In C, E, and G values are expressed as percentage of the control. In neurons these structures appeared as small structures, distributed especially along the neurites and at their terminal tips. After 30 min in the incubator, the cells were monitored by phase-contrast microscopy, with images taken at 0, 18, 24, and 48 h. Please review our privacy policy. FM fluorescence was assayed as described by Bertrand et al.

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In other processes, such as exo- and a03p, the role of the protein appears viseo important. In the A30P Syn-expressing cells, the cytoskeleton, already disrupted at rest, during recovery from LatA exhibited a number of discrete, subplasmalemma puncta, composed by a core of A30P synuclein surrounded by a shell of compacted actin defined from hereon as actin-rich foci. For electron microscopy analysis, grids were washed twice with water and incubated with a saturated solution of uranyl acetate for 5 min.

Double-knockout mice for alpha- and beta-synucleins: Exo- and endocytic traffic was stopped with ice-cold PBS before fixation. After treatment, the drug was washed out and the cells were reincubated in conventional medium to let their cytoskeleton repolymerize.

Wash out of the drug was followed by reorganization of the cytoskeleton, which viceo was still incomplete after 20 min. Please review our privacy policy.

A rat brain homogenate, prepared in a bounce homogenizer in 0. For depolymerization experiments actin was polymerized as above for 1 h. Cells were then washed, incubated in complete medium for 0, 10, 20, 40, or 60 min and processed for immunofluorescence. The ultrastructural distribution of alpha-synuclein-like protein in normal mouse brain. However, this did not appear to be the case, because the levels of actin phosphorylation were similarly low in all MDCK clones Supplementary Figure S2C.


α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

Pellets and supernatants were analyzed by Coomassie blue staining of SDS gels. After washing to eliminate the noninternalized dye the cells were fixed and analyzed for FM fluorescence. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded.

The subsequent disappearance of these structures was slower than in the fixed samples Figure 5 Cwith a few foci still detectable at 60 min. Depletion of the reserve pool of synaptic vesicles has been reported by one group Cabin et al.

The marks present in every image were aligned, and the migration distance was measured, keeping the marks as reference, in 10 different points of five fields. Alpha-synuclein and Parkinson’s disease. vieo

The two proteins have been observed to partially colocalize in vjdeo neuronal cell lines Esposito et al. Nucleofection of primary neurons. The role of local actin instability in axon formation.

LatA was used to analyze separately, within the cell, the local depolymerization of actin, taking place by its action, from the repolymerization taking place after drug washout.

Right and bottom panels, the Y-Z and X-Z projections at positions marked by the vertical and horizontal gray lines, respectively. Fiber length was calculated with the Neuron J a3p0 Meijering et al. Protein expression w30p a Drosophila model of Parkinson’s disease. Filamentous actin structures formed during the first 3 min of incubation with LatA were no longer evident after 10 min, when the actin cytoskeleton was completely disassembled. Figure 4 A shows filamentous and soluble fractions of actin separated by high-speed centrifugation, in either the absence or presence of LatA.


α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

Open in a separate window. Immunofluorescence of cells fixed and analyzed at various time points during LatA experiments Figure 4 B revealed a clear disruption of the actin cytoskeleton in the cells treated with the drug. The following antibodies were used: Notice in the top row the appearance of lamellipodia guiding the cell movement during wash out of LatA; in the low bottom row the appearance, already at 2 min, of large subplasmalemma GFP-actin aggregates persisting up to the end of the experiment.

In control MDCK cells, LatA induced a disruption of the actin network into fragments scattered throughout the cytoplasm, and a partial release of the cell-to-cell contacts that were complete after min treatment.

Coverslips were mounted and photographed as for immunofluorescence. As control samples, either beads alone or beads incubated with the preadsorbed anti-actin antibody were used. Alternatively, undiluted actin was seeded on carbon-coated copper grids at room temperature for 30 min.