Nepal, P. | bioRxiv Science

bioRxiv2022

Small-angle x-ray microdiffraction from fibrils embedded in tissue thin sections

Small-angle x-ray scattering (SAXS) from fibrils embedded in a fixed, thin section of tissue includes contributions from the fibrils; the polymeric matrix surrounding the fibrils; other constituents of the tissue; and cross-terms due to the spatial correlation between fibrils and neighbouring molecules. This complex mixture severely limits the amount of information that can be extracted from scattering studies. However, availability of micro- and nano-beams has made possible measurement of scattering from very small volumes which, in some cases, may be dominated by a single fibrillar constituent. In those cases, information about the predominant species may be accessible. Nevertheless, even in these cases, the correlations between the positions of fibrils and other constituents have significant impact on the observed scattering. Here, we propose strategies to extract partial information about fibril structure and tissue organization on the basis of SAXS from samples of this type. We show that the spatial correlation function of the fibril in the direction perpendicular to the fibril axis can be computed and contains information about the predominant fibril structure and the organization of the surrounding tissue matrix. It has significant advantages over approaches based on techniques developed for x-ray solution scattering. We present examples of the calculation of correlation in different types of samples to demonstrate the kinds of information that can be obtained from these measurements. SynopsisThe availability of micro- and nano- x-ray beams is making possible measurement of scattering from very small volumes, opening possibilities for deriving in situ structural information on fibrillar constituents in complex materials and tissues. This work outlines a set of strategies for confronting the formidable technical obstacles to extracting useful structural information from scattering derived from these materials.

biophysics↗

bioRxiv2019

Folding latency of fluorescent proteins affects the mitochondrial localization of fusion protein

The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (aqGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type aqGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.

cell biology↗