Covalent labeling of cell-surface proteins for in-vivo FRET studies

Cell surface proteins anchored to membranes via covalently attached glycosyl-phosphatidylinositol (GPI) have been implicated in neuronal adhesion, promotion of neurite outgrowth and directed cell migration. Treatment of grasshopper embryos with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves the GPI anchor, often induced disruptions in the highly stereotyped migrations of peripheral pioneer growth cones and afferent neuron cell bodies. In distal limb regions of embryos treated with PI-PLC at early stages of pioneer axon outgrowth, growth cones lost their proximal orientation toward the central nervous system (CNS) and turned distally. Pioneer growth cones in treated limbs also failed to make a characteristic ventral turn along the trochanter-coxa (Tr-Cx) segment boundary, and instead continued to grow proximally across the boundary. Treatment at an earlier stage of development caused pre-axonogenesis Cx1 neurons to abandon their normal circumferential migration and reorient toward the CNS. None of these abnormal phenotypes were observed in limbs of untreated embryos or embryos exposed to other phospholipases that do not release GPI-anchored proteins. Incubation of embryos with PI-PLC effectively removed immunoreactivity for fasciclin I, a GPI-anchored protein expressed on a subset of neuronal surfaces. These results suggest that cell surface GPI-anchored proteins are involved in pioneer growth cone guidance and in pre-axonogenesis migration of neurons in the grasshopper limb bud in vivo.

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Ligatin from embryonic chick neural retina.

The Journal of Cell Biology ◽

10.1083/jcb.80.3.642 ◽

1979 ◽

Vol 80 (3) ◽

pp. 642-650 ◽

Cited By ~ 25

Author(s):

E R Jakoi ◽

R B Marchase

Keyword(s):

Cell Surface ◽

Plasma Membranes ◽

Protein A ◽

Neural Retina ◽

Surface Proteins ◽

Embryonic Chick ◽

Cell Surface Proteins ◽

Rat Ileum ◽

Discrete Band

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.

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Discriminating gene expression profiles of memory B cell subpopulations

Journal of Experimental Medicine ◽

10.1084/jem.20072682 ◽

2008 ◽

Vol 205 (8) ◽

pp. 1807-1817 ◽

Cited By ~ 111

Author(s):

Götz R.A. Ehrhardt ◽

Atsushi Hijikata ◽

Hiroshi Kitamura ◽

Osamu Ohara ◽

Ji-Yang Wang ◽

...

Keyword(s):

Transcription Factors ◽

Cell Surface ◽

Intracellular Signaling ◽

Expression Profiles ◽

Surface Proteins ◽

Epithelial Tissue ◽

Cell Surface Proteins ◽

Cell Subpopulations

Morphologically and functionally distinct subpopulations of human memory B (BMem) cells are identifiable by either their expression of CD27 or Fc receptor–like 4 (FCRL4), an immunoglobulin domain containing a receptor with strong inhibitory potential. We have conducted comparative transcriptome and proteome analyses of FCRL4+ and FCRL4− BMem cells and found that these two subsets have very distinctive expression profiles for genes encoding transcription factors, cell-surface proteins, intracellular signaling molecules, and modifiers of the cell-cycle status. Among the differentially expressed transcription factors, runt-related transcription factor 1 (RUNX1) transcript levels were up-regulated in FCRL4− cells, whereas RUNX2 transcripts were preferentially detected in FCRL4+ cells. In vitro evidence for FCRL4 promoter responsiveness and in vivo promoter occupancy suggested that RUNX transcription factors are involved in the generation of these BMem cell subpopulations. A distinctive signature profile was defined for the FCRL4+ BMem cells by their expression of CD11c, receptor activator for nuclear factor κB ligand, and FAS cell-surface proteins, in combination with increased levels of SOX5, RUNX2, DLL1, and AICDA expression. We conclude that this recently identified subpopulation of BMem cells, which normally resides in epithelial tissue-based niches, may serve a unique role in mucosal defense and, conversely, as a target for neoplastic transformation events.

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Membrane protein redistribution during differentiation of cultured human erythroleukemic cells.

Molecular and Cellular Biology ◽

10.1128/mcb.1.12.1150 ◽

1981 ◽

Vol 1 (12) ◽

pp. 1150-1162 ◽

Cited By ~ 8

Author(s):

R C Hunt ◽

L M Marshall

Keyword(s):

Cell Surface ◽

K562 Cells ◽

Erythroid Differentiation ◽

Surface Proteins ◽

Cell Surface Proteins ◽

Mature Erythrocyte ◽

Transmission Electron ◽

Immune Precipitation

Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium. In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin. However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation. Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membranes. A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 micrometers in diameter, is divided from the nucleus-containing part of the cell. With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin.

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Membrane protein redistribution during differentiation of cultured human erythroleukemic cells

Molecular and Cellular Biology ◽

10.1128/mcb.1.12.1150-1162.1981 ◽

1981 ◽

Vol 1 (12) ◽

pp. 1150-1162

Author(s):

R C Hunt ◽

L M Marshall

Keyword(s):

Cell Surface ◽

K562 Cells ◽

Erythroid Differentiation ◽

Surface Proteins ◽

Cell Surface Proteins ◽

Mature Erythrocyte ◽

Transmission Electron ◽

Immune Precipitation

Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium. In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin. However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation. Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membranes. A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 micrometers in diameter, is divided from the nucleus-containing part of the cell. With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin.

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In vivo interaction of antibodies with cell surface proteins used as antigens

Tissue and Cell ◽

10.1016/0040-8166(86)90039-x ◽

1986 ◽

Vol 18 (6) ◽

pp. 809-816 ◽

Cited By ~ 5

Author(s):

Peter R.B. Caldwell ◽

Jan R. Brentjens ◽

Giovanni Camussi ◽

Giuseppe Andres

Keyword(s):

Cell Surface ◽

Surface Proteins ◽

Cell Surface Proteins

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What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140300 ◽

1995 ◽

Vol 53 ◽

pp. 786-787

Author(s):

Watt W. Webb

Keyword(s):

Cell Surface ◽

Lipid Membranes ◽

Surface Proteins ◽

Protein Diffusion ◽

Coated Pits ◽

Cell Surface Proteins ◽

Membrane Heterogeneity ◽

Fluorescence Photobleaching Recovery ◽

Photobleaching Recovery ◽

Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

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