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Every Petri dish was marked, and the confluent cell monolayer was scratched along the marked line using a micropipette tip. The latter process resembles the in vitro de novo actin polymerization. FM fluorescence was assayed as described by Bertrand et al. S, supernatant of the P2 centrifugation half of the supernatant was loaded. Cells were then washed, incubated in complete medium for 0, 10, 20, 40, or 60 min and processed for immunofluorescence. Wash out of the drug was followed by reorganization of the cytoskeleton, which however was still incomplete after 20 min.

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Pellets and supernatants were analyzed by Coomassie blue staining of SDS gels. Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion. 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. Exo- and endocytic traffic was stopped with ice-cold PBS before fixation.

The reduction of cell size, that was already noticed at steady state in the A30P cells see also Figure 3B and Dwas even more pronounced upon disassembly of the actin cytoskeleton and during the first phases of reassembly Figure 4 C. 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.

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Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.

In C, E, and G values are expressed as percentage of the control. 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.

Please review our privacy policy. Figure 7 C z30p the cells mobility, assayed by taking the center of the nucleus as a reference point.

The two proteins have been observed to partially colocalize in two neuronal cell lines Esposito et al. C Negative staining of actin bundles prepared as in A. Alpha-synuclein and Parkinson’s disease. How is actin polymerization nucleated in vivo?

The subsequent disappearance of these structures was slower than in the fixed samples Figure 5 Cwith a few foci still detectable at 60 min.

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

Reduced neuritic outgrowth and cell adhesion in viveo cells transfected with human alpha-synuclein. Activity-related redistribution of presynaptic proteins at the active zone.

Cytoskeleton recovery was analyzed at 1 and 2 h after LatA washout.

F Kinetics of actin depolymerization. The number of foci was normalized for the number of neurons. Rare genetic mutations shed light on the pathogenesis of Parkinson disease.

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

For electron microscopy analysis, grids were washed twice with water and incubated with a saturated solution of uranyl acetate for 5 min. Journal List Mol Biol Cell v. The presence of the foci was quantified by measuring the areas exhibiting actin fluorescence intensity above an arbitrary threshold Figure 5 E. This structure, in fact, appears to decrease the probability of spontaneous vesicle exocytosis in resting cells. Protein expression in a Drosophila model of Parkinson’s disease.

The following antibodies were used: The cytoplasm volume was calculated subtracting the nucleus volume from the total cell volume, assuming spherical shapes.

Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. Neural activity controls the synaptic accumulation of alpha-synuclein.

In fact, the disruption of the actin cytoskeleton visible after 3-min treatment with LatA was similar to that seen in control cells after 10 min. Occludin is a functional component of the tight junction. Coverslips were mounted and photographed as for immunofluorescence.

Methods for detecting internalized, FM stained particles in epithelial cells and monolayers.