Fluorescence light microscopy is an indispensable approach for the investigation of cell biological mechanisms. With the development of cutting-edge tools such as genetically encoded fluorescent proteins and superresolution methods, light microscopy is more powerful than ever at providing insight into a broad range of phenomena, from bacterial fission to cancer metastasis. However, as with all experimental approaches, care must be taken to ensure reliable and reproducible data collection, analysis, and reporting. Each step of every imaging experiment, from design to execution to communication to data management, should be critically assessed for bias, rigor, and reproducibility. This Perspective provides a basic “best practices” guide for designing and executing fluorescence imaging experiments, with the goal of introducing researchers to concepts that will help empower them to acquire images with rigor.
SignificanceReactive oxygen species (ROS) increase with age and have been shown to negatively impact age-related diseases. However, the physiological roles they might play in development have not been extensively characterized. Here, we show that ROS have essential functions as oocytes complete meiosis, the specialized cell division that generates haploid eggs, and transition to embryonic development. Meiotic progression and early embryonic divisions are defective when ROS is misregulated. Furthermore, we document the effects of ROS on specific proteins. Our identification of proteins altered in redox state as the oocyte transitions to an embryo provides a valuable resource to guide future exploration of ROS functions in early development. The regulatory system described here has important implications for female fertility.
Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.
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