Abstract
BackgroundMicroglia migrate during brain development and after CNS injury, but it is not known how they degrade the extracellular matrix (ECM) to accomplish this. Podosomes are tiny structures with the unique ability to adhere to and dissolve ECM. Podosomes have a two-part architecture: a core that is rich in F-actin and actin-regulatory molecules (for example, Arp2/3), surrounded by a ring with adhesion and structural proteins (for example, talin, vinculin). We recently discovered that the lamellum at the leading edge of migrating microglia contains a large F-actin-rich superstructure (‘podonut’) composed of many podosomes. Microglia that expressed podosomes could degrade ECM molecules. Finely tuned Ca2+ signaling is important for cell migration, cell-substrate adhesion and contraction of the actomyosin network. Here, we hypothesized that podosomes contain Ca2+-signaling machinery, and that podosome expression and function depend on Ca2+ influx and specific ion channels.MethodsHigh-resolution immunocytochemistry was used on rat microglia to identify podosomes and novel molecular components. A pharmacological toolbox was applied to functional assays. We analyzed roles of Ca2+-entry pathways and ion channels in podosome expression, microglial migration into a scratch-wound, transmigration through pores in a filter, and invasion through Matrigel™-coated filters.ResultsMicroglial podosomes were identified using well-known components of the core (F-actin, Arp2) and ring (talin, vinculin). We discovered four novel podosome components related to Ca2+ signaling. The core contained calcium release activated calcium (CRAC; Orai1) channels, calmodulin, small-conductance Ca2+-activated SK3 channels, and ionized Ca2+ binding adapter molecule 1 (Iba1), which is used to identify microglia in the CNS. The Orai1 accessory molecule, STIM1, was also present in and around podosomes. Podosome formation was inhibited by removing external Ca2+ or blocking CRAC channels. Blockers of CRAC channels inhibited migration and invasion, and SK3 inhibition reduced invasion.ConclusionsMicroglia podosome formation, migration and/or invasion require Ca2+ influx, CRAC, and SK3 channels. Both channels were present in microglial podosomes along with the Ca2+-regulated molecules, calmodulin, Iba1 and STIM1. These results suggest that the podosome is a hub for sub-cellular Ca2+-signaling to regulate ECM degradation and cell migration. The findings have broad implications for understanding migration mechanisms of cells that adhere to, and dissolve ECM.
Highlights
In the healthy adult Central nervous system (CNS), microglia constantly survey the environment, extending and retracting their ramified processes, but without overall cell displacement [1,2]
We made the surprising discovery that the small-conductance Ca2+-activated K+ channel, SK3, is enriched at the leading edge of migrating microglia
TRP melastatin 7 (TRPM7) can be blocked by 2-aminoethyl diphenylborinate (2-APB) ([17]; and see Discussion), and we found that 50 μM 2-APB nearly abolished podonuts (0.2 ± 0.2% of cells; Figure 4D,G)
Summary
In the healthy adult CNS, microglia constantly survey the environment, extending and retracting their ramified processes, but without overall cell displacement [1,2]. Outside the CNS, two related structures identified in several cell types have the unique ability to both adhere to and degrade ECM molecules. Podosomes (non-cancer cells) and invadopodia (cancer cells) differ from other cell adhesion structures in this dual functionality, and in their composition, architecture and dynamics (recently reviewed in [7,8,9]). Microglia migrate during brain development and after CNS injury, but it is not known how they degrade the extracellular matrix (ECM) to accomplish this. Podosomes are tiny structures with the unique ability to adhere to and dissolve ECM. Podosomes have a two-part architecture: a core that is rich in F-actin and actin-regulatory molecules (for example, Arp2/3), surrounded by a ring with adhesion and structural proteins (for example, talin, vinculin). Microglia that expressed podosomes could degrade ECM molecules. We hypothesized that podosomes contain Ca2+-signaling machinery, and that podosome expression and function depend on Ca2+ influx and specific ion channels
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