Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy.

OBJECTIVE: Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening. RESULTS: GmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

Optical control of the Ca2+ concentration in a live specimen with a genetically encoded Ca2+-releasing molecular tool.

Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.

Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen-stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

Time-lapse imaging of self- and cross-pollinations in Brassica rapa.

BACKGROUND AND AIMS: Pollination is an important process in the life cycle of plants and is the first step in bringing together the male and female gametophytes for plant reproduction. While pollination has been studied for many years, accurate knowledge of the morphological aspects of this process is still far from complete. This study therefore focuses on a morphological characterization of pollination, using time-series image analysis of self- and cross-pollinations in Brassica rapa. METHODS: Time-lapse imaging of pollen behaviour during self- and cross-pollinations was recorded for 90 min, at 1 min intervals, using a stereoscopic microscope. Using time-series digital images of pollination, characteristic features of pollen behaviours during self- and cross-pollinations were studied. KEY RESULTS: Pollen exhibited various behaviours in both self- and cross-pollinations, and these were classified into six representative patterns: germination, expansion, contraction, sudden contraction, pulsation and no change. It is noteworthy that in 'contraction' pollen grains shrunk within a short period of 30-50 min, and in 'pulsation' repeated expansion and contraction occurred with an interval of 10 min, suggesting that a dehydration system is operating in pollination. All of the six patterns were observed on an individual stigma with both self- and cross-pollinations, and the difference between self- and cross-pollinations was in the ratios of the different behaviours. With regard to water transport to and from pollen grains, this occurred in multiple steps, before, during and after hydration. Thus, pollination is regulated by a combination of multiple components of hydration, rehydration and dehydration systems. CONCLUSIONS: Regulated hydration of pollen is a key process for both pollination and self-incompatibility, and this is achieved by a balanced complex of hydration, dehydration and nutrient supply to pollen grains from stigmatic papilla cells.