A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision.

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision (fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture

Atomic Force Microscopy Characterization and Lithography of Cu-Ligated Mercaptoalkanoic Acid “Molecular Ruler” Multilayers

Hybrid chemical patterning strategies that combine the sophistication of lithography with the intrinsic precision of molecular self-assembly are of broad interest for applications including nanoelectronics and bioactive surfaces. This approach is exemplified by the molecular-ruler process where the sequential deposition of mercaptoalkanoic acid molecules and coordinated metal ions is integrated with conventional lithographic techniques to fabricate registered, nanometer-scale spacings. Herein, we illustrate the capabilities of atomic force microscopy characterization and lithography to investigate the morphology, quality, and local thickness of Cu-ligated mercaptohexadecanoic acid multilayers on Au{111} substrates. These multilayers are a key component utilized in the molecular-ruler process. The rich and varied topographic features of each layer are investigated via contact-mode atomic force microscopy. Using nanoshaving, an atomic force microscopy lithographic strategy that reveals the underlying Au{111} substrate via tip-induced desorption of a molecular film, the local thicknesses of these multilayers are ascertained; these thicknesses are consistent with the anticipated heights for Cu-ligated mercaptohexadecanoic acid multilayers as well as previous ensemble surface analytical measurements. By regulating the force set point utilized during nanoshaving, the upper layer of a Cu-ligated mercaptohexadecanoic acid bilayer is removed, revealing the carboxyl moiety of the lower mercaptohexadecanoic acid layer. This selective nanoshaving demonstrates a simple and practical means to generate three-dimensional multilayers and to reveal buried chemical functionalities within metal-ligated multilayers. © 2014 American Chemical Society

Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)

Abstract Background Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians. Methods Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay. Results We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker. Conclusions In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies