Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Proc Natl Acad Sci U S A
2021 Mar 16;11811:. doi: 10.1073/pnas.2019071118.
Show Gene links
Show Anatomy links
POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.
Sugizaki A
,
Sato K
,
Chiba K
,
Saito K
,
Kawagishi M
,
Tomabechi Y
,
Mehta SB
,
Ishii H
,
Sakai N
,
Shirouzu M
,
Tani T
,
Terada S
.
???displayArticle.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.
Amann,
The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments.
2001, Pubmed
Amann,
The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments.
2001,
Pubmed
Arai,
Design of the linkers which effectively separate domains of a bifunctional fusion protein.
2001,
Pubmed
Azoury,
Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments.
2008,
Pubmed
Backer,
Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements.
2016,
Pubmed
Barondeau,
Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation.
2006,
Pubmed
Bedford,
Alternative reagents to antibodies in imaging applications.
2017,
Pubmed
Brejc,
Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein.
1997,
Pubmed
DeMay,
Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals.
2011,
Pubmed
Farina,
The centrosome is an actin-organizing centre.
2016,
Pubmed
Farina,
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis.
2019,
Pubmed
Field,
Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs.
2019,
Pubmed
Field,
Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs.
2015,
Pubmed
Forkey,
Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization.
2003,
Pubmed
Hamaguchi,
Quantitative analysis of cortical actin filaments during polar body formation in starfish oocytes.
2007,
Pubmed
,
Echinobase
Hesketh,
Affimer reagents as tools in diagnosing plant virus diseases.
2019,
Pubmed
Huang,
Application of Fluorescence Polarization in HTS Assays.
2016,
Pubmed
Hughes,
Generation of specific inhibitors of SUMO-1- and SUMO-2/3-mediated protein-protein interactions using Affimer (Adhiron) technology.
2017,
Pubmed
INOUE,
[Polarization optical studies of the mitotic spindle. I. The demonstration of spindle fibers in living cells].
1953,
Pubmed
Inoue,
Actin filaments regulate microtubule growth at the centrosome.
2019,
Pubmed
Inoué,
Cell division and the mitotic spindle.
1981,
Pubmed
Kampmann,
Mapping the orientation of nuclear pore proteins in living cells with polarized fluorescence microscopy.
2011,
Pubmed
Kearney,
Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.
2019,
Pubmed
Kita,
Spindle-F-actin interactions in mitotic spindles in an intact vertebrate epithelium.
2019,
Pubmed
Klont,
Affimers as an Alternative to Antibodies in an Affinity LC-MS Assay for Quantification of the Soluble Receptor of Advanced Glycation End-Products (sRAGE) in Human Serum.
2018,
Pubmed
Kyle,
Exploration of the HIF-1α/p300 interface using peptide and Adhiron phage display technologies.
2015,
Pubmed
Lamason,
Actin-based motility and cell-to-cell spread of bacterial pathogens.
2017,
Pubmed
Lopata,
Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.
2018,
Pubmed
Lénárt,
A contractile nuclear actin network drives chromosome congression in oocytes.
2005,
Pubmed
,
Echinobase
Mattheyses,
Polarized fluorescence resonance energy transfer microscopy.
2004,
Pubmed
Mehta,
Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells.
2016,
Pubmed
Michel,
Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.
2017,
Pubmed
Miller,
Regulation of cytokinesis by Rho GTPase flux.
2009,
Pubmed
Mogessie,
Actin protects mammalian eggs against chromosome segregation errors.
2017,
Pubmed
Mori,
An Arp2/3 nucleated F-actin shell fragments nuclear membranes at nuclear envelope breakdown in starfish oocytes.
2014,
Pubmed
,
Echinobase
Mori,
Intracellular transport by an anchored homogeneously contracting F-actin meshwork.
2011,
Pubmed
,
Echinobase
Nakai,
Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy.
2019,
Pubmed
Nishizaka,
Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.
2004,
Pubmed
Nolen,
Characterization of two classes of small molecule inhibitors of Arp2/3 complex.
2009,
Pubmed
Nordenfelt,
Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.
2017,
Pubmed
Ohmachi,
Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V.
2012,
Pubmed
Ojha,
Imaging of fluorescence anisotropy during photoswitching provides a simple readout for protein self-association.
2020,
Pubmed
Oura,
Polarization-dependent fluorescence correlation spectroscopy for studying structural properties of proteins in living cell.
2016,
Pubmed
Pettersen,
UCSF Chimera--a visualization system for exploratory research and analysis.
2004,
Pubmed
Pruyne,
Role of formins in actin assembly: nucleation and barbed-end association.
2002,
Pubmed
RAPPAPORT,
Experiments concerning the cleavage stimulus in sand dollar eggs.
1961,
Pubmed
,
Echinobase
Richter,
Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.
2018,
Pubmed
Riedl,
Lifeact: a versatile marker to visualize F-actin.
2008,
Pubmed
Rizvi,
Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly.
2009,
Pubmed
Robinson,
Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action.
2018,
Pubmed
Rosell,
Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions.
2003,
Pubmed
Santella,
Maturation and fertilization of echinoderm eggs: Role of actin cytoskeleton dynamics.
2018,
Pubmed
,
Echinobase
Schuh,
A new model for asymmetric spindle positioning in mouse oocytes.
2008,
Pubmed
Sosa,
ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy.
2001,
Pubmed
Spira,
Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments.
2017,
Pubmed
Swaminathan,
Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.
2017,
Pubmed
Terada,
Visualization of slow axonal transport in vivo.
1996,
Pubmed
Terada,
Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons.
2000,
Pubmed
Tiede,
Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications.
2014,
Pubmed
Tiede,
Affimer proteins are versatile and renewable affinity reagents.
2017,
Pubmed
Valades Cruz,
Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy.
2016,
Pubmed
Vrabioiu,
Structural insights into yeast septin organization from polarized fluorescence microscopy.
2006,
Pubmed
Vrabioiu,
Symmetry of septin hourglass and ring structures.
2007,
Pubmed
Wesolowska,
Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes.
2020,
Pubmed
,
Echinobase
Winder,
Utrophin actin binding domain: analysis of actin binding and cellular targeting.
1995,
Pubmed
Woolner,
Myosin-10 and actin filaments are essential for mitotic spindle function.
2008,
Pubmed
Woolner,
Unconventional myosins acting unconventionally.
2009,
Pubmed
Yamamoto,
Rotational diffusion measurements using polarization-dependent fluorescence correlation spectroscopy based on superconducting nanowire single-photon detector.
2015,
Pubmed
Zhanghao,
Super-resolution dipole orientation mapping via polarization demodulation.
2016,
Pubmed
Zhanghao,
Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy.
2019,
Pubmed
von Dassow,
Concurrent cues for cytokinetic furrow induction in animal cells.
2009,
Pubmed