The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling

The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling

Function and regulation of Drosophila Epsin in notch signaling

textEpsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. How Epsin promotes ligand endocytosis and thus Notch signaling is unclear. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, I tested each of the five protein or lipid interaction modules of Epsin for a role in Notch activation by each of the two Drosophila ligands, Serrate and Delta. here are five main results of this work that impact present thinking about endocytic machinery/Epsin, Epsin/ligand, or ligand/receptor interactions at the plasma membrane. First, I discovered that deletion or mutation of both UIMs destroys Epsin’s function in Notch signaling and has a greater negative effect on Epsin’s ability to function than removal of any other module type. Second, only one of the two UIMs of Epsin is essential. Third, the lipid-binding function of the ENTH domain is required for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins are necessary collectively for Epsin activity, their functions are highly redundant. Finally, I detected no ligand-specific requirements for Epsin modules. Most unexpected was the finding that Epsin’s Clathrin binding motifs were dispensable. All of these observations are consistent with a model where Epsin’s essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins.Cellular and Molecular Biolog

Mammoth : gearing Hadoop towards memory-intensive MapReduce applications

The MapReduce platform has been widely used for large-scale data processing and analysis recently. It works well if the hardware of a cluster is well configured. However, our survey has indicated that common hardware configurations in small and medium-size enterprises may not be suitable for such tasks. This situation is more challenging for memory-constrained systems, in which the memory is a bottleneck resource compared with the CPU power and thus does not meet the needs of large-scale data processing. The traditional high performance computing (HPC) system is an example of the memory-constrained system according to our survey. In this paper, we have developed Mammoth, a new MapReduce system, which aims to improve MapReduce performance using global memory management. In Mammoth, we design a novel rule-based heuristic to prioritize memory allocation and revocation among execution units (mapper, shuffler, reducer, etc.), to maximize the holistic benefits of the Map/Reduce job when scheduling each memory unit. We have also developed a multi-threaded execution engine, which is based on Hadoop but runs in a single JVM on a node. In the execution engine, we have implemented the algorithm of memory scheduling to realize global memory management, based on which we further developed the techniques such as sequential disk accessing, multi-cache and shuffling from memory, and solved the problem of full garbage collection in the JVM. We have conducted extensive experiments with comparison against the native Hadoop platform. The results show that the Mammoth system can reduce the job execution time by more than 40% in typical cases, without requiring any modifications of the Hadoop programs. When a system is short of memory, Mammoth can improve the performance by up to 5.19 times, as observed for I/O intensive applications, such as PageRank. Given the growing importance of supporting large-scale data processing and analysis and the proven success of the MapReduce platform, the Mammoth system can have a promising potential and impact

LoomIO : object-level coordination in distributed file systems

Device-level interference is recognized as a major cause of the performance degradation in distributed file systems. Although the approaches of mitigating interference through coordination at application-level, middleware-level, and server-level have shown beneficial results in previous studies, we find their effectiveness is largely reduced since I/O requests are re-arranged by underlying object file systems. In this research study, we prove that object-level coordination is critical and often the key to address the interference issue, as the scheduling of object requests determines the device-level accesses and thus determines the actual I/O bandwidth and latency. This article proposes an object-level coordination system, LoomIO, which uses an OBOP (One-Broadcast-One-Propagate) method and a time-limited coordination process to deliver highly efficient coordination service. Specifically, LoomIO enables object requests to achieve an optimized scheduling decision within a few milliseconds and largely mitigates the device-level interference. We have implemented a LoomIO prototye and integrated it into Ceph file system. The evaluation results show that LoomIO achieved the considerable improvements in resource utilization (by up to 35%), in I/O throughput (by up to 31%), and in 99th percentile latency (by up to 54%) compared to the K-optimal method which uses the same scheduling algorithm as LoomIO but does not have the coordination support

The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling.

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling

Analyses of Genetic Structure of Tibeto-Burman Populations Reveals Sex-Biased Admixture in Southern Tibeto-Burmans

An unequal contribution of male and female lineages from parental populations to admixed ones is not uncommon in the American continents, as a consequence of directional gene flow from European men into African and Hispanic Americans in the past several centuries. However, little is known about sex-biased admixture in East Asia, where substantial migrations are recorded. Tibeto-Burman (TB) populations were historically derived from ancient tribes of northwestern China and subsequently moved to the south, where they admixed with the southern natives during the past 2,600 years. They are currently extensively distributed in China and Southeast Asia. In this study, we analyze the variations of 965 Y chromosomes and 754 mtDNAs in >20 TB populations from China. By examining the haplotype group distributions of Y-chromosome and mtDNA markers and their principal components, we show that the genetic structure of the extant