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Molecular interaction of S-100 proteins with microtubule proteins in vitro
R Donato1, I Giambanco, M C Aisa
1Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy.
Abstract:
Several procedures were employed to examine the in vitro interaction between S-100 proteins and microtubule proteins. Binding of S-100 to tau factors was observed under all experimental conditions. S-100 binding to microtubule-associated protein 2 (MAP2) was best detected by exposing nitrocellulose-immobilized MAP2 or MAPs to either 125I-labeled S-100 or biotinylated S-100. S-100 binding to tubulin was detected when the two protein fractions were first incubated with each other followed by exposure to the bifunctional cross-linker disuccinimidylsuberate, and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfered onto nitrocellulose paper. By this procedure, complex formation between S-100 and tubulin, as well as between S-100 and a relatively low-molecular-weight MAP, was evidenced by immunoblotting using an anti-S-100 antiserum. Alternatively, complex formation between biotinylated S-100 and either tubulin or MAPs was visualized by means of avidin-peroxidase, after SDS-PAGE of the complex mixtures and transfer of the separated proteins onto nitrocellulose. The interaction between S-100 and tubulin was strictly Ca2+ dependent, and resistant to high concentrations of KCl, colchicine, or vinblastine.
Insights
S-100 proteins bind to microtubule proteins, including tau and MAP2. This interaction with tubulin is calcium-dependent and unaffected by common microtubule-disrupting agents.
Area of Science:
- Biochemistry
- Cell Biology
Background:
- S-100 proteins are a family of calcium-binding proteins involved in various cellular functions.
- Microtubule proteins, including tubulin and microtubule-associated proteins (MAPs), are crucial for cellular structure and dynamics.
Purpose of the Study:
- To investigate the in vitro interactions between S-100 proteins and key microtubule components.
- To characterize the binding of S-100 proteins to tubulin, tau, and MAP2.
Main Methods:
- Utilized various biochemical assays, including nitrocellulose binding assays with labeled S-100.
- Employed cross-linking with disuccinimidylsuberate followed by SDS-PAGE and immunoblotting.
- Investigated calcium dependency and resistance to specific inhibitors.
Main Results:
- S-100 proteins demonstrated binding to tau factors under all tested conditions.
- S-100 binding to microtubule-associated protein 2 (MAP2) was successfully detected.
- Complex formation between S-100 and tubulin was evidenced, particularly under calcium-dependent conditions.
Conclusions:
- S-100 proteins interact with multiple microtubule protein components, including tubulin and MAPs.
- The interaction between S-100 and tubulin is specifically regulated by calcium ions.
- These findings suggest a potential role for S-100 proteins in modulating microtubule functions.