Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.
Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca2+, we characterize three kinetically different pools of vesicles in rat pancreatic β-cells, namely, a highly calcium-sensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is ∼20 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca2+, we find that the HCSP is a distinct population of vesicles from those colocalized with Ca2+ channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca2+.
A critical part of the functional development of our peripheral balance system is the embryonic formation of otoconia, composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the gravity receptor in the inner ear. To date neither the functions of otoconial proteins nor the processes of crystal formation are clearly defined. Using gene targeting and protein analysis strategies, we demonstrate that the predominant mammalian otoconin, otoconin-90/95 (Oc90), is essential for formation of the organic matrix of otoconia by specifically recruiting other matrix components, which includes otolin, a novel mammalian otoconin that we identified to be in wildtype murine otoconia. We show that this matrix controls otoconia growth and morphology by embedding the crystallites during seeding and growth. During otoconia development, the organic matrix forms prior to CaCO3 deposition and provides optimal calcification efficiency. Histological and ultrastructural examinations show normal inner ear epithelial morphology but reduced acellular matrices, including otoconial, cupular and tectorial membranes, in Oc90 null mice, likely due to an absence of Oc90 and a profound reduction of otolin. Our data demonstrate the critical roles of otoconins in otoconia seeding, growth and anchoring and suggest mechanistic similarities and differences between otoconia and bone calcification.
Our results suggest that the SARS outbreak was significantly associated with DAWV, and that DAAP, DARH and DHS may also have influenced the SARS outbreak to some extent. However, because of ecological fallacy and uncontrolled confounding effects that may have biased the results, the association between the SARS outbreak and these meteorological factors and air pollution deserve further investigation.
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