scholarly journals Unusual Centrosome Cycle inDictyostelium: Correlation of Dynamic Behavior and Structural Changes

1999 ◽  
Vol 10 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Masahiro Ueda ◽  
Manfred Schliwa ◽  
Ursula Euteneuer
Keyword(s):  
Green Fluorescent Protein ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Ultrastructural Level ◽  
Central Importance ◽  
Green Fluorescent Protein Construct ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Inside Out

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a γ-tubulin–green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyosteliumcentrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of γ-tubulin–green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.

scholarly journals Two Point Mutations Produce Infectious Retrovirus Bearing a Green Fluorescent Protein-SU Fusion Protein

2001 ◽  
Vol 75 (23) ◽  
pp. 11881-11885 ◽  
Author(s):  
Krishnakumar Kizhatil ◽  
Adam Gromley ◽  
Lorraine M. Albritton
Keyword(s):  
Green Fluorescent Protein ◽  
Envelope Protein ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Fundamental Problem ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Slight Reduction ◽  
Virus Envelope ◽  
Green Fluorescent

ABSTRACT Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.

scholarly journals In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

2004 ◽  
Vol 70 (5) ◽  
pp. 3073-3081 ◽  
Author(s):  
Zexun Lu ◽  
Riccardo Tombolini ◽  
Sheridan Woo ◽  
Susanne Zeilinger ◽  
Matteo Lorito ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Critical Role ◽  
Pythium Ultimum ◽  
Confocal Scanning Laser Microscopy ◽  
Green Fluorescent

ABSTRACT Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.

Visualization of individual astrocytes in three-dimensional cerebellar tissues using green fluorescent protein

1996 ◽  
Vol 54 ◽  
pp. 908-909
Author(s):  
M. D. Andersen ◽  
D. H. Szarowski ◽  
J. N. Turner ◽  
W. Shain
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Complex Structure ◽  
Three Dimensional ◽  
Cellular Interactions ◽  
Organotypic Cultures ◽  
Stable Transfectants ◽  
The Central Nervous System ◽  
Green Fluorescent

The central nervous system is a complex structure traditionally studied through selective stains and two-dimensional images of fixed tissues. A long term goal of this laboratory is the study of dynamic cellular interactions in three dimensional tissues such as organotypic cultures. Due to the large volume of tissue, traditional microscopy methods contain excessive out-of-focus data making it difficult to observe clearly specific cellular interactions. The visualization of morphological changes in specific cells in living, three-dimensional tissues is commonly done with vital dyes. An alternate approach is labeling individual astrocytes with Green Fluorescent Protein (GFP) and injecting them into organotypic cultures.GFP is a naturally fluorescent protein originally isolated from the jellyfish Aequorea victorea. To label individual astrocytes, LRM55 astroglial cells were transfected with GFP using calcium phosphate. The percentage of stable transfectants was low (< 1%) resulting in expression of varying amounts of GFP, as seen in Figs 5 and 6.

scholarly journals Dictyostelium EB1 Is a Genuine Centrosomal Component Required for Proper Spindle Formation

2002 ◽  
Vol 13 (7) ◽  
pp. 2301-2310 ◽  
Author(s):  
Markus Rehberg ◽  
Ralph Gräf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Spindle Formation ◽  
Microtubule Associated Proteins ◽  
Spindle Poles ◽  
Associated Proteins ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Green Fluorescent

EB1 proteins are ubiquitous microtubule-associated proteins involved in microtubule search and capture, regulation of microtubule dynamics, cell polarity, and chromosome stability. We have cloned a complete cDNA of Dictyostelium EB1 (DdEB1), the largest known EB1 homolog (57 kDa). Immunofluorescence analysis and expression of a green fluorescent protein-DdEB1 fusion protein revealed that DdEB1 localizes along microtubules, at microtubule tips, centrosomes, and protruding pseudopods. During mitosis, it was found at the spindle, spindle poles, and kinetochores. DdEB1 is the first EB1-homolog that is also a genuine centrosomal component, because it was localized at isolated centrosomes that are free of microtubules. Furthermore, centrosomal DdEB1 distribution was unaffected by nocodazole treatment. DdEB1 colocalized with DdCP224, the XMAP215 homolog, at microtubule tips, the centrosome, and kinetochores. Furthermore, both proteins were part of the same cytosolic protein complex, suggesting that they may act together in their functions. DdEB1 deletion mutants expressed as green fluorescent protein or maltose-binding fusion proteins indicated that microtubule binding requires homo-oligomerization, which is mediated by a coiled-coil domain. A DdEB1 null mutant was viable but retarded in prometaphase progression due to a defect in spindle formation. Because spindle elongation was normal, DdEB1 seems to be required for the initiation of the outgrowth of spindle microtubules.

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