The residue at position 5 of the N-terminal region of Src and Fyn modulates their myristoylation, palmitoylation, and membrane interactions

The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.

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Src-mediated caveolin-1 phosphorylation affects the targeting of active Src to specific membrane sites

Molecular Biology of the Cell ◽

10.1091/mbc.e13-03-0163 ◽

2013 ◽

Vol 24 (24) ◽

pp. 3881-3895 ◽

Cited By ~ 35

Author(s):

Efrat Gottlieb-Abraham ◽

Dmitry E. Shvartsman ◽

John C. Donaldson ◽

Marcelo Ehrlich ◽

Orit Gutman ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Cell Transformation ◽

Quantitative Imaging ◽

Focal Adhesions ◽

Sh2 Domain ◽

Size Analysis ◽

Caveolin 1 ◽

Green Fluorescent ◽

Specific Membrane

Src interactions with the plasma membrane are an important determinant of its activity. In turn, Src activity modulates its association with the membrane through binding of activated Src to phosphotyrosylated proteins. Caveolin-1 (Cav-1), a major component of caveolae, is a known Src phosphorylation target, and both were reported to regulate cell transformation. However, the nature of Src-Cav-1 interactions, a potential mechanism of their coregulation, remained unclear. Here we used fluorescence recovery after photobleaching beam-size analysis, coimmunoprecipitation, quantitative imaging, and far-Western studies with cells expressing wild type, as well as structural and activity mutants of Src–green fluorescent protein and Cav-1–monomeric red fluorescent protein, to measure their interactions with the membrane and with each other. We show dynamic Src–plasma membrane interactions, which are augmented and stabilized by Cav-1. The mechanism involves phosphorylation of Cav-1 at Tyr-14 by Src and subsequent binding of the Src SH2 domain to phospho–Cav-1, leading to accumulation of activated Src in focal adhesions. This novel Cav-1 function potentially modulates focal adhesion dynamics.

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Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0211 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3085-3094 ◽

Cited By ~ 67

Author(s):

Ken Sato ◽

Miyuki Sato ◽

Anjon Audhya ◽

Karen Oegema ◽

Peter Schweinsberg ◽

...

Keyword(s):

Cell Cycle ◽

Plasma Membrane ◽

Caenorhabditis Elegans ◽

Fluorescent Protein ◽

Germ Line ◽

Cortical Granule ◽

Major Protein ◽

Dynamic Regulation ◽

Caveolin 1 ◽

Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

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Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA

International Journal of Molecular Sciences ◽

10.3390/ijms20071648 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1648 ◽

Cited By ~ 4

Author(s):

Felix Umrath ◽

Heidrun Steinle ◽

Marbod Weber ◽

Hans-Peter Wendel ◽

Siegmar Reinert ◽

...

Keyword(s):

Stem Cells ◽

Fluorescent Protein ◽

Osteogenic Potential ◽

Safety Standards ◽

Stem Cell Source ◽

Periosteal Cells ◽

Induced Pluripotent ◽

Green Fluorescent ◽

First Time

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

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The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study

The Journal of Cell Biology ◽

10.1083/jcb.137.7.1525 ◽

1997 ◽

Vol 137 (7) ◽

pp. 1525-1535 ◽

Cited By ~ 134

Author(s):

Jianwei Liu ◽

Thomas E. Hughes ◽

William C. Sessa

Keyword(s):

Nitric Oxide ◽

Amino Acids ◽

Fluorescent Protein ◽

3T3 Cells ◽

Fatty Acylation ◽

Golgi Region ◽

Green Fluorescent ◽

Oxide Synthase ◽

Nih 3T3 ◽

Endothelial Nitric Oxide

Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS–green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS–GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)5 repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS–GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.

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Switching from a Unicellular to Multicellular Organization in an Aspergillus niger Hypha

mBio ◽

10.1128/mbio.00111-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 16

Author(s):

Robert-Jan Bleichrodt ◽

Marc Hulsman ◽

Han A. B. Wösten ◽

Marcel J. T. Reinders

Keyword(s):

