Background: p87 and p101 represent non-catalytic subunits of class I B PI3K␥. Results: Expression and activity of PI3K␥ is modified differently by p87 and p101 in vitro and in living cells. Conclusion: Non-catalytic subunits of PI3K␥ represent two different regulators in the absence of G␥ or Ras. Significance: p87 and p101 determine diversity within class I B PI3K␥ and allow integration in distinct PI3K␥ signaling pathways.
Background/Aims: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary (“hair”) bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. Methods: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. Results: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. Conclusion: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes.
The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic-SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B-1 cells, the major natural IgM-producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL-5 receptor α chain on B-1 cells, resulting in decreased B-1 cell numbers and decreased differentiation into Ab-secreting cells. Since B-1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal-specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.
Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.
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