Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

• Keywords
• G protein-coupled receptor kinase-interacting protein 2 (GIT2), centrosome, mast cells, microtubule nucleation, protein kinase C (PKC),
• MeSH
• Centrosome metabolism   MeSH
• Bone Marrow * MeSH
• Mast Cells * metabolism   MeSH
• Microtubules * metabolism   MeSH
• Mice MeSH
• GTPase-Activating Proteins * metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Git2 protein, mouse MeSH Browser
• GTPase-Activating Proteins * MeSH

A better understanding of the molecular mechanisms leading to mast cell migration and chemotaxis is the long-term goal in mast cell research and is essential for comprehension of mast cell function in health and disease. Various techniques have been developed in recent decades for in vitro and in vivo assessment of mast cell motility and chemotaxis. In this chapter, three microscopy assays facilitating real-time quantification of mast cell chemotaxis and migration are described, focusing on individual cell tracking and data analysis.

• Keywords
• Cell migration, Cell tracking, Chemoattractant, Chemokine, Chemotaxis, Mast cells,
• MeSH
• Cell Migration Assays methods   MeSH
• Biological Assay methods   MeSH
• Cell Tracking methods   MeSH
• Chemotaxis physiology   MeSH
• Fibronectins metabolism   MeSH
• Humans MeSH
• Mast Cells cytology   physiology   MeSH
• Microscopy methods   MeSH
• Mice MeSH
• Computer Systems MeSH
• Cell Movement physiology   MeSH
• Environment, Controlled MeSH
• Sepharose MeSH
• Software MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fibronectins MeSH
• Sepharose MeSH

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

• Keywords
• SHP-1 tyrosine phosphatase, bone marrow-derived mast cells, cell activation, microtubule nucleation, γ-tubulin complexes,
• MeSH
• Cell Degranulation MeSH
• HEK293 Cells MeSH
• Syk Kinase metabolism   MeSH
• Humans MeSH
• Mast Cells cytology   metabolism   MeSH
• MCF-7 Cells MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Tubulin metabolism   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 antagonists & inhibitors   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Syk Kinase MeSH
• Ptpn6 protein, mouse MeSH Browser
• Syk protein, mouse MeSH Browser
• Tubulin MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 MeSH