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

Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.

• Keywords
• microtubule nucleation, αβ-tubulin dimer, γ-tubulin functions, γ-tubulin isotypes, γ-tubulin ring complexes (γ-TuRC),
• Publication type
• Journal Article MeSH
• Review MeSH

ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.

• Keywords
• CDK5RAP3, ER stress, UFL1, microtubule nucleation, γ-tubulin,
• MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Endoplasmic Reticulum Stress physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK5RAP3 protein, human MeSH Browser
• Tumor Suppressor Proteins MeSH
• Cell Cycle Proteins MeSH

In cells, microtubules typically nucleate from microtubule organizing centers, such as centrosomes. γ-Tubulin, which forms multiprotein complexes, is essential for nucleation. The γ-tubulin ring complex (γ-TuRC) is an efficient microtubule nucleator that requires additional centrosomal proteins for its activation and targeting. Evidence suggests that there is a dysfunction of centrosomal microtubule nucleation in cancer cells. Despite decades of molecular analysis of γ-TuRC and its interacting factors, the mechanisms of microtubule nucleation in normal and cancer cells remains obscure. Here, we review recent work on the high-resolution structure of γ-TuRC, which brings new insight into the mechanism of microtubule nucleation. We discuss the effects of γ-TuRC protein dysregulation on cancer cell behavior and new compounds targeting γ-tubulin. Drugs inhibiting γ-TuRC functions could represent an alternative to microtubule targeting agents in cancer chemotherapy.

• Keywords
• cancers, microtubule nucleation, γ-tubulin complexes,
• Publication type
• Journal Article MeSH
• Review MeSH

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

• MeSH
• Actins metabolism   MeSH
• Caco-2 Cells MeSH
• Centrosome metabolism   MeSH
• Formins metabolism   MeSH
• Gene Knockout Techniques MeSH
• Humans MeSH
• Melanoma, Experimental metabolism   pathology   MeSH
• Microfilament Proteins metabolism   MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Skin Neoplasms metabolism   pathology   MeSH
• Polymerization MeSH
• Profilins genetics   metabolism   MeSH
• Signal Transduction genetics   MeSH
• Transfection MeSH
• Tubulin metabolism   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
• Actins MeSH
• Formins MeSH
• Microfilament Proteins MeSH
• PFN1 protein, human MeSH Browser
• Pfn1 protein, mouse MeSH Browser
• Profilins MeSH
• Tubulin 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

• Publication type
• Editorial MeSH