scholarly journals Arrival, Reversal, and Departure of Neurofilaments at the Tips of Growing Axons

2004 ◽  
Vol 15 (9) ◽  
pp. 4215-4225 ◽  
Author(s):  
Atsuko Uchida ◽  
Anthony Brown
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Imaging ◽  
Growth Cone ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Growth Cones ◽  
Reverse Direction ◽  
Preferential Site ◽  
Reversal Frequency ◽  
Green Fluorescent

We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent, and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that 1) growth cones are a preferential site of neurofilament reversal in distal axons, 2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, 3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and 4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency.

scholarly journals Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid

1998 ◽  
Vol 9 (9) ◽  
pp. 2463-2476 ◽  
Author(s):  
Janis E. Lochner ◽  
Mary Kingma ◽  
Samuel Kuhn ◽  
C. Daniel Meliza ◽  
Bryan Cutler ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Plasminogen Activator ◽  
Axonal Transport ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Time Lapse ◽  
Growth Cones ◽  
Differentiated Cells ◽  
Tissue Plasminogen ◽  
Green Fluorescent

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

scholarly journals Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

1999 ◽  
Vol 73 (5) ◽  
pp. 4110-4119 ◽  
Author(s):  
Gillian Elliott ◽  
Peter O’Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Live Cell ◽  
Cell Periphery ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.

scholarly journals Feasibility, sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

2003 ◽  
Vol 7 (6) ◽  
pp. 765-773 ◽  
Author(s):  
Severine Wack ◽  
Amor Hajri ◽  
Francine Heisel ◽  
Malgorzata Sowinska ◽  
Cedric Berger ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Imaging ◽  
Fluorescent Protein ◽  
Laser Induced Fluorescence ◽  
Green Fluorescent

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

scholarly journals GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein

1997 ◽  
Vol 327 (3) ◽  
pp. 637-642 ◽  
Author(s):  
B. Paru OATEY ◽  
David H. J. VAN WEERING ◽  
P. Stephen DOBSON ◽  
W. Gwyn GOULD ◽  
Jeremy M. TAVARÉ
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Target Cells ◽  
Green Fluorescent ◽  
Vesicle Movement ◽  
Vesicular Compartment ◽  
Intracellular Structure

Insulin stimulates glucose uptake into its target cells by a process which involves the translocation of the GLUT4 isoform of glucose transporter from an intracellular vesicular compartment(s) to the plasma membrane. The step(s) at which insulin acts in the vesicle trafficking pathway (e.g. vesicle movement or fusion with the plasma membrane) is not known. We expressed a green-fluorescent protein-GLUT4 (GFP-GLUT4) chimaera in 3T3 L1 adipocytes. The chimaera was expressed in vesicles located throughout the cytoplasm and also close to the plasma membrane. Insulin promoted a substantial translocation of GFP-GLUT4 to the plasma membrane. Time-lapse confocal microscopy demonstrated that the majority of GFP-GLUT4-containing vesicles in the basal state were relatively static, as if tethered (or attached) to an intracellular structure. A proportion (approx. 5%) of the vesicles spontaneously lost their tether, and were observed to move rapidly within the cell. Other vesicles appear to be tethered only on one edge and were observed in a rapid stretching motion. The data support a model in which GLUT4-containing vesicles are tightly tethered to an intracellular structure(s), and indicate that a primary site of insulin action must be to release these vesicles, allowing them to then translocate to and fuse with the plasma membrane.

scholarly journals Two-Color Green Fluorescent Protein Time-Lapse Imaging

BioTechniques ◽  
1998 ◽  
Vol 25 (5) ◽  
pp. 838-846 ◽  
Author(s):  
Jan Ellenberg ◽  
Jennifer Lippincott-Schwartz ◽  
John F. Presley
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Green Fluorescent ◽  
Time Lapse Imaging

scholarly journals The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes

2004 ◽  
Vol 381 (3) ◽  
pp. 581-585 ◽  
Author(s):  
Francesco M. LASORSA ◽  
Pasquale SCARCIA ◽  
Ralf ERDMANN ◽  
Ferdinando PALMIERI ◽  
Hanspeter ROTTENSTEINER ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Imaging ◽  
Fluorescent Protein ◽  
Adenine Nucleotide ◽  
Ph Sensitive ◽  
Peroxisomal Membrane ◽  
Transport Modes ◽  
Proton Movement ◽  
Green Fluorescent

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a ΔpH across the peroxisomal membrane, acidic inside.

scholarly journals Spindle Formation inAspergillusIs Coupled to Tubulin Movement into the Nucleus

2003 ◽  
Vol 14 (5) ◽  
pp. 2192-2200 ◽  
Author(s):  
Yulia Ovechkina ◽  
Paul Maddox ◽  
C. Elizabeth Oakley ◽  
Xin Xiang ◽  
Stephen A. Osmani ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Interphase Nuclei ◽  
Spindle Formation ◽  
Spinning Disk ◽  
Green Fluorescent

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/α-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.

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