scholarly journals Dynamics of myosin, microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells

2009 ◽  
Vol 186 (5) ◽  
pp. 727-738 ◽  
Author(s):  
Ronald D. Vale ◽  
James A. Spudich ◽  
Eric R. Griffis
Keyword(s):  
Fluorescent Protein ◽  
De Novo ◽  
Cell Cortex ◽  
S2 Cells ◽  
Contractile Ring ◽  
Anaphase Onset ◽  
Myosin Filaments ◽  
Drosophila S2 Cells ◽  
Guanosine Triphosphatase ◽  
Green Fluorescent

Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.

Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1227-1240 ◽  
Author(s):  
R. Neujahr ◽  
R. Albrecht ◽  
J. Kohler ◽  
M. Matzner ◽  
J.M. Schwartz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Myosin Ii ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Mitotic Apparatus ◽  
Alpha Tubulin ◽  
Multinucleate Cells ◽  
Green Fluorescent

To study centrosome motility and the interaction of microtubules with the cell cortex in mitotic, post-mitotic and interphase cells, (alpha)-tubulin was tagged in Dictyostelium discoideum with green fluorescent protein. Multinucleate cells formed by myosin II-null mutants proved to be especially suited for the analysis of the control of cleavage furrow formation by the microtubule system. After docking of the mitotic apparatus onto the cell cortex during anaphase, the cell surface is activated to form ruffles on top of the asters of microtubules that emanate from the centrosomes. Cleavage furrows are initiated at spaces between the asters independently of the positions of spindles. Once initiated, the furrows expand as deep folds without a continued connection to the microtubule system. Occurrence of unilateral furrows indicates that a closed contractile ring is dispensable for cytokinesis in Dictyostelium. The progression of cytokinesis in the multinucleate cells underlines the importance of proteins other than myosin II in specifying a cleavage furrow. The analysis of centrosome motility suggests a major role for a minus-end directed motor protein, probably cytoplasmic dynein, in applying traction forces on guiding microtubules that connect the centrosome with the cell cortex.

scholarly journals Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

2021 ◽  
Vol 22 (4) ◽  
pp. 2127
Author(s):  
Jakub Suchodolski ◽  
Anna Krasowska
Keyword(s):  
Candida Albicans ◽  
Multidrug Resistance ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Pathogenic Fungus ◽  
Serum Levels ◽  
Efflux Transporters ◽  
Fluconazole Resistance ◽  
Green Fluorescent

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Nematode Infection Triggers the de Novo Formation of Unloading Phloem That Allows Macromolecular Trafficking of Green Fluorescent Protein into Syncytia

2005 ◽  
Vol 138 (1) ◽  
pp. 383-392 ◽  
Author(s):  
Stefan Hoth ◽  
Alexander Schneidereit ◽  
Christian Lauterbach ◽  
Joachim Scholz-Starke ◽  
Norbert Sauer
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Nematode Infection ◽  
Macromolecular Trafficking ◽  
Green Fluorescent

Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes

1999 ◽  
Vol 112 (22) ◽  
pp. 4101-4112 ◽  
Author(s):  
K. Ojima ◽  
Z.X. Lin ◽  
Z.Q. Zhang ◽  
T. Hijikata ◽  
S. Holtzer ◽  
...  
Keyword(s):  
Binding Sites ◽  
Thin Filament ◽  
Fluorescent Protein ◽  
De Novo ◽  
Actin Binding ◽  
Double Staining ◽  
Green Fluorescent ◽  
Short Time ◽  
Alpha Actin ◽  
De Novo Assembling

While over a dozen I-Z-I proteins are expressed in postmitotic myoblasts and myotubes it is unclear how, when, or where these first assemble into transitory I-Z-I bodies (thin filament/Z-band precursors) and, a short time later, into definitive I-Z-I bands. By double-staining the growth tips of transfected myotubes expressing (a) MYC-tagged s-alpha-actinins (MYC/s-alpha-actinins) or (b) green fluorescent protein-tagged titin cap (GFP/T-cap) with antibodies against MYC and I-Z-I band proteins, we found that the de novo assembly of I-Z-I bodies and their maturation into I-Z-I bands involved relatively concurrent, cooperative binding and reconfiguration of, at a minimum, 5 integral Z-band molecules. These included s-alpha-actinin, nebulin, titin, T-cap and alpha-actin. Resolution of the approximately 1.0 microm polarized alpha-actin/nebulin/tropomyosin/troponin thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration. Of particular interest is finding that mutant MYC/s-alpha-actinin peptides (a) lacking spectrin-like repeats 1–4, or consisting of spectrin-like repeats 1–4 only, as well as (b) mutants/fragments lacking titin or alpha-actin binding sites, were promptly and exclusively incorporated into de novo assembling I-Z-I bodies and definitive I-Z-I bands as was exogenous full length MYC/s-alpha-actinin or GFP/T-cap.

scholarly journals G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

2002 ◽  
Vol 156 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Francesca Santini ◽  
Ibragim Gaidarov ◽  
James H. Keen
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Membrane Skeleton ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
Cortical Actin ◽  
Endocytic Machinery ◽  
Green Fluorescent ◽  
G Protein Coupled

Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing β2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

scholarly journals Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex

1997 ◽  
Vol 138 (3) ◽  
pp. 629-641 ◽  
Author(s):  
Janet L. Carminati ◽  
Tim Stearns
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Dynamic Instability ◽  
Dynamic Properties ◽  
Yeast Cells ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Animal Cells ◽  
Green Fluorescent ◽  
Mutant Cells

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)–tubulin fusion protein to observe microtubules in living yeast cells. GFP–tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

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