Micrometric lipid compartmentation at the plasma membrane is disputed. Using live confocal imaging, we found that three unrelated fluorescent sphingomyelin (SM) analogs spontaneously clustered at the outer leaflet into micrometric domains, contrasting with homogeneous labelling by DiIC18 and TMA-DPH. In erythrocytes, these domains were round, randomly distributed, and reversibly coalesced under hypotonicity. BODIPY-SM and -glucosylceramide showed distinct temperature-dependence, in the same ranking as Tm for corresponding natural lipids, indicating phase behaviour. Scanning electron microscopy excluded micrometric surface structural features. In CHO cells, similar surface micrometric patches were produced by either direct BODIPY-SM insertion or intracellular processing from BODIPY-ceramide, ruling out aggregation artefacts. BODIPY-SM surface micrometric patches were refractory to endocytosis block or actin depolymerization and clustered upon cholesterol deprivation, indicating self-clustering at the plasma membrane. BODIPY-SM excimers further suggested clustering in ordered domains. Segregation of BODIPY-SM and -lactosylceramide micrometric domains showed coexistence of distinct phases. Consistent with micrometric domain boundaries, fluorescence recovery after photobleaching (FRAP) revealed restriction of BODIPY-SM lateral diffusion over long-range, but not short-range, contrasting with comparable high mobile fraction of BODIPY-lactosylceramide in both ranges. Controlled perturbations of endogenous SM pool similarly affected BODIPY-SM domain size by confocal imaging and its mobile fraction by FRAP. The latter evidence supports the hypothesis that, as shown for BODIPY-SM, endogenous SM spontaneously clusters at the plasmalemma outer leaflet of living cells into ordered micrometric domains, defined in shape by liquid-phase coexistence and in size by membrane tension and cholesterol. This proposal remains speculative and calls for further investigations.

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v‐src mutant. Shifting monolayers established at 40 °C (non‐permissive temperature) to 34 °C (permissive temperature) rapidly reactivated v‐Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v‐src was recruited on apical vesicles, induced cortactin‐associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3‐kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 °C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule‐dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v‐src, Rab11, and rabankyrin‐5 but not early endosome antigen 1, thus distinguishing two separate Rab5‐dependent apical pathways. The mechanisms of Src‐induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.

No abstract

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

Shaken baby syndrome, a rotational acceleration injury, is most common between 3 and 6 months of age and causes death in about 10 to 40% of cases and permanent neurological abnormalities in survivors. We developed a mouse model of shaken baby syndrome to investigate the pathophysiological mechanisms underlying the brain damage. Eight‐day‐old mouse pups were shaken for 15 seconds on a rotating shaker. Animals were sacrificed at different ages after shaking and brains were processed for histology. In 31‐day‐old pups, mortality was 27%, and 75% of survivors had focal brain lesions consisting of hemorrhagic or cystic lesions of the periventricular white matter, corpus callosum, and brainstem and cerebellar white matter. Hemorrhagic lesions were evident from postnatal day 13, and cysts developed gradually between days 15 and 31. All shaken animals, with or without focal lesions, had thinning of the hemispheric white matter, which was significant on day 31 but not earlier. Fragmented DNA labeling revealed a significant increase in cell death in the periventricular white matter, on days 9 and 13. White matter damage was reduced by pre‐treatment with the NMDA receptor antagonist MK‐801. This study showed that shaking immature mice produced white matter injury mimicking several aspects of human shaken baby syndrome and provided evidence that excess release of glutamate plays a role in the pathophysiology of the lesions.