Activation of microtubule dynamics increases neuronal growth via the nerve growth factor (NGF)- and Gαs-mediated signaling pathways.

Signals that activate the G protein Gαs and promote neuronal differentiation evoke Gαs internalization in rat pheochromocytoma (PC12) cells. These agents also significantly increase Gαs association with microtubules, resulting in an increase in microtubule dynamics because of the activation of tubulin GTPase by Gαs. To determine the function of Gαs/microtubule association in neuronal development, we used real-time trafficking of a GFP-Gαs fusion protein. GFP-Gαs concentrates at the distal end of the neurites in differentiated living PC12 cells as well as in cultured hippocampal neurons. Gαs translocates to specialized membrane compartments at tips of growing neurites. A dominant-negative Gα chimera that interferes with Gαs binding to tubulin and activation of tubulin GTPase attenuates neurite elongation and neurite number both in PC12 cells and primary hippocampal neurons. This effect is greatest on differentiation induced by activated Gαs. Together, these data suggest that activated Gαs translocates from the plasma membrane and, through interaction with tubulin/microtubules in the cytosol, is important for neurite formation, development, and outgrowth. Characterization of neuronal G protein dynamics and their contribution to microtubule dynamics is important for understanding the molecular mechanisms by which G protein-coupled receptor signaling orchestrates neuronal growth and differentiation.

Effect of a Stannous Fluoride Dentifrice on Biofilm Composition, Gene Expression and Biomechanical Properties.

An in situ study was conducted to examine the mode of action of a 0.454% stannous fluoride (SnF2)-containing dentifrice in controlling the composition and properties of oral biofilm. Thirteen generally healthy individuals participated in the study. Each participant wore an intra-oral appliance over a 48-h period to measure differences in the resulting biofilm's architecture, mechanical properties, and bacterial composition after using two different toothpaste products. In addition, metatranscriptomics analysis of supragingival plaque was conducted to identify the gene pathways influenced. The thickness and volume of the microcolonies formed when brushing with the SnF2 dentifrice were dramatically reduced compared to the control 0.76% sodium monofluorophosphate (MFP)-containing toothpaste. Similarly, the biophysical and nanomechanical properties measured by atomic force microscopy (AFM) demonstrated a significant reduction in biofilm adhesive properties. Metatranscriptomic analysis identified pathways associated with biofilm formation, cell adhesion, quorum sensing, and N-glycosylation that are significantly downregulated with SnF2. This study provides a clinically relevant snapshot of how the use of a stabilized, SnF2 toothpaste formulation can change the spatial organization, nanomechanical, and gene expression properties of bacterial communities.

Heterotrimeric G-proteins associate with microtubules during differentiation in PC12 pheochromocytoma cells.

Tubulin modifies G-protein signaling and heterotrimeric G-proteins regulate microtubule assembly. Here we report an interplay among G-protein-coupled receptor and receptor tyrosine kinase (such as nerve growth factor-NGF) signaling systems in PC12 pheochromocytoma cells that resulted in a translocation of Galpha(s), Galpha(i1), and Galpha(o) from cell bodies to cellular processes where they appear to localize with tubulin-containing structures. This relocation appeared to depend on the integrity of microtubules, as it was blocked and reversed by nocodazole. Latrunculin, which promotes actin filament depolymerization, had no effect. Both deconvolution microscopy and immunoprecipitation showed a significant increase of Galpha association with microtubules that was coincident with the extension of "neurites." There were distinctions among the Galpha subtypes, with Galpha(s) showing the most profound NGF-induced colocalization with tubulin. Translocation of Galpha was blocked by agents that inhibit the MAP kinases required for neuronal differentiation, suggesting that G-protein relocation is triggered by the intracellular signals for differentiation. Consistent with this, Galpha in Neuro-2A cells, which spontaneously differentiate, showed a similar translocation coincident with differentiation. Thus, diverse signals that promote neuronal differentiation and changes in cell morphology may use specific G-proteins to evoke cytoskeletal rearrangement.

Internalization and axonal transport of the HIV glycoprotein gp120.

The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity.

Cytosolic G{alpha}s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth.

It is now evident that Galpha(s) traffics into cytosol following G protein-coupled receptor activation, and alpha subunits of some heterotrimeric G-proteins, including Galpha(s) bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein alpha subunits remain unclear. In this study, several biochemical approaches demonstrated that activated Galpha(s) directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Galpha(s) colocalized with microtubules. The activated, GTP-bound, Galpha(s) mimicked tubulin in serving as a GTPase activator for beta-tubulin. As a result, activated Galpha(s) made microtubules more dynamic, both in vitro and in cells, decreasing the pool of insoluble microtubules without changing total cellular tubulin content. The amount of acetylated tubulin (an indicator of microtubule stability) was reduced in the presence of Galpha(s) activated by mutation. Previous studies showed that cholera toxin and cAMP analogs may stimulate neurite outgrowth in PC12 cells. However, in this study, overexpression of a constitutively activated Galpha(s) or activation of Galpha(s) with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated Galpha(s) acts as an intracellular messenger to regulate directly microtubule dynamics and promote neurite outgrowth. These data serve to link G-protein signaling with modulation of the cytoskeleton and cell morphology.