PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P2 in yeast and mouse

The signalling lipid PI(3,5)P2 is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P2 levels. Mutations that lower PI(3,5)P2 cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P2 was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P2 regulatory complex by direct contact with the known regulators of PI(3,5)P2: Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P2 regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P2. Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse

Acute increase of alpha-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation

This work was supported by grants from the NIH/National Institute of Neurological Disorder and Stroke RO1 NS078165 (to J.R.M.), the Morton Cure Paralysis Fund (to J.R.M.), and the Branfman Family Foundation (to J.M.G.) and by a Dorothea Bennett graduate fellowship (to D.J.B.)

Mouse Hepatitis Coronavirus RNA Replication Depends on GBF1-Mediated ARF1 Activation

Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected

The ongoing pursuit of neuroprotective therapies in Parkinson disease

Many agents developed for neuroprotective treatment of Parkinson disease (PD) have shown great promise in the laboratory, but none have translated to positive results in patients with PD. Potential neuroprotective drugs, such as ubiquinone, creatine and PYM50028, have failed to show any clinical benefits in recent high-profile clinical trials. This 'failure to translate' is likely to be related primarily to our incomplete understanding of the pathogenic mechanisms underlying PD, and excessive reliance on data from toxin-based animal models to judge which agents should be selected for clinical trials. Restricted resources inevitably mean that difficult compromises must be made in terms of trial design, and reliable estimation of efficacy is further hampered by the absence of validated biomarkers of disease progression. Drug development in PD dementia has been mostly unsuccessful; however, emerging biochemical, genetic and pathological evidence suggests a link between tau and amyloid-β deposition and cognitive decline in PD, potentially opening up new possibilities for therapeutic intervention. This Review discusses the most important 'druggable' disease mechanisms in PD, as well as the most-promising drugs that are being evaluated for their potential efficiency in treatment of motor and cognitive impairments in PD