Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization ofArabidopsisPIN-FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear. Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze-fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains. Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell-wall components as well as connections between the cell wall and the plasma membrane. This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems. ; European Research Council (ERC) 742985 Comision Nacional de Investigacion Cientifica y Tecnologica CONICYT-PAI 82130047 European Union (EU) 291734
In the replacement of genetic probes, there is increasing interest in labeling living cells with high-quality extrinsic labels, which avoid over-expression artifacts and are available in a wide spectral range. This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells. Here, we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique. We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation. We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses, allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells. We demonstrate high-quality time-lapse imaging with confocal, total internal reflection fluorescence (TIRF), and Airyscan super-resolution microscopy. As the entire procedure is readily compatible with fluorescence (super resolution) microscopy, photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging. ; K.B. acknowledges financial support from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 648124) and from the Ghent University Special Research Fund (01B04912) with gratitude. J.L. gratefully acknowledges the financial support from the China Scholarship Council (CSC) (201506750012) and the Special Research Fund from Ghent University (01SC1416). R.X. gratefully acknowledges the financial support from the China Scholarship Council (CSC) (2010634103). H.B. acknowledges funding from the Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek, FWO) for a doctoral fellowship (11ZB115N). E.T. acknowledges funding from the Agency for Innovation by Science and Technology (IWT). S.S. and R.B. acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the University of Lille, the Hauts-de-France region, the CPER "Photonics for Society", and the EU union through FLAG-ERA JTC 2015-Graphtivity and the Marie Sklodowska-Curie action (H2020-MSCA-RISE-2015, PANG-690836). M.A. acknowledges the support by the FWO Research Community "Scanning and Wide Field Microscopy of (Bio)-organic Systems" and the Province of Limburg (Belgium) for the financial support within the tUL IMPULS FASE II program, allowing for the upgrade of the laser source used in this work. We would thank the VIB BioImaging Core for the use of the microscopy equipment.
