This study explores the general utility of a new class of biosensor that allows one to selectively visualize molecules of a chosen membrane protein that are at the cell surface. These biosensors make use of recently described bipartite fluoromodules comprised of a fluorogen-activating protein (FAP) and a small molecule (fluorogen) whose fluorescence increases dramatically when noncovalently bound by the FAP (SzentGyorgyi et al., Nat Biotechnol 2008;26:235-240 CELLS that express single-pass recombinant membrane proteins, each presenting a FAP on the exterior face of the plasma membrane and a standard fluorescent protein (EGFP or mRFP) on the interior face, were generated and examined by fluorescence microscopy. In each case, fluorescent signal was observed exclusively at the cell surface when the FAP domain was imaged using membrane-impermeant fluorogen but was observed in additional intracellular locations when the fluorescent protein domain was imaged. Cells that expressed external N-terminal FAP-fusions to three wellstudied human membrane proteins-the b2 adrenergic receptor (b 2 AR), the insulinregulated glucose transporter (GLUT4), and the cystic fibrosis transmembrane conductance regulator (CFTR)-were also generated and examined; these too showed fluorescent signal exclusively at the cell surface after exposure to membrane-impermeant fluorogen. Further, when endocytosis of tagged b 2 AR was stimulated by agonist treatment in the presence of fluorogen, fluorescent signal was seen to transit from the surface to the cell interior. FAP tagging thus provides a means for selectively visualizing plasma membrane proteins and for monitoring the trafficking of these proteins to and from the cell surface.Plasma membrane proteins play roles in thousands of cellular processes and are the targets of more than half of all therapeutic drugs. Many of these proteins exhibit regulated translocation between the cell surface and the cell interior. For example, most G-protein coupled receptors (GPCRs) and receptor tyrosine kinases are internalized by endocytosis after exposure to agonists (1,2), and numerous ion and metabolite transporters traffic to the membrane in response to particular physiological stimuli (3).Many methods are available to selectively label particular plasma membrane proteins in living cells with fluorescent molecules so as to study membrane protein trafficking. These include immunofluorescence with specific antibodies or antibodies to epitope tags, labeling of tetracysteine (TC) tagged fusion proteins with FlAsH or ReAsH reagents, covalent attachment of fluorophores using SNAP-tag or Halo Tag
Echinonectin (EN) is a galactose-binding lectin present in eggs and embryos of the sea urchin Lytechinus variegatus. Recent studies have suggested that EN is a hyaline layer protein that may function as a substrate adhesion molecule (SAM) during development. We have used monoclonal and affinitypurified polyclonal antibodies that specifically recognize this protein to determine its spatial and temporal expression during embryogenesis. EN is stored in granules or vesicles in the unfertilized egg. After fertilization, these granules are rapidly redistributed to the apical cytoplasm of the zygote. Our results show that at subsequent stages of development the lectin is expressed by cells of all three germ layers, including cells of the developing gut, coelomic pouches, and ectoderm, and by both primary and secondary rnesenchyme cells. In contrast to previous observations based solely upon light level immunofluorescent staining, immunoelectron microscopy demonstrates that EN is localized in intracellular, membrane-bounded vesicles. In epithelia1 cell types these vesicles have a highly polarized distribution and are found in the apical cortical cytoplasm. In rnesenchyrne cells the distribution of EN-containing vesicles is not obviously polarized. Steady-state levels of EN protein in the embryo remain almost constant from fertilization to the pluteus larva stage. Metabolic labeling studies show that synthesis of EN in L. variegatus begins immediately after fertilization and continues throughout embryogenesis. Monospecific antibodies raised against L. variegatus EN have also been used to determine whether this lectin is expressed in other echinoid species.
Protein subcellular location is one of the most important determinants of protein function during cellular processes. Changes in protein behavior during the cell cycle are expected to be involved in cellular reprogramming during disease and development, and there is therefore a critical need to understand cell-cycle dependent variation in protein localization which may be related to aberrant pathway activity. With this goal, it would be useful to have an automated method that can be applied on a proteomic scale to identify candidate proteins showing cell-cycle dependent variation of location. Fluorescence microscopy, and especially automated, high-throughput microscopy, can provide images for tens of thousands of fluorescently-tagged proteins for this purpose. Previous work on analysis of cell cycle variation has traditionally relied on obtaining time-series images over an entire cell cycle; these methods are not applicable to the single time point images that are much easier to obtain on a large scale. Hence a method that can infer cell cycle-dependence of proteins from asynchronous, static cell images would be preferable. In this work, we demonstrate such a method that can associate protein pattern variation in static images with cell cycle progression. We additionally show that a one-dimensional parameterization of cell cycle progression and protein feature pattern is sufficient to infer association between localization and cell cycle.
W. L. 1981. Anatomical and physiological characteristics of the petiole of Abutilon theophrasti in relation to circadian leaf movements. -Physiol. Plant. 51: 309-313.Leaf movements in Abutilon theophrasti Medic, were monitored manually and by a continuous electronic recording device. Plants entrained to a daily regime of a 15 h light span followed by 9 h of darkness showed rhythmic movements that persisted under conditions of continuous illumination and constant temperature with a circadian period. The rhythmic change in orientation of the leaf from a near horizontal (day) to a near vertical (night) position was attributed to movement of the blade and not the petiole. The end of the petiole next to the blade functions as a joint or pulvinus. Anatomical confirmation of the existence of a pulvinus in the Abutilon leaf was provided by light microscopy. Vascular tissue in this region forms a solid cylinder with no pith, and the cortex is parenchymatous. In the main part of the petiole, the vascular tissue is arranged in four to six bundles, a pith is present, and the cortex contains a sub-epidermal ring of collenchyma. Both the functional and anatomical evidence indicate the presence of a pulvinus that functions in circadian leaf movements of Abutilon,
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