A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell-cell contacts.

Junctions between the ER and plasma membrane (PM) are implicated in calcium homeostasis, non-vesicular lipid transfer, and other cellular functions. Two ER proteins that function both as tethers to the PM via a polybasic C-terminus motif and as phospholipid transporters are brain-enriched TMEM24 (C2CD2L) and its paralog C2CD2. We report that both proteins also form a complex with band 4.1 family members, which in turn bind PM proteins including cell adhesion molecules such as SynCAM 1. This complex enriches TMEM24 and C2CD2 containing ER/PM junctions at sites of cell contacts. Dynamic properties of TMEM24-dependent ER/PM junctions are impacted when band 4.1 is part of the junction, as TMEM24 at cell-adjacent ER/PM junctions is not shed from the PM by calcium rise, unlike TMEM24 at non-cell adjacent junctions. Lipid transport between the ER and the PM by TMEM24 and C2CD2 at sites where cells, including neurons, contact other cells may participate in adaptive responses to cell contact-dependent signaling.

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes.

As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already competent to collect proteins found on mature autophagosomes, including LC3-II. Further, we use styrene-maleic acid lipid particles to reveal the nanoscale organization of protein on LC3-II membranes; ATG9 and LC3-II are each fully integrated into expanding autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.

Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P(2) breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P(2) breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P(2) depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P(2) in clathrin coat dynamics and Arp2/3-dependent actin regulation.

Rapid Ca2+-dependent decrease of protein ubiquitination at synapses.

Protein ubiquitination has been implicated in the regulation of axonal growth and synaptic plasticity as well as in the pathogenesis of neurodegenerative diseases. Here we show that depolarization-dependent Ca2+ influx into synaptosomes produces a global, rapid (range of seconds), and reversible decrease of the ubiquitinated state of proteins, which correlates with the Ca2+-dependent dephosphorylation of several synaptic proteins. A similar general decrease in protein ubiquitination was observed in nonneuronal cells on Ca2+ entry induced by ionomycin. Both in synaptosomes and in nonneuronal cells, this decrease was blocked by FK506 (a calcineurin antagonist). Proteins whose ubiquitinated state was decreased include epsin 1, a substrate for the deubiquitinating enzyme fat facets/FAM, which we show here to be concentrated at synapses. These results reveal a fast regulated turnover of protein ubiquitination. In nerve terminals, protein ubiquitination may play a role both in the regulation of synaptic function, including vesicle traffic, and in the coordination of protein turnover with synaptic use.