Reversible optogenetic control of kinase activity during differentiation and embryonic development.

A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

Bulk-state and single-particle imaging are central to understanding carbon dot photo-physics and elucidating the effects of precursor composition and reaction temperature.

Carbon dots have garnered attention for their strong multi-color luminescence properties and unprecedented biocompatibility. Despite significant progress in the recent past, a fundamental understanding of their photoluminescence and structure-properties relationships, especially at the bulk vs. single-particle level, has not been well established. Here we present a comparative study of bulk- and single-particle properties as a function of precursor composition and reaction temperature. The synthesis and characterization of multicolored inherently functionalized carbon dots were achieved from a variety of carbon sources, and at synthesis temperatures of 150 °C and 200 °C. Solvothermal synthesis at 200 °C led to quantum yields as high as 86%, smaller particle sizes, and a narrowed fluorescence emission, while synthesis at 150 °C resulted in a greater UV-visible absorbance, increase in nanoparticle stability, red-shifted fluorescence, and a greater resistance to bulk photobleaching. These results suggest the potential for synthesis temperature to be utilized as a simple tool for modulating carbon dot photophysical properties. Single-particle imaging resolved that particle brightness was determined by both the instantaneous intensity and the on-time duty cycle. Increasing the synthesis temperature caused an enhancement in blinking frequency, which led to an increase in on-time duty cycle in three out of four precursors.

Multiplex gene removal by two-step polymerase chain reactions.

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.

Optogenetic Delineation of Receptor Tyrosine Kinase Subcircuits in PC12 Cell Differentiation.

Nerve growth factor elicits signaling outcomes by interacting with both its high-affinity receptor, TrkA, and its low-affinity receptor, p75NTR. Although these two receptors can regulate distinct cellular outcomes, they both activate the extracellular-signal-regulated kinase pathway upon nerve growth factor stimulation. To delineate TrkA subcircuits in PC12 cell differentiation, we developed an optogenetic system whereby light was used to specifically activate TrkA signaling in the absence of nerve growth factor. By using tyrosine mutants of the optogenetic TrkA in combination with pathway-specific pharmacological inhibition, we find that Y490 and Y785 each contributes to PC12 cell differentiation through the extracellular-signal-regulated kinase pathway in an additive manner. Optogenetic activation of TrkA eliminates the confounding effect of p75NTR and other potential off-target effects of the ligand. This approach can be generalized for the mechanistic study of other receptor-mediated signaling pathways.

Carbon dots with induced surface oxidation permits imaging at single-particle level for intracellular studies.

For robust single particle optical detection, a high sensitivity in photoluminescence (PL) of Carbon Dots (CDs) must be achieved. PL sensitivity can be successfully correlated with their surface chemistry but requires high synthetic control without altering their basic surface properties. Here we describe conditions for the controlled synthesis of CDs that resulted in a PL sensitivity at the single-particle level. We report that a stoichiometric catalyst N-methyl morpholine-N-oxide (NMMO) can be used as a 'sacrificial' single additive to aid nanoscale surface oxidation. A 24 h NMMO-mediated oxidation increased coverage of oxidized nanoscale surface 3% to 20.9%. NMMO-oxidized CDs (CD-NMMOs) display superior particle brightness, as evidenced by the increase of light absorbance and an enhancement of quantum yield which is characterized by a series of physicochemical and biophysical experiments. We also demonstrate that CD-NMMOs is well suited for intracellular and single-particle imaging.

Drive the Car(go)s-New Modalities to Control Cargo Trafficking in Live Cells.

Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.

Applications of Optobiology in Intact Cells and Multicellular Organisms.

Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Polymerase chain reaction-based gene removal from plasmids.

This data article contains supplementary figures and methods to the research article entitled, "Multiplex gene removal by two-step polymerase chain reactions" (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033), which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116). Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.