Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells.

Engineered proteins must be phenotypically selected for function in the appropriate physiological context. Here, we present a versatile approach that allows generating panels of mammalian cells that express diversified heterologous protein libraries in the cytosol or subcellular compartments under stable conditions and in a single-variant-per-cell manner. To this end we adapt CRISPR/Cas9 editing technology to diversify targeted stretches of a protein of interest in situ. We demonstrate the utility of the approach by in situ engineering and intra-lysosome specific selection of an extremely pH-resistant long Stokes shift red fluorescent protein variant. Tailoring properties to specific conditions of cellular sub-compartments or organelles of mammalian cells can be an important asset to optimize various proteins, protein-based tools, and biosensors for distinct functions.

Imaging-Based Screening Platform Assists Protein Engineering.

Protein engineering involves generating and screening large numbers of variants for desired properties. While modern DNA technology has made it easy to create protein diversity on the DNA level, the selection and validation of candidate proteins from large libraries remains a challenge. We built a screening platform that integrates high-quality fluorescence-based image analysis and robotic picking of bacterial colonies. It allows tracking each individual colony in a large population and collecting quantitative information on library composition during the protein evolution process. We demonstrate the power of the screening platform by optimizing a dim far-red-emitting fluorescent protein whose brightness increased several fold using iterative cycles of mutagenesis and platform-based screening. The resulting protein variant mCarmine is useful for imaging cells and structures within live tissue as well as for molecular tagging. Overall, the platform presented provides powerful, flexible, and low-cost instrumentation to accelerate many fluorescence-based protein optimization projects.

Regulation of Shootin1 Gene Expression Involves NGF-induced Alternative Splicing during Neuronal Differentiation of PC12 Cells.

Shootin1 is a protein involved in neuronal polarization, and has been shown to be a key molecule for the positive/negative feedback loop for axon induction required during neuronal symmetry breaking. To better understand the molecular basis of shootin1 dynamics, we analysed the regulatory pathways and the expressional status of shootin1 gene during NGF-induced neuronal differentiation. We demonstrated that the isoform-1 and isoform-2 of shootin1 is differentially expressed during neuronal differentiation. By blocking individual downstream pathways of NGF signalling, we found that PI3K/Akt pathway plays a major role in the expression of shootin1 isoform-2. Western blot and RT-PCR results showed that the isoform-1 of shootin1 is constitutively expressed, while the isoform-2 is expressed in a manner that is strictly dependent on NGF-stimulation. Isoform-specific RT-PCR results demonstrated that the differential expression of the isoform-1 and isoform-2 of shootin1 is a consequence of alternative splicing of shootin1 pre-mRNA, in response to NGF-signalling. Collectively these findings provide the first information on the molecular mechanisms regulating the expression of shootin1 gene and represent the first example of NGF-induced alternative splicing process that has a regulatory role in neuritogenesis.

4,5-dianilinophtalimide protects neuroendocrine cells against serum deprivation-induced stress and apoptosis.

OBJECTIVE: The aim of this study was to reveal the effects of 4,5-dianilinophthalimide (DAPH), which inhibits amyloid ß fibrillization, against serum deprivation (SD)-induced apoptosis and the possible mechanisms in differentiated PC12 neuron cells. METHODS: Firstly, we evaluated whether DAPH protects cell viability exposed to SD by MTT assay. Next, we examined the changes of phospho-p38 MAPK (Thr180/Tyr182), phospho-HSP27 (Ser82), phospho-c-JUN (Ser73) and cleaved-CASP3 (Asp175) profiles by immunoblotting, in PC12 cells exposed to SD. Intracellular reactive oxygen species (ROS) level was also measured. RESULTS: SD induced apoptosis accompanied by up-regulation of phospho-p38 MAPK (Thr180/Tyr182), phospho-HSP27 (Ser82), phospho-c-JUN (Ser73), cleaved-CASP3 (Asp175) and intracellular ROS content. Co-treatment with non-toxic doses of DAPH prevented apoptosis by the attenuation of activated proteins and reduction of ROS level. These results suggest that serum deprivation-induced apoptosis inhibited by DAPH administration. CONCLUSION: We have provided for the first evidence that DAPH has a neuroprotective effect on SD-caused stress, probably via contributing the re-establishment of redox homeostasis.

Fiber laser-microscope system for femtosecond photodisruption of biological samples.

We report on the development of a ultrafast fiber laser-microscope system for femtosecond photodisruption of biological targets. A mode-locked Yb-fiber laser oscillator generates few-nJ pulses at 32.7 MHz repetition rate, amplified up to ∼125 nJ at 1030 nm. Following dechirping in a grating compressor, ∼240 fs-long pulses are delivered to the sample through a diffraction-limited microscope, which allows real-time imaging and control. The laser can generate arbitrary pulse patterns, formed by two acousto-optic modulators (AOM) controlled by a custom-developed field-programmable gate array (FPGA) controller. This capability opens the route to fine optimization of the ablation processes and management of thermal effects. Sample position, exposure time and imaging are all computerized. The capability of the system to perform femtosecond photodisruption is demonstrated through experiments on tissue and individual cells.

Texturing of titanium (Ti6Al4V) medical implant surfaces with MHz-repetition-rate femtosecond and picosecond Yb-doped fiber lasers.

We propose and demonstrate the use of short pulsed fiber lasers in surface texturing using MHz-repetition-rate, microjoule- and sub-microjoule-energy pulses. Texturing of titanium-based (Ti6Al4V) dental implant surfaces is achieved using femtosecond, picosecond and (for comparison) nanosecond pulses with the aim of controlling attachment of human cells onto the surface. Femtosecond and picosecond pulses yield similar results in the creation of micron-scale textures with greatly reduced or no thermal heat effects, whereas nanosecond pulses result in strong thermal effects. Various surface textures are created with excellent uniformity and repeatability on a desired portion of the surface. The effects of the surface texturing on the attachment and proliferation of cells are characterized under cell culture conditions. Our data indicate that picosecond-pulsed laser modification can be utilized effectively in low-cost laser surface engineering of medical implants, where different areas on the surface can be made cell-attachment friendly or hostile through the use of different patterns.