title: Calmodulin inhibition regulates morphological and functional changes related to the actin cytoskeleton in pure microglial cells.
abstract: The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells derived from mixed primary cultures from embryonal forebrains of rats, were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filopodia and podosome formation. In summary, these CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.
authors.affiliation: Department of Cell Biology and Molecular Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Melinda
authors.initials: M
authors.lastname: Szabo
authors.affiliation: Department of Cell Biology and Molecular Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Karolina
authors.initials: K
authors.lastname: Dulka
authors.affiliation: Department of Cell Biology and Molecular Medicine, University of Szeged, Szeged, Hungary. Electronic address: gulyak@bio.u-szeged.hu.
authors.forename: Karoly
authors.initials: K
authors.lastname: Gulya
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002491:Central Nervous System Agents / Q000494:pharmacology; D005625:Frontal Lobe / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D007093:Imidazoles / Q000494:pharmacology; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008070:Lipopolysaccharides; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology; D017207:Rats, Sprague-Dawley; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002491:Central Nervous System Agents; D007093:Imidazoles; D019394:Ki-67 Antigen; D008070:Lipopolysaccharides; D008840:Microfilament Proteins; D014268:Trifluoperazine; C031938:calmidazolium
keywords: Calmidazolium; Cell viability; Ionized calcium-binding adaptor molecule 1; Lipopolysaccharide; Phagocytosis; Phalloidin; Proliferation; Trifluoperazine