Microglia were fixed at 30 min and podonuts were visua lized by staining for F actin and talin. Representative images Sorafenib are shown in Figure 4A to Inhibitors,Modulators,Libraries 4F, and the proportion of cells with a podonut is summarized in Figure 4G. Omitting external Ca2 reduced podonut prevalence by more than tenfold, from 10. 9 1. 2% of cells in con trol solution to 1. 0 0. 2% in Ca2 free so lution. The broad spectrum Ca2 channel blocker, 5 uM Gd3, decreased podonut prevalence to a similar degree, to 0. 7 0. 2% of cells. Rat microglia express several Ca2 permeable transient receptor Inhibitors,Modulators,Libraries potential channels, including TRP mela statin 7. TRPM7 can be blocked by 2 APB, and we found that 50 uM 2 APB nearly abolished podonuts. Importantly, this concentration of 2 APB also blocks the highly Ca2 selective CRAC chan nel produced by the pore forming subunit, Orai1.

We previously showed that CRAC is a major component of store operated Ca2 entry in rat microglia, and is Inhibitors,Modulators,Libraries effectively blocked by 2 APB without Inhibitors,Modulators,Libraries toxicity. Therefore, we next tested BTP2, a more selective CRAC channel blocker with reported IC50 values ranging from 0. 1 to 2. 2 uM. At 10 uM, BTP2 decreased podo nut prevalence to 3. 5 1. 0% of cells. At 1 uM, there was no inhibition by BTP2. This result rules out the main potential side effect of BTP2. That is, Ca2 entry through TRPM4 channels in lymphocytes was enhanced by low nanomolar concen trations of BTP2. This was a concern because TRPM4 is expressed in murine microglia. Finally, to further distinguish between CRAC and TRPM7 chan nels, we applied spermine.

We previously showed that the concentration used effectively blocks TRPM7 in rat microglia, without toxicity. Podonut prevalence was not reduced by 100 uM spermine, it remained at 9. 9 2. 0% of cells. This provides evidence against non specific effects or toxicity of spermine. To gether, these results show that podosomes require Ca2 entry, most likely through CRAC Inhibitors,Modulators,Libraries channels. CRAC current is produced by the pore forming sub unit, Orai1, and requires transient interaction with STIM1, a Ca2 sensor protein that is primarily localized to the endoplasmic reticulum membrane. Orai1 immunoreactivity was highly enriched in podonuts and in the core of individual podo somes, where it co localized with Arp2. As expected for an ER associated protein, STIM1 was enriched near the nucleus, and was wide spread throughout the cell.

STIM1 was also prevalent in the podonut, near the podosome ring component, more info vinculin. High magnification images show a close association between STIM1 and vinculin, with some co localization in podosome rings. An earlier study over expressed TRPM7 in the N1E 115 neuroblastoma cell line, and showed that it induced podosomes and was present in them. In contrast, in microglia, block of native TRPM7 channels with spermine had no effect and the channel was not enriched in podonuts. Microtubules regu late podosome dynamics and localization in monocytic cells.