Nonlinearity of Mechanochemical Motions in Motor Proteins

The assumption of linear response of protein molecules to thermal noise or structural perturbations, such as ligand binding or detachment, is broadly used in the studies of protein dynamics. Conformational motions in proteins are traditionally analyzed in terms of normal modes and experimental data on thermal fluctuations in such macromolecules is also usually interpreted in terms of the excitation of normal modes. We have chosen two important protein motors - myosin V and kinesin KIF1A - and performed numerical investigations of their conformational relaxation properties within the coarse-grained elastic network approximation. We have found that the linearity assumption is deficient for ligand-induced conformational motions and can even be violated for characteristic thermal fluctuations. The deficiency is particularly pronounced in KIF1A where the normal mode description fails completely in describing functional mechanochemical motions. These results indicate that important assumptions of the theory of protein dynamics may need to be reconsidered. Neither a single normal mode, nor a superposition of such modes yield an approximation of strongly nonlinear dynamics.Comment: 10 pages, 6 figure

Covert Genetic Selections to Optimize Phenotypes

In many high complexity systems (cells, organisms, institutions, societies, economies, etc.), it is unclear which components should be regulated to affect overall performance. To identify and prioritize molecular targets which impact cellular phenotypes, we have developed a selection procedure (“SPI”–single promoting/inhibiting target identification) which monitors the abundance of ectopic cDNAs. We have used this approach to identify growth regulators. For this purpose, complex pools of S. cerevisiae cDNA transformants were established and we quantitated the evolution of the spectrum of cDNAs which was initially present. These data emphasized the importance of translation initiation and ER-Golgi traffic for growth. SPI provides functional insight into the stability of cellular phenotypes under circumstances in which established genetic approaches cannot be implemented. It provides a functional “synthetic genetic signature” for each state of the cell (i.e. genotype and environment) by surveying complex genetic libraries, and does not require specialized arrays of cDNAs/shRNAs, deletion strains, direct assessment of clonal growth or even a conditional phenotype. Moreover, it establishes a hierarchy of importance of those targets which can contribute, either positively or negatively, to modify the prevailing phenotype. Extensions of these proof-of-principle experiments to other cell types should provide a novel and powerful approach to analyze multiple aspects of the basic biology of yeast and animal cells as well as clinically-relevant issues

Chlamydia trachomatis Co-opts GBF1 and CERT to Acquire Host Sphingomyelin for Distinct Roles during Intracellular Development

The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche

Roles of Dynein and Dynactin in Early Endosome Dynamics Revealed Using Automated Tracking and Global Analysis

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85–98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150Glued to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150Glued remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms−1 that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes

Data for: Regioselective hydrogenolysis of alga-derived squalane over silica-supported ruthenium-vanadium catalyst

- Detail of XAS measurement and analysis - Detailed data of Table 1 - Detailed data of Table 2 - Detailed data of Figure 2 - Detailed data of Figure 3 - Detailed data of Figure 4 - TG-DTA profiles of Ru-VOx/SiO2 catalyst after hydrogenolysis of squalane. - XRD patterns of catalysts without pretreatment - Enlarged view of Figure 6 - XRD patterns of the catalysts after reuse test