Response of rigor cross-bridges to stretch detected by fluorescence lifetime imaging microscopy of myosin essential light chain in skeletal muscle fibers

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.     

A two-step search and run response to gradients shapes leukocyte navigation in vivo

Migrating cells must interpret chemical gradients to guide themselves within tissues. A long-held principle is that gradients guide cells via reorientation of leading-edge protrusions. However, recent evidence indicates that protrusions can be dispensable for locomotion in some contexts, raising questions about how cells interpret endogenous gradients in vivo and whether other mechanisms are involved. Using laser wound assays in zebrafish to elicit acute endogenous gradients and quantitative analyses, we demonstrate a two-stage process for leukocyte chemotaxis in vivo: first a “search” phase, with stimulation of actin networks at the leading edge, cell deceleration, and turning. This is followed by a “run” phase, with fast actin flows, cell acceleration, and persistence. When actin dynamics are perturbed, cells fail to resolve the gradient, suggesting that pure spatial sensing of the gradient is insufficient for navigation. Our data suggest that cell contractility and actin flows provide memory for temporal sensing, while expansion of the leading edge serves to enhance gradient sampling.     

Positive genetic interactors of HMG2 identify a new set of genetic perturbations for improving sesquiterpene production in Saccharomyces cerevisiae

Background

Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood.

Results

We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins.

Conclusions

This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.     

A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips

The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures 相似文献   

Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines

Polycystic Kidney Disease (PKD), which is attributable to mutations in the PKD1 and PKD2 genes encoding polycystin‐1 (PC1) and polycystin‐2 (PC2) respectively, shares common cellular defects with cancer, such as uncontrolled cell proliferation, abnormal differentiation and increased apoptosis. Interestingly, PC1 regulates many signalling pathways including Jak/STAT, mTOR, Wnt, AP‐1 and calcineurin‐NFAT which are also used by cancer cells for sending signals that will allow them to acquire and maintain malignant phenotypes. Nevertheless, the molecular relationship between polycystins and cancer is unknown. In this study, we investigated the role of PC1 in cancer biology using glioblastoma (GOS3), prostate (PC3), breast (MCF7), lung (A549) and colorectal (HT29) cancer cell lines. Our in vitro results propose that PC1 promotes cell migration in GOS3 cells and suppresses cell migration in A549 cells. In addition, PC1 enhances cell proliferation in GOS3 cells but inhibits it in MCF7, A549 and HT29 cells. We also found that PC1 up‐regulates mTOR signalling and down‐regulates Jak signalling in GOS3 cells, while it up‐regulates mTOR signalling in PC3 and HT29 cells. Together, our study suggests that PC1 modulates cell proliferation and migration and interacts with mTOR and Jak signalling pathways in different cancer cell lines. Understanding the molecular details of how polycystins are associated with cancer may lead to the identification of new players in this devastating disease.     

The <Emphasis Type="Italic">Bactrocera dorsalis</Emphasis> species complex: comparative cytogenetic analysis in support of Sterile Insect Technique applications

Background

The Bactrocera dorsalis species complex currently harbors approximately 90 different members. The species complex has undergone many revisions in the past decades, and there is still an ongoing debate about the species limits. The availability of a variety of tools and approaches, such as molecular-genomic and cytogenetic analyses, are expected to shed light on the rather complicated issues of species complexes and incipient speciation. The clarification of genetic relationships among the different members of this complex is a prerequisite for the rational application of sterile insect technique (SIT) approaches for population control.

Results

Colonies established in the Insect Pest Control Laboratory (IPCL) (Seibersdorf, Vienna), representing five of the main economic important members of the Bactrocera dorsalis complex were cytologically characterized. The taxa under study were B. dorsalis s.s., B. philippinensis, B. papayae, B. invadens and B. carambolae. Mitotic and polytene chromosome analyses did not reveal any chromosomal characteristics that could be used to distinguish between the investigated members of the B. dorsalis complex. Therefore, their polytene chromosomes can be regarded as homosequential with the reference maps of B. dorsalis s.s.. In situ hybridization of six genes further supported the proposed homosequentiallity of the chromosomes of these specific members of the complex.

Conclusions

The present analysis supports that the polytene chromosomes of the five taxa under study are homosequential. Therefore, the use of the available polytene chromosome maps for B. dorsalis s.s. as reference maps for all these five biological entities is proposed. Present data provide important insight in the genetic relationships among the different members of the B. dorsalis complex, and, along with other studies in the field, can facilitate SIT applications targeting this complex. Moreover, the availability of 'universal' reference polytene chromosome maps for members of the complex, along with the documented application of in situ hybridization, can facilitate ongoing and future genome projects in this complex.

     

Structure-property evaluation of thermally and chemically gelling injectable hydrogels for tissue engineering

The impact of synthesis and solution formulation parameters on the swelling and mechanical properties of a novel class of thermally and chemically gelling hydrogels combining poly(N-isopropylacrylamide)-based thermogelling macromers containing pendant epoxy rings with polyamidoamine-based hydrophilic and degradable diamine cross-linking macromers was evaluated. Through variation of network hydrophilicity and capacity for chain rearrangement, the often problematic tendency of thermogelling hydrogels to undergo significant syneresis was addressed. The demonstrated ability to tune postformation dimensional stability easily at both the synthesis and formulation stages represents a significant novel contribution toward efforts to utilize poly(N-isopropylacrylamide)-based polymers as injectable biomaterials. Furthermore, the cytocompatibility of the hydrogel system under relevant conditions was established while demonstrating time- and dose-dependent cytotoxicity at high solution osmolality. Such injectable in situ forming degradable hydrogels with tunable water content are promising candidates for many tissue-engineering applications, particularly for cell delivery to promote rapid tissue regeneration in non-load-bearing defects.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

Evaluation of the environmental stress index (ESI) for the southern hemisphere

The environmental stress index (ESI), constructed from ambient temperature, relative humidity, and solar radiation, was further evaluated from databases collected in Israel and New Zealand. High correlations were found between ESI and WBGT (R²=0.959 and R²=0.973 for Israel and New Zealand, respectively). However, for the New Zealand database, residuals were not distributed symmetrically around the zero line, which might be due to the difference in the global radiation spectrum. The ultraviolet radiation measurements were significantly higher in New Zealand than in Israel. As a consequence, a correction factor might be needed for ESI to be used in the southern hemisphere.