multi-color-localization-microscopy-single-membrane-proteins

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

University of Osnabrück

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

The biomechanical and ecophysiological properties of plant seed/fruit structures are fundamental to survival in distinct environments. Dispersal of fruits with hard pericarps (fruit coats) encasing seeds has evolved many times independently within taxa that have seed dispersal as their default strategy. The mechanisms by which the constraint of a hard pericarp determines germination timing in response to the environment are currently unknown. Here, we show that the hard pericarp of Lepidium didymum controls germination solely by a biomechanical mechanism. Mechanical dormancy is conferred by preventing full phase-II water uptake of the encased non-dormant seed. The lignified endocarp has biomechanically and morphologically distinct regions that serve as predetermined breaking zones. This pericarp-imposed mechanical dormancy is released by the activity of common fungi, which weaken these zones by degrading non-lignified pericarp cells. We propose that the hard pericarp with this biomechanical mechanism contributed to the global distribution of this species in distinct environments.

Fruit fracture biomechanics and the release of Lepidium didymum pericarp-imposed mechanical dormancy by fungi.

Stress granules (SGs) are cytosolic, nonmembranous RNA-protein complexes. In vitro experiments suggested that they are formed by liquid-liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs. Within SGs, single-molecule localization microscopy revealed distributed hotspots of immobilized G3BP1 and IMP1 that reflect the presence of relatively immobile nanometer-sized nanocores. We demonstrate alternating binding in nanocores and anomalous diffusion in the liquid phase with similar characteristics for both RBPs. Reduction of low-complexity regions in G3BP1 resulted in less detectable mobile molecules in the liquid phase without change in binding in nanocores. The data provide direct support for liquid droplet behavior of SGs in living cells and reveal transient binding of RBPs in nanocores. Our study uncovers a surprising disconnect between SG partitioning and internal diffusion and interactions of RBPs.

Article	Year
Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport. Molecular biology of the cell\| PubMed ID: 25657322	2015
Fruit fracture biomechanics and the release of Lepidium didymum pericarp-imposed mechanical dormancy by fungi. Nature communications\| PubMed ID: 29192192	2017
Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules. The Journal of cell biology\| PubMed ID: 29463567	2018

Rainer Kurre

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