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.

Simulation of P2X‐mediated calcium signalling in microglia

“…In microglia, CaM is linked to cytoskeletal remodelling (Szabo et al . ) and the activation of CN (Furman & Norris, ). The CN phosphatase is well established to play a significant role in glial physiological function, as reviewed by Furman & Norris ().…”

A novel pleiotropic effect of aspirin: Beneficial regulation of pro- and anti-inflammatory mechanisms in microglial cells

“…The fluid-phase phagocytic capabilities of the control and variously treated pure microglial cell cultures were determined via the uptake of fluorescent microspheres (carboxylate-modified polystyrene beads, fluorescent yellow-green (λex ∼ 470 nm; λem ∼ 505 nm), aqueous suspension, 2.0 μm mean particle size; L4530, Sigma) as we described previously (Szabo and Gulya, 2013;Szabo et al, 2016). Unchallenged (control) and LPS-challenged pure microglial cell cultures (subDIV4) with or without aspirin treatments were tested for 24 h. At the end of the treatment period, 1 μl of a 2.5% aqueous suspension of fluorescent microspheres per ml was added to the culture, which was then incubated for 60 min at 37°C.…”

The Nuclear Proteome of White and Gray Matter from Schizophrenia Postmortem Brains

“…A few recent studies have associated calcium/calmodulin-dependent protein with glial cells in functions such as actin cytoskeleton remodeling, glutamate and glycine transport, and regulation of neurotrophic factors [59,60,61,62]. This involves the activation of the PI3K protein which, according to Pérez-Garcia et al [62], is regulated by calmodulin proteins associated with calcium.…”