title: Regulation of podosome formation, microglial migration and invasion by Ca(2+)-signaling molecules expressed in podosomes.
abstract: BACKGROUND
  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). 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 Ca(2+) signaling is important for cell migration, cell-substrate adhesion and contraction of the actomyosin network. Here, we hypothesized that podosomes contain Ca(2+)-signaling machinery, and that podosome expression and function depend on Ca(2+) influx and specific ion channels.
  
  
  METHODS
  High-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 Ca(2+)-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.
  
  
  RESULTS
  Microglial 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 Ca(2+) signaling. The core contained calcium release activated calcium (CRAC; Orai1) channels, calmodulin, small-conductance Ca(2+)-activated SK3 channels, and ionized Ca(2+) 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 Ca(2+) or blocking CRAC channels. Blockers of CRAC channels inhibited migration and invasion, and SK3 inhibition reduced invasion.
  
  
  CONCLUSIONS
  Microglia podosome formation, migration and/or invasion require Ca(2+) influx, CRAC, and SK3 channels. Both channels were present in microglial podosomes along with the Ca(2+)-regulated molecules, calmodulin, Iba1 and STIM1. These results suggest that the podosome is a hub for sub-cellular Ca(2+)-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.
authors.affiliation: Toronto Western Research Institute, 399 Bathurst Street, Toronto, Ontario, M5T 2S8, Canada.
authors.forename: Tamjeed A
authors.initials: TA
authors.lastname: Siddiqui
authors.forename: Starlee
authors.initials: S
authors.lastname: Lively
authors.forename: Catherine
authors.initials: C
authors.lastname: Vincent
authors.forename: Lyanne C
authors.initials: LC
authors.lastname: Schlichter
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002921:Cicatrix / Q000378:metabolism / Q000473:pathology; D022083:Cytoplasmic Structures / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000166:cytology* / Q000502:physiology*; D000071740:ORAI1 Protein; D051381:Rats; D017207:Rats, Sprague-Dawley; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism; D050053:TRPM Cation Channels / Q000378:metabolism
chemical_list: D000199:Actins; C100745:Aif1 protein, rat; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005353:Fibronectins; C496901:Kcnn3 protein, rat; D008840:Microfilament Proteins; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; D051640:Small-Conductance Calcium-Activated Potassium Channels; D050053:TRPM Cation Channels; C486584:Trpm7 protein, rat; D002118:Calcium