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

Abstract

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.

Disc and Actin-Associated Protein 1 Influence Attachment in the Intestinal Parasite Giardia lamblia

“…Actomyosin interactions influence the orientation of the mitotic spindle [38,39] and F-actin associates directly with mitotic spindles in Xenopus and starfish embryos [40,41]. Actin is also involved in microtubule-based host interaction in other parasites.…”

“…Actin monomers are a key component of the flagella through interaction with the inner dynein arm [35][36][37][38] and recent evidence indicates actin is a component of the gamma-tubulin ring complex [39]. Actomyosin interactions influence the orientation of the mitotic spindle [40,41] and F-actin associates directly with mitotic spindles in Xenopus and starfish embryos [42,43].…”

Disc and Actin Associated Protein 1 influences attachment in the intestinal parasite Giardia lamblia

“…Actin monomers are a key component of the flagella through interaction with the inner dynein arm [ 34 – 37 ] and recent evidence indicates actin is a component of the gamma-tubulin ring complex [ 38 ]. Actomyosin interactions influence the orientation of the mitotic spindle [ 39 , 40 ] and F-actin associates directly with mitotic spindles in Xenopus and starfish embryos [ 41 , 42 ]. Actin is also involved in microtubule- or tubulin-based host interaction in other parasites.…”

Improved two-photon imaging of GPCR-based optogenetic neurotransmitter sensors using orthogonally polarized excitation

“…This architecture orients the GFP barrel more or less parallel to the cell membrane, depending on the length of the linkers [21]. Based on available structural information [20,[22][23][24], we propose that in highly anisotropic sensors, the dipole moment of cpGFP is oriented normal to the membrane plane (Fig. 7).…”

“…If the fluorophore can rotate in a cone, its dipole orientation relative to the direction of polarization is randomized. To obtain polarization-sensitive probes with single-ended tether, the linker has to form a continuous α-helix to prevent angular movements [20].…”