Imaging of mRNAs in living cells is an effective approach to elucidate the molecular mechanism of biomolecules and cellular functions. Here we introduce methods to fluorescently label a specific mRNA using antisense oligonucleotide probes or RNA binding proteins. Next, we introduce examples of imaging dynamics of mRNAs in living cells. The first example is the analysis of dynamics of mRNAs in stress granules(SGs). mRNAs in COS7 cells were labeled with a poly(U)22 2'-O-methyl RNA and SGs were formed by arsenite. The analysis of mRNA dynamics using FRAP showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)+ mRNAs. The second example is single-molecule imaging of β-actin mRNAs in a chicken fibroblast. β-Actin mRNAs were labeled with MS2-GFP and their movement was analyzed. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA(DMACRO) at the leading edge was 0.3μm2/s. On the other hand, DMACRO in the perinuclear region was 0.02μm2/s. The destruction of microfilaments with cytochalasin D led to an increase in DMACRO to 0.2μm2/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of β-actin mRNA.
Genetically-Encoded Voltage Indicators (GEVIs) draw neuroscientist's attention since it enables to simultaneously monitor direct electrical activity from lots of neurons, and which is not achievable by using electrodes. However many GEVIs reported so far often confuse users. Actually since each GEVIs has pros and cons, we have to carefully select GEVIs which is the most suitable for one's experiments according to the accurate information. Here, we describe the trend of GEVIs from ion channel based GEVIs reported as prototypes in the early stage to current rhodopsin-fluorescent protein based GEVIs. Also we note the feature of representative GEVIs and which GEVIs should be used for one's experiments.
Nitric oxide(NO) is now recognized as an endogenous signaling molecule involved in various physiological events such as vasodilation. Since NO is a gaseous molecule and has a short half-life due to high chemical reactivity under ambient conditions, NO itself is practically hard to be handled in biological systems. So, NO chemical donors which release NO under physiological conditions are now indispensable chemical tools in NO biological research. Especially, “caged” NO, which releases NO in response to photoirradiation, are advantageous in NO treatment with spatiotemporal control. We have been developed caged NOs, which have unique photochemical reaction mechanisms for NO release, and adopted them to tissue and in vivo experiments. It was demonstrated that some are responsible for visible light and applicable cellular and tissue experiments, and others are responsible to near infrared pulse laser and applicable for in vivo experiments.
Cellular or molecular dynamics and function are continuously changing in vivo. Their properties also vary under the influence of tissue micro-environment. Dynamic information of a cell or a molecule obtained through intravital imaging allows spatio-temporal analysis of various phenomena in a living body. However, a common hurdle in achieving high resolution imaging is the motion artifact which is caused by motion of organs, such as breathing, cardiac contractions, pulsatile blood flow or peristalsis. In this section, we will introduce the techniques for the fluorescence imaging in a living mouse in real time at cellular level resolution.
[Serial] Front line of DDS development in pharmaceutical industries
Exenatide extended-release for injectable suspension, BYDUREON® 2mg, an injectable glucagon-like peptide-1(GLP-1)receptor agonist, has been shown to improve glycemic control with positive effect on body weight in patients with type 2 diabetes. This long acting formulation contains exenatide encapsulated in microspheres of PLG. It is injected subcutaneously by patients once weekly, with no dose titration required. However, it was complicated to prepare for injection. Therefore, BYDUREON® 2mg Pen, a single-use, dual-chamber pen was developed to improve the convenience of exenatide once weekly delivery.
[Serial] Reviews on useful reagents for DDS research and development