Heterotrimeric G Proteins and the Regulation of Microtubule Assembly

Microtubules (MTs), a major component of cell cytoskeleton, exhibit diverse cellular functions including cell motility, intracellular transport, cell division, and differentiation. These functions of MTs are critically dependent on their ability to polymerize and depolymerize. Although a significant progress has been made in identifying cellular factors that regulate microtubule assembly and dynamics, the role of signal transducing molecules in this process is not well understood. It has been demonstrated that heterotrimeric G proteins, which are components of G protein-coupled receptor (GPCR) signaling pathway, interact with microtubules and play important roles in regulating assembly/dynamics of this cytoskeletal filament. While α subunit of G proteins (Gα) inhibits microtubule assembly and accelerates microtubule dynamics, Gβγ promotes tubulin polymerization. In this chapter, we review the current status of G-protein modulation of microtubules and cellular and physiological aspects of this regulation. Molecular, biochemical, and cellular methodologies that have been used to advance this field of research are discussed. Emphasis has been given on G-protein-microtubule interaction in neuronal differentiation as significant progress has been made in this field. The outcome from this research reflects the importance of GPCRs in transducing extracellular signals to regulate a variety of microtubule-associated cellular events

G Protein α subunits activate tubulin GTPase and modulate microtubule polymerization dynamics

G proteins serve many functions involving the transfer of signals from cell surface receptors to intracellular effector molecules. Considerable evidence suggests that there is an interaction between G proteins and the cytoskeleton. In this report, G protein α subunits Gi1α, Gsα, and Goα are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. Giα inhibited polymerization of tubulin-GTP into microtubules by 80-90% in the absence of exogenous GTP. Addition of exogenous GTP, but not guanylylimidodiphosphate, which is resistant to hydrolysis, overcame the inhibition. Analysis of the dynamics of individual microtubules by video microscopy demonstrated that Gi1α increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, Gα may play a role in modulating microtubule dynamic instability, providing a mechanism for the modification of the cytoskeleton by extracellular signals

Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction

Background: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. Results: Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells. Conclusions: Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement. Electronic supplementary material The online version of this article (doi:10.1186/s12868-014-0132-4) contains supplementary material, which is available to authorized users

Zinc-induced self-assembly of goat brain tubulin: some novel aspects

Unlike normal microtubule assembly, the in vitro assembly of DEAE-purified goat brain tubulin in presence of Zn(II) is not inhibited by suprastoichiometric concentrations of antimicrotubular drugs like colchicine and podophyllotoxin. However, assembly in the presence of Zn(II) is inhibited by vinblastine. Vinblastine sensitivity of the assembly process depends on the Mg(II) concentration in the assembly medium. Like normal microtubules, Zn(II)-induced polymers are sensitive to cold. The polymers assembled in presence of Zn(II) are readily disassembled on treatment with Zn(II)-chelators like EDTA or o-phenanthroline, indicating that the binding of Zn(II) to tubulin is essential for maintaining the polymeric structure. MAPs, Microtubule-associated proteins; MES, 2-(N-Morpholino) ethane sulfonic acid; MT, Microtubule; CD, Colchicine-tubulin dimer complex; GTP, Guanosine-5'-triphosphate; SDS, Sodium dodecyl sulphate; EDTA, Ethylenediamine-tetraacetic acid; Buffer A, 0.1 M MES (pH 6.4), 0.5 mM MgCl2, 1 mM GTP; Buffer B, 4 M glycerol in buffer A

Gβγ-Microtubule Interaction and Neurodegeneration

Aberrant organization of microtubules (MTs) is known to be a feature of neurodegeneration, which occurs in many neurological disorder including Alzheimer’s disease, Parkinson’s disease, neuropsychiatric illness such as schizophrenia, and drug addiction. Previously, Gβγ, an important component of G protein-coupled signaling pathways, has been shown to regulate neurite outgrowth by interacting with MTs. The aim of the present study is to understand the Gβγ regulation of MT assembly and its relation to neurodegeneration, using GRK-ct peptide (consists of the carboxy terminus of G protein-coupled-receptor kinase) which is known to inhibit Gβγ-dependent signaling by binding to and sequestering Gβγ. PC12 cells were used to conduct the study because they respond to nerve growth factor (NGF) with growth arrest and exhibit a typical phenotype of neuronal cells sending out neurites. PC12 cells were treated with NGF over the course of three days followed by treatment with GRK-ct peptide for 1h, and the result was evaluated by subcellular fractionation, and confocal microscopy. Confocal microscopy revealed that the GRK-ct peptide has a very dramatic effect on morphology and survival of NGF-differentiated PC12 cells, causing cellular aggregation, neurite and MT disruption, and severe degeneration. Interestingly, the levels of MAP2, a cytoskeletal protein that stabilizes MTs, dramatically decreased in the presence of GRK-ct peptide. The results indicate that the inhibition of Gβγ-dependent signaling induce cytoskeletal alterations and neurodegeneration. Future investigation will address whether Gβγ signaling could be targeted for developing novel drugs against drug addiction and other neurodegenerative disorders

Inhibitors of Polyisoprenylated Methylated Protein Methyl Esterase (PMPMEase) cause Neurodegeneration by altering Microtubules and Gβγ in PC12 Cells

Neurodegeneration, a progressive loss of nerve cells (neurons), occurs in many neurological disorders including Alzheimer’s disease, Parkinson’s disease, Schizophrenia and drug addiction. Disruption of Microtubules (MTs), a major component of cytoskeleton and aggregation of proteins associated with them is the hallmark of neurodegeneration. Gβγ, an important component of G protein signaling has been shown to induce neurite outgrowth of PC12 cells by interacting with microtubules. The goal of the present study is to understand whether interfering with Gβγ-MT mediated pathway causes neurodegeneration. Because prenylation of γ subunits is important for the interaction of Gβγ with MTs, we used inhibitors (L-23 and L-28) for PMPMEase (polyisoprenylated methylated protein methyl esterase), an enzyme involved in the prenylation pathway to conduct the study. PC12 cells were treated with NGF over the course of three days, followed by overnight treatment with L-28 or L-23. Confocal microscopy was used to analyze the results. We found that more than 70% of PC12 cells exhibit neurite formation in the presence of NGF. Neurite formation was not affected significantly in the presence of PMSF or L-23 (100mM). L-28 (10μM), on the other hand significantly reduced neurite formation as well as MTs and Gβγ labeling. In addition, severe cellular degeneration was observed (more than 60% areas in the slides). The result suggests that inhibitors of PMPMEase could be used as valuable tools to study the mechanism of neurodegeneration and design strategies to develop effective drugs against drug addiction and other neurodegenerative disorders

Glucosylceramide Transferase Activity is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan, Giardia lamblia

The production of viable cysts by Giardia is essential for its survival in the environment and spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst-wall proteins to trophozoite’s plasma membrane before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by anti-sense morpholino oligonucleotide abolished ESV production and generated mostly non-viable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapie