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Microtubule-mediated cellular events such as intracellular transport and the maintenance of cell polarity are highly dependent upon microtubule stability, which is controlled by a repertoire of microtubule-associated proteins (MAPs) in the cell. MAP7 domain-containing protein 3 (Mdp3) has recently been identified as a critical regulator of microtubule stability. However, it remains elusive how Mdp3 carries out this function. In this study, by examination of tubulin partitioning between the polymer and soluble dimer forms, we found that Mdp3 could protect microtubules from cold- or nocodazole-induced depolymerization. Immunoblotting and immunofluorescence microscopy showed that knockdown of Mdp3 expression significantly reduced the level of tubulin acetylation. In vitro tubulin polymerization assays revealed that the amino-terminal region of Mdp3 was necessary for its ability to stabilize microtubules. Immunoprecipitation and pulldown experiments showed that the amino-terminal region mediated the interaction of Mdp3 with histone deacetylase 6 (HDAC6), in addition to its association with tubulin and microtubules. Immunofluorescence microscopy further demonstrated that endogenous Mdp3 and HDAC6 colocalized in the cytoplasm. Moreover, depletion of Mdp3 dramatically increased the activity of HDAC6 toward tubulin deacetylation. These findings suggest that Mdp3 controls microtubule stability through its binding to tubulin and microtubules as well as its regulation of HDAC6 activity. [ABSTRACT FROM AUTHOR]

The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners. [ABSTRACT FROM AUTHOR]

The post-translational modifications (PTMs) occurring on microtubules have been implicated in the regulation of microtubule properties and functions. Acetylated K40 of α-tubulin, a hallmark of long-lived stable microtubules, is known to be negatively controlled by histone deacetylase 6 (HDAC6). However, the vital roles of HDAC6 in microtubule-related processes such as cell motility and cell division cannot be fully explained by the only known target site on tubulin. Here, we attempt to comprehensively map lysine acetylation sites on tubulin purified from mouse brain tissues. Furthermore, mass spectrometry-based quantitative comparison of acetylated peptides from wild-type vs HDAC6 knockout mice allowed us to identify six new deacetylation sites possibly mediated by HDAC6. Thus, adding new sites to the repertoire of HDAC6-mediated tubulin deacetylation events would further our understanding of the multi-faceted roles of HDAC6 in regulating microtubule stability and cellular functions. [ABSTRACT FROM AUTHOR]

Cilia play important roles in sensing extracellular signals and directing fluid flow. Ciliary dysfunction is associated with a variety of diseases known as ciliopathies. Histone deacetylase 6 (HDAC6) has recently emerged as a major driver of ciliary disassembly, but little is known about the downstream players. Here we provide the first evidence that HDAC6-mediated deacetylation of α-tubulin and cortactin is critical for its induction of ciliary disassembly. HDAC6 is localized in the cytoplasm and enriched at the centrosome and basal body. Overexpression of HDAC6 decreases the levels of acetylated α-tubulin and cortactin without affecting the expression or localization of known ciliary regulators. We also find that overexpression of α-tubulin or cortactin or their acetylation-deficient mutants enhances the ability of HDAC6 to induce ciliary disassembly. In addition, acetylation-mimicking mutants of α-tubulin and cortactin counteract HDAC6-induced ciliary disassembly. Furthermore, HDAC6 stimulates actin polymerization, and inhibition of actin polymerization abolishes the activity of HDAC6 to trigger ciliary disassembly. These findings provide mechanistic insight into the ciliary role of HDAC6 and underscore the importance of reversible acetylation in regulating ciliary homeostasis. [ABSTRACT FROM AUTHOR]

Lentiviruses are a class of genetically unique retroviruses that share similar features, despite their wide variety of host species. Transactivator of transcription (Tat) proteins of lentiviruses are critical for the regulation of viral transcription and replication. Recent studies demonstrate that in addition to mediating transactivation, Tat binds to the microtubule cytoskeleton of the host cell and interferes with microtubule dynamics, ultimately triggering apoptosis. This non-canonical function of Tat appears to be critical for the pathogenesis of lentiviral diseases, such as acquired immunodeficiency syndrome. Here, we compare the structure and activity of Tat proteins from three different types of lentiviruses, focusing on the roles of these proteins in the alteration of host microtubule dynamics and induction of apoptosis. We propose that further investigation of the Tat-microtubule interaction will provide important insight into the process of lentiviral pathogenesis and elucidate new avenues for the development of antiviral therapies. [ABSTRACT FROM AUTHOR]

[Display omitted] Endothelial cilia are microtubule-based hair-like protrusions in the lumen of blood vessels that function as fluid mechanosensors to regulate vascular hemodynamics. However, the functions of endothelial cilia in vascular development remain controversial. In this study, depletion of several key proteins responsible for ciliogenesis allows us to identify a cilium-independent role for intraflagellar transport 88 (IFT88) in mammalian angiogenesis. Disruption of primary cilia by heat shock does not affect the angiogenic process. However, depletion of IFT88 significantly inhibits angiogenesis both in vitro and in vivo. IFT88 mediates angiogenesis by regulating the migration, polarization, proliferation, and oriented division of vascular endothelial cells. Further mechanistic studies demonstrate that IFT88 interacts with γ-tubulin and microtubule plus-end tracking proteins and promotes microtubule stability. Our findings indicate that IFT88 regulates angiogenesis through its actions in microtubule-based cellular processes, independent of its role in ciliogenesis. [ABSTRACT FROM AUTHOR]

The cell cycle is key to life. After decades of research, it is unclear whether any parts of this process have yet to be identified. Fam72a is a poorly characterized gene and is evolutionarily conserved across multicellular organisms. Here, we have found that Fam72a is a cell-cycle-regulated gene that is transcriptionally and post-transcriptionally regulated by FoxM1 and APC/C, respectively. Functionally, Fam72a directly binds to tubulin and both the Aα and B56 subunits of PP2A-B56 to modulate tubulin and Mcl1 phosphorylation, which in turn affects the progression of the cell cycle and signaling of apoptosis. Moreover, Fam72a is involved in early responses to chemotherapy, and it efficiently antagonizes various anticancer compounds such as CDK and Bcl2 inhibitors. Thus, Fam72a switches the tumor-suppressive PP2A to be oncogenic by reprogramming its substrates. These findings identify a regulatory axis of PP2A and a protein member in the cell cycle and tumorigenesis regulatory network in human cells. [Display omitted] • Fam72a is a cell-cycle-regulated gene • Fam72a interacts with the PP2A-B56 complex • Fam72a regulates tubulin phosphorylation • Fam72a controls Mcl-1 phosphorylation Fu et al. find a cell cycle gene, Fam72a, that supports cell proliferation, antagonizes apoptosis, and controls tubulin and Mcl-1 phosphorylation during the G2/M phase. This work defines the function of Fam72 in regulating PP2A in a cell cycle and tumorigenesis regulatory network. [ABSTRACT FROM AUTHOR]

Microtubule-binding proteins provide an alternative and vital pathway to the functional diversity of microtubules. Considerable work is still required to understand the complexities of microtubule-associated cellular processes and to identify novel microtubule-binding proteins. In this study, we identify Bcl2-associated athanogene cochaperone 6 (BAG6) as a novel microtubule-binding protein and reveal that it is crucial for primary ciliogenesis. By immunofluorescence we show that BAG6 largely colocalizes with intracellular microtubules and by co-immunoprecipitation we demonstated that it can interact with α-tubulin. Additionally, both the UBL and BAG domains of BAG6 are indispensable for its interaction with α-tubulin. Moreover, the assembly of primary cilia in RPE-1 cells is significantly inhibited upon the depletion of BAG6. Notably, BAG6 inhibition leads to an abnormal G0/G1 transition during the cell cycle. In addition, BAG6 colocalizes and interactes with the centrosomal protein γ-tubulin, suggesting that BAG6 might regulate primary ciliogenesis through its action in centrosomal function. Collectively, our findings suggest that BAG6 is a novel microtubule-bindng protein crucial for primary ciliogenesis. [ABSTRACT FROM AUTHOR]