Assessment of cellular actin dynamics by measurement of fluorescence anisotropy.

To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylenediaminetetraacetate or well-known effectors of the polymerization. This assay gives the overall status of actin dynamics in cell extracts which are the closest conditions to in vivo, implying most of the regulating proteins that are missing in purified actin measurements. It can be used in a large-scale screening for chemical compounds which modulate actin polymerization.

TSH receptor interaction with the extracellular matrix: role on constitutive activity and sensitivity to hormonal stimulation.

Using immunocytochemistry, we have observed that the TSH receptor (TSHR) is concentrated at the leading edge of lamellipodia in both cultured human thyroid cells and in various transfected cells. This segregation of the receptor is due to its interaction with extracellular matrix (ECM) and specially with fibronectin. The TSHR, which interacts with the ECM, is known to undergo cleavage by a matrix metalloprotease. The homologous LH receptor, which does not interact with ECM, is not cleaved. The attachment to the ECM modifies the functional properties of the receptor: it increases adenylate cyclase stimulation by hormone, whereas PLC stimulation is not modified. Furthermore, the constitutive activity of the TSHR is only observed in attached cells, suggesting that it is dependent on TSHR interaction with the ECM. Thus, aside from its classical properties of hormone binding and signalization through G proteins, the TSHR is also involved in cell-matrix interactions, which modulate its functional properties.