scholarly journals Introduction of Green Fluorescent Protein (GFP) into Hippocampal Neurons through Viral Infection

2010 ◽  
Vol 2010 (4) ◽  
pp. pdb.prot5406-pdb.prot5406 ◽  
Author(s):  
R. Malinow ◽  
Y. Hayashi ◽  
M. Maletic-Savatic ◽  
S. H. Zaman ◽  
J.-C. Poncer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Viral Infection ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes

2008 ◽  
Vol 181 (6) ◽  
pp. 1027-1039 ◽  
Author(s):  
Junghoon Ha ◽  
Kevin W.-H. Lo ◽  
Kenneth R. Myers ◽  
Tiffany M. Carr ◽  
Michael K. Humsi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Receptor Tyrosine Kinase ◽  
Pc12 Cell ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Neurotrophin Receptor ◽  
Immunoaffinity Purification ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.

scholarly journals Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal Neurons

1999 ◽  
Vol 10 (9) ◽  
pp. 2945-2953 ◽  
Author(s):  
Martin Köhrmann ◽  
Ming Luo ◽  
Christoph Kaether ◽  
Luc DesGroseillers ◽  
Carlos G. Dotti ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Hippocampal Neurons ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Maximum Velocity ◽  
Time Lapse ◽  
Mrna Transport ◽  
Transport Pathway ◽  
Green Fluorescent ◽  
Dependent Transport

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

scholarly journals Analysis of Cell-to-Cell and Long-Distance Movement of a Bean Dwarf Mosaic Geminivirus-Green Fluorescent Protein Reporter in Host and Nonhost Species: Identification of Sites of Resistance

1999 ◽  
Vol 12 (4) ◽  
pp. 345-355 ◽  
Author(s):  
H. L. Wang ◽  
M. R. Sudarshana ◽  
R. L. Gilbertson ◽  
W. J. Lucas
Keyword(s):  
Green Fluorescent Protein ◽  
Viral Infection ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Systemic Infection ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Long Distance ◽  
Green Fluorescent ◽  
Long Distance Movement

A bean dwarf mosaic geminivirus-green fluorescent protein (BDMV-GFP) reporter system was employed to analyze the viral infection process in host and nonhost species. Five classes of BDMV/host interaction were identified: (i) adapted hosts (susceptible Phaseolus vulgaris cultivars) permissive for systemic infection; (ii) adapted hosts (resistant P. vulgaris cv. Othello) displaying the development of a hypersensitive response (HR) associated with resistance to systemic infection; (iii) adapted (resistant P. vulgaris cv. Black Turtle Soup T-39) and nonadapted (Vigna unguiculata) hosts in which cell-to-cell, but not long-distance, movement was permitted; (iv) nonadapted hosts (Glycine max) in which systemic infection was coat protein-dependent; and (v) nonhosts (Cucurbita maxima, Gossypium barbadense, and Zea mays) in which the virus was confined to inoculated cells. Confocal laser scanning microscopy, fluorescence microscopy, and histochemical analyses were used to identify the cellular distribution of BDMV-GFP and the host response to viral infection. With this approach, the HR in P. vulgaris cv. Othello was visualized within cells of the epidermis, cortex, and phloem of inoculated hypocotyls. Infection studies performed with four begomoviruses and infectious BDMV/tomato mottle geminivirus pseudorecombinants revealed that the HR determinant(s) mapped to the BDMV DNA-B component.

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