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Synaptic vesicle recycling has long served as a model for the general mechanisms of cellular trafficking. We used an integrative approach, combining quantitative immunoblotting and mass spectrometry to determine protein numbers; electron microscopy to measure organelle numbers, sizes, and positions; and super-resolution fluorescence microscopy to localize the proteins. Using these data, we generated a three-dimensional model of an \"average\" synapse, displaying 300,000 proteins in atomic detail. The copy numbers of proteins involved in the same step of synaptic vesicle recycling correlated closely. In contrast, copy numbers varied over more than three orders of magnitude between steps, from about 150 copies for the endosomal fusion proteins to more than 20,000 for the exocytotic ones.

A mechanism for phosphoinositide conversion at endosomes to enable exit from the endosomal system, suggesting that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy. Phosphoinositide conversion during endosome exit Directional membrane traffic requires regulated conversion of phosphoinositides (PIs) — membrane phospholipids that act as determinants of membrane identity — by PI metabolizing enzymes. Volker Haucke and co-workers studied the mechanism of PI identity shifts during trafficking from the endosomal system — defined by phosphatidylinositol 3-phosphate (PI(3)P) — to the secretory compartments and the plasma membrane, dominated by phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). The authors find that endosomal cargo en route to intracellular destinations can change direction and make its way back to the cell surface by the action of two enzymes. Specifically, PI(3)P on the membrane of these compartments is hydrolysed by the phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy in humans. This hydrolysis of PI(3)P is accompanied by the generation of PI(4)P through the action of phosphatidylinositol 4-kinase, as well as the recruitment of the exocyst tethering complex to enable subsequent membrane fusion. Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity 1 , 2 . Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) are concentrated at the plasma membrane, on secretory organelles 3 , and on lysosomes 4 , whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P) 5 , are a hallmark of the endosomal system 1 , 2 . Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans 6 . Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

Phosphoinositides (PIs) have long been known to have important roles in cell signalling. During the past decade, it has become clear that these lipids also act as constitutive signals that aid in defining organelle identity, and are short‐lived recruiters and regulators of cytoskeletal and membrane dynamics. Recent studies have provided important clues as to how regulated activation of PI‐metabolizing enzymes and recruitment of their binding proteins might cooperate in targeting distinct pools of PIs to different cell physiological functions.

Phosphoinositides are important regulators of intracellular membrane traffic, and although the role of PI(4,5)P 2 has been well characterised, the function of PI(3,4)P 2 remains unclear; here the formation of PI(3,4)P 2 by the class II phosphatidylinositol-3-kinase C2α enzyme is shown to control clathrin-mediated endocytosis. Endocytosis control by lipid switch Phosphoinositides are important regulators of intracellular membrane traffic. Although the role of phosphatidylinositol-4,5-bisphosphate has been well characterized, that of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) remains unclear. In this study, Volker Haucke and colleagues show that formation of PI(3,4)P 2 by the class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) enzyme spatiotemporally controls clathrin-mediated endocytosis. These findings present a novel function of PI(3,4)P 2 in membrane traffic. Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic 1 , 2 . Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits 3 , 4 , 5 , 6 . No phosphatidylinositol other than PI(4,5)P 2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P) 7 . How phosphatidylinositol conversion from PI(4,5)P 2 -positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P 2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P 2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P 2 in endocytosis and unravel a novel discrete function of PI(3,4)P 2 in a central cell physiological process.

Cytokinesis mediates the final separation of a mother cell into two daughter cells. Septins are recruited to the cleavage furrow at an early stage. During cytokinetic progression the septin cytoskeleton is constantly rearranged, ultimately leading to a concentration of septins within the intercellular bridge (ICB), and to the formation of two rings adjacent to the midbody that aid ESCRT-dependent abscission. The molecular mechanisms underlying this behavior are poorly understood. Based on observations that septins can associate with actin, microtubules and associated motors, we review here established roles of septins in mammalian cytokinesis, and discuss, how septins may support cytokinetic progression by exerting their functions at particular sites. Finally, we discuss how this might be assisted by phosphoinositide-metabolizing enzymes.

Septins constitute a family of GTP-binding proteins, which assemble into non-polar filaments in a nucleotide-dependent manner. These filaments can be recruited to negatively charged membrane surfaces. When associated with membranes septin filaments can act as diffusion barriers, which confine subdomains of distinct biological functions. In addition, they serve scaffolding roles by recruiting cytosolic proteins and other cytoskeletal elements. Septins have been implicated in a large variety of membrane-dependent processes, including cytokinesis, signaling, cell migration, and membrane traffic, and several family members have been implicated in disease. However, surprisingly little is known about the molecular mechanisms underlying their biological functions. This review summarizes evidence in support of regulatory roles of septins during endo-lysosomal sorting, with a particular focus on phosphoinositides, which serve as spatial landmarks guiding septin recruitment to distinct subcellular localizations.

Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P₂] is an important factor for a variety of cellular functions ranging from cell signaling to actin cytoskeletal dynamics and endocytic membrane traffic. Here, we have identified the clathrin adaptor complex AP-2 as a regulator of phosphatidylinositol 4-phosphate 5-kinase (PIPK)mediated PI(4,5)P₂ synthesis. AP-2 directly interacts with the kinase core domain of type I PIPK isozymes via its μ2-subunit in vitro and in native protein extracts. Endocytic cargo protein binding to μ2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-2μ, and PIPK type I which may provide a specific pool of PI(4,5)P₂ dedicated to clathrin/AP-2-dependent receptor internalization.

Water is used in dairy farming for producing feed, watering the animals, and cleaning and disinfecting barns and equipment. The objective of this study was to investigate the drinking and cleaning water use in a dairy cow barn. The water use was measured on a well-managed commercial dairy farm in North-East Germany. Thirty-eight water meters were installed in a barn with 176 cows and two milking systems (an automatic milking system and a herringbone parlour). Their counts were logged hourly over 806 days. On average, the cows in the automatic milking system used 91.1 (SD 14.3) L drinking water per cow per day, while those in the herringbone parlour used 54.4 (SD 5.3) L per cow per day. The cows drink most of the water during the hours of (natural and artificial) light in the barn. Previously published regression functions of drinking water intake of the cows were reviewed and a new regression function based on the ambient temperature and the milk yield was developed (drinking water intake (L per cow per day) = −27.937 + 0.49 × mean temperature + 3.15 × milk yield (R2 = 0.67)). The cleaning water demand had a mean of 28.6 (SD 14.8) L per cow per day in the automatic milking system, and a mean of 33.8 (SD 14.1) L per cow per day in the herringbone parlour. These findings show that the total technical water use in the barn makes only a minor contribution to water use in dairy farming compared with the water use for feed production.

The author, former president of St. Johns College in Santa Fe, New Mexico, and former acting chancellor of the American University in Iraq, is concerned over the future of Americas 240-year-old experiment in liberal democracy. Among the superb insights the interested reader will glean from Rediscovering America are: the difference between wealth and justice (the latter is what we should seek; the former is a by-product); the limits of democracy and the reasons why the Founders were somewhat suspicious Michael I. Krauss is professor of law at the Antonin Scalia Law School of George Mason University, Arlington, VA 22201; mkrauss@gmu.edu. 484 Reviews of it; the correct meaning of equality in our founding documents (hintit has little to nothing to do with material condition); the reason why the Declaration of Independence is a vital founding document to be read along with our Constitution; and the inner character of small r American republicanism.

Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the \"classical\" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.