Aspergillus Niger ◽

Fluorescent Protein ◽

Stress Conditions ◽

Physical Contact ◽

Effective Control ◽

Mobility Rate ◽

Content Type ◽

Green Fluorescent ◽

First Time ◽

Compound Concentration

ABSTRACT Pores in fungal septa enable cytoplasmic streaming between hyphae and their compartments. Consequently, the mycelium can be considered unicellular. However, we show here that Woronin bodies close ~50% of the three most apical septa of growing hyphae of Aspergillus niger. The incidence of closure of the 9th and 10th septa was even ≥94%. Intercompartmental streaming of photoactivatable green fluorescent protein (PA-GFP) was not observed when the septa were closed, but open septa acted as a barrier, reducing the mobility rate of PA-GFP ~500 times. This mobility rate decreased with increasing septal age and under stress conditions, likely reflecting a regulatory mechanism affecting septal pore diameter. Modeling revealed that such regulation offers effective control of compound concentration between compartments. Modeling also showed that the incidence of septal closure in A. niger had an even stronger impact on cytoplasmic continuity. Cytoplasm of hyphal compartments was shown not to be in physical contact when separated by more than 4 septa. Together, data show that apical compartments of growing hyphae behave unicellularly, while older compartments have a multicellular organization. IMPORTANCE The hyphae of higher fungi are compartmentalized by porous septa that enable cytosolic streaming. Therefore, it is believed that the mycelium shares cytoplasm. However, it is shown here that the septa of Aspergillus niger are always closed in the oldest part of the hyphae, and therefore, these compartments are physically isolated from each other. In contrast, only part of the septa is closed in the youngest part of the hyphae. Still, compartments in this hyphal part are physically isolated when separated by more than 4 septa. Even open septa act as a barrier for cytoplasmic mixing. The mobility rate through such septa reduces with increasing septal age and under stress conditions. Modeling shows that the septal pore width is set such that its regulation offers maximal control of compound concentration levels within the compartments. Together, we show for the first time that Aspergillus hyphae switch from a unicellular to multicellular organization.

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Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.4013 ◽

2001 ◽

Vol 12 (12) ◽

pp. 4013-4029 ◽

Cited By ~ 106

Author(s):

Marie-Neige Cordonnier ◽

Daniel Dauzonne ◽

Daniel Louvard ◽

Evelyne Coudrier

Keyword(s):

Long Range ◽

Molecular Motors ◽

Actin Filaments ◽

Fluorescent Protein ◽

Time Lapse ◽

Myosin I ◽

Latrunculin A ◽

Living Mouse ◽

Green Fluorescent ◽

First Time

An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.

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The Dynamics of Postmitotic Reassembly of the Nucleolus

The Journal of Cell Biology ◽

10.1083/jcb.150.3.433 ◽

2000 ◽

Vol 150 (3) ◽

pp. 433-446 ◽

Cited By ~ 173

Author(s):

Miroslav Dundr ◽

Tom Misteli ◽

Mark O.J. Olson

Keyword(s):

Fluorescence Recovery After Photobleaching ◽

Fluorescent Protein ◽

Living Cells ◽

M Phase ◽

Specific Proteins ◽

Distribution Of Components ◽

Green Fluorescent ◽

Related Proteins ◽

First Time ◽

Rrna Sequences

Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.

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Monitoring disulfide bond formation in the eukaryotic cytosol

The Journal of Cell Biology ◽

10.1083/jcb.200402120 ◽

2004 ◽

Vol 166 (3) ◽

pp. 337-345 ◽

Cited By ~ 222

Author(s):

Henrik Østergaard ◽

Christine Tachibana ◽

Jakob R. Winther

Keyword(s):

Fluorescent Protein ◽

Redox Status ◽

Reduced And Oxidized Glutathione ◽

Cytosolic Glutathione ◽

Green Fluorescent ◽

Genetically Manipulated ◽

First Time ◽

Glutathione Redox

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein–based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be −289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.

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Functional Characterization of Candida albicans ABC Transporter Cdr1p

Eukaryotic Cell ◽

10.1128/ec.2.6.1361-1375.2003 ◽

2003 ◽

Vol 2 (6) ◽

pp. 1361-1375 ◽

Cited By ~ 101

Author(s):

Suneet Shukla ◽

Preeti Saini ◽

Smriti ◽

Sudhakar Jha ◽

Suresh V. Ambudkar ◽

...

Keyword(s):

Binding Sites ◽

Fluorescent Protein ◽

Mutational Analysis ◽

Substrate Binding ◽

Functional Characterization ◽

Drug Binding ◽

Transmembrane Segment ◽

Green Fluorescent ◽

Substrate Binding Sites ◽

First Time

ABSTRACT In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).

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Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae

Development ◽

10.1242/dev.124.22.4649 ◽

1997 ◽

Vol 124 (22) ◽

pp. 4649-4659 ◽

Cited By ~ 2

Author(s):

M.I. Arnone ◽

L.D. Bogarad ◽

A. Collazo ◽

C.V. Kirchhamer ◽

R.A. Cameron ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Sea Urchin ◽

Fluorescent Protein ◽

Regulatory Systems ◽

Transcriptional Regulatory ◽

Experimental Approaches ◽

Regulatory Analysis ◽

Green Fluorescent ◽

First Time

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected.

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