Development of nanobody-based POLArIS orientation probes enabled multi-color/multi-target orientation imaging in living cells.

Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms.

Coexistence of Dense and Sparse Areas in the Longitudinal Smooth Muscle of the Anal Canal: Anatomical and Histological Analyses Inspired by Magnetic Resonance Images.

Magnetic resonance images of the anal canal show small, circular, low-intensity areas arranged in a row and a high-intensity area surrounding them internally and externally in the longitudinal muscle layer that cannot be explained by current anatomical findings. The purpose of this study was to elucidate the detailed structure of the longitudinal smooth muscle of the anal canal and to interpret the magnetic resonance image of the longitudinal muscle. Specimens for macroscopic anatomy and histology were obtained from six and seven cadavers, respectively. The histological nature of the longitudinal muscle was examined by staining serial transverse and coronal sections of the lateral wall of the anal canal with Masson's trichrome stain and using immunohistochemistry for smooth and skeletal muscle fibers. Dense and sparse areas of smooth muscle fibers coexisted in the longitudinal muscle layer. The dense areas formed columnar muscle bundles approximately 1.0-1.5 mm in diameter, and they continued from the longitudinal muscle bundles of the rectum. The columnar muscle bundles of the longitudinal anal muscle were internally and externally surrounded by sparsely arranged smooth muscle fibers that ran longitudinally. The coexistence of dense and sparse areas of smooth muscle fibers suggests that the structure of the smooth muscle is optimized for its function. This histological nature is probably reflected in the magnetic resonance image of the longitudinal muscle as the coexistence of low- and high-intensity areas. Clin. Anat. 33:619-626, 2020. © 2019 Wiley Periodicals, Inc.