Microtubule Nucleation in Mouse Bone Marrow–Derived Mast Cells Is Regulated by the Concerted Action of GIT1/βPIX Proteins and Calcium

Abstract

Ag-mediated activation of mast cells initiates signaling events leading to Ca2+ response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow–derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein–coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca2+ affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca2+ level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/βPIX proteins, and Ca2+ in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.

The actin regulator profilin 1 is functionally associated with the mammalian centrosome

“…Next we again took advantage of the KO27 cells, to determine de novo formation of microtubules from interphase centrosomes. To that end, nocodazole-washout experiments were performed as described previously (35,36). As demonstrated by immunofluorescence microscopy of α-tubulin after drug removal and 2 min of recovery in fresh medium at 37°C, the microtubule array reformed more rapidly in KO27 than in the profilin-expressing control cells ( Fig 3A).…”

TUBG1 missense variants underlying cortical malformations disrupt neuronal locomotion and microtubule dynamics but not neurogenesis

“…Construct was verified by sequencing. Lentiviral vector pmTubg1-TagRF with puromycin resistance, encoding mouse γ-tubulin 1 fused to TagRFP was prepared as described 57 . Shortly, cassette encoding mouse γ-tubulin fused to TagRFP was digested out from pmTubg1-TagRFP construct 19 (see above) by Eco RI/ Not I and ligated to pCDH-CMV-MCS-EF1-puro vector (System Biosciences) resulting in the lentiviral construct pmTubg1-TagRFP-puro.…”

Antimicrobial agent triclosan suppresses mast cell signaling via phospholipase D inhibition

“…Even though PLD activity is inhibited by TCS by 15 minutes post-antigen, translocation of PLD 1 is not grossly hindered, likely because its movement toward the plasma membrane begins within~2 minutes post-antigen and has occurred to a large degree with the first 10 minutes. Below we discuss how dampened cytosolic Ca 2+ could cause the inhibitory events cited in (Sulimenko et al, 2015). Taken together, these PKC and PLD studies strongly suggest that a significant accumulated decrease in cytosolic Ca 2+ concentration, which does not occur until~15 minutes post-antigen (Weatherly et al, 2018), along with PLD inhibition ( Figure 5A), are the key events leading to inhibition of degranulation.…”

“…Taken together, these PKC and PLD studies strongly suggest that a significant accumulated decrease in cytosolic Ca 2+ concentration, which does not occur until~15 minutes post-antigen (Weatherly et al, 2018), along with PLD inhibition (Figure 5A), are the key events leading to inhibition of degranulation. Below we discuss how dampened cytosolic Ca 2+ could cause the inhibitory events cited in (Sulimenko et al, 2015).…”