POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

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.

Challenges in the diagnosis and management of vitreoretinal lymphoma - Clinical and basic approaches.

Vitreoretinal lymphoma (VRL) is a subtype of diffuse large B-cell lymphoma and is sight- and life-threatening in the vast majority of patients. Lymphoma cells infiltrate the vitreous body and/or subretinal space and exhibit clinical signs of vitreous opacities and creamy white subretinal lesions. Although the intraocular signs can serve as clues to suspect VRL, they are nonspecific and may be misdiagnosed as uveitis. Histopathological evidence of malignant cells on vitreous biopsy, for instance, is the gold standard for diagnosis of VRL; however, cytological examination of the vitreous often results in a low success rate owing to the small quantity and poor quality of tissues and cells in the sample. Recent advancements in immunological, molecular, and gene analyses using intraocular samples have made it possible to accurately diagnose VRL. As for the management of VRL, local treatments with irradiation and/or intravitreal injections of anti-tumor agents (methotrexate or rituximab) are effective in suppressing intraocular VRL lesions. However, the effect of systemic chemotherapy, with or without brain irradiation, on preventing central nervous system involvements remains controversial. In this review article, we discuss the following concepts based on previous literature and our unpublished results: current ocular imaging examinations such as optical coherence tomography and fundus autofluorescence; immunological, molecular, and gene expression characterization of intraocular biopsies with special attention to flow cytometry; immunoglobulin gene rearrangement assays that use the polymerase chain reaction test; cytokine assays; gene mutations (MYD88, CD79B); and current local and systemic treatments of VRL.

Discrimination of dissociated lymphoma cells from leukocytes by Raman spectroscopy.

Diagnosis of intraocular lymphoma is difficult. Among the hurdles in the diagnosis are the variety of reactive inflammatory and ischemic changes among intraocular lymphoma patients. Thus, a novel diagnostic method is desired such that lymphoma cells can be distinguished by the signals intrinsic to the cells, not by those from the surrounding tissues with reactive changes. Raman spectroscopy is a technique that can detect intrinsic signals from each cell. Therefore, Raman spectroscopy is a good candidate for an intraocular evaluation technology that could contribute to improve the diagnosis of intraocular lymphoma. In this study, we tested whether the intrinsic Raman signals from malignant lymphoma cells, in the absence of surrounding tissue, were sufficient for the discrimination of malignant lymphoma cells from leukocytes. We acquired spectra from dissociated lymphoma cells, along with spectra from normal B cells and other leukocytes involved in intraocular inflammatory diseases. We analysed the spectra using principal component analyses and quadratic discriminant analyses. We found that Raman spectra from dissociated cells without confounding tissues showed high discriminating ability, regardless of the variation due to day-to-day differences and donor differences. The present study demonstrates the possible effectiveness of Raman spectroscopy as a tool for intraocular evaluation.

Direct label-free measurement of the distribution of small molecular weight compound inside thick biological tissue using coherent Raman microspectroscopy.

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 cm(-1) inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.

Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites.

In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. Here we show that an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein.