process

We start from reviewing different epistemological constructions used for explaining morphogenesis. Among them, we explore the explanatory power of a law-centered approach which includes top-down causation and the basic concepts of a self-organization theory. Within such a framework, we discuss the morphomechanical models based upon the presumption of feedbacks between mechanical stresses imposed onto a given embryo part from outside and those generated within the latter as a kind of active response. A number of elementary morphogenetic events demonstrating that these feedbacks are directed towards hyper-restoration (restoration with an overshoot) of the initial state of mechanical stresses are described. Moreover, we show that these reactions are bound together into the larger scale feedbacks. That permits to suggest a reconstruction of morphogenetic successions in early Metazoan development concentrated around two main archetypes distinguished by the blastopores geometry. The perspectives of applying the same approach to cell differentiation are outlined. By discussing the problem of positional information we suggest that the developmental pathway of a given embryo part depends upon its preceded deformations and the corresponding mechanical stresses rather than upon its static position at any moment of development.     

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Lateral organization of biological membranes is frequently studied using fluorescence microscopy. One of the most widely used probes for these studies is 2-dimethylamino-6-lauroylnaphthalene (laurdan). The fluorescence of this probe is sensitive to the environment polarity, and thus laurdan reports the local penetration of water when inserted in membranes. Unfortunately, this probe can only be used under two-photon excitation due to its low photostability. This is a very important limitation, because there are not too many laboratories with capability for two-photon microscopy. In this work, we explored the performance of 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), a carboxyl-modified version of laurdan, for imaging biological membranes using a conventional confocal microscopy setup. We acquired generalized polarization (GP) images of C-laurdan inserted in giant unillamelar vesicles composed of binary mixtures of lipids and verified that the probe allows observing the coexistence of different phases. We also tested the performance of the probe for measurement with living cells and registered GP images of melanophore cells labeled with C-laurdan in which we could observe highly ordered regions such as filopodia. These findings show that C-laurdan can be successfully employed for studies of membrane lateral organization using a conventional confocal microscope and can open the possibility of studying a wide variety of membrane-related processes.     

Synergistic Binding of the Phosphorylated S233- and S259-Binding Sites of C-RAF to One 14-3-3ζ Dimer

C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen‐activated protein kinase/extracellular signal‐regulated kinase)-ERK (extracellular signal‐regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single‐site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single‐site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins.     

Structural Interactions between Lipids, Water and S1-S4 Voltage-Sensing Domains

Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here, we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains and that these interactions have lifetimes on the timescale of ≤ 10^− 3 s. Arg residues within S1-S4 domains are well hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid headgroups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane yet are well hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage.     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM642-1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM642-1290解旋酶与dsDNA的结合和解链特性．结果表明,BLM642-1290解旋酶与dsDNA的结合和解链和dsDNA3’末端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA3’末端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM642-1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3’末端ssDNA的dsDNA．推测BLM642-1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

多聚亚基蛋白质在固-液界面团聚的测量与表征

以血红蛋白和乙肝表面抗原(HBsAg)为模型,联用双偏振光干涉分析仪(DPI)和原子力显微镜(AFM)研究在固-液界面上的吸附行为.结果发现:当溶液环境中血红蛋白的质量浓度为1.08 mg/mL时,测得在硅羟基表面吸附的平均厚度为3.54 nm,蛋白颗粒直径达60～ 150 nm,远高于血红蛋白的分子尺寸,说明血红蛋白在固-液界面上发生团聚.当HBsAg质量浓度为0.67 mg/mL时,测得在硅羟基表面及DEAE琼脂糖表面吸附的平均厚度分别为1.06和23.69 nm,表面吸附的蛋白颗粒直径分别为288～401和520 ～ 971 nm,是单分子HBsAg尺寸的13.1～44.1倍,表明在模型蛋白表面都形成了大的团聚体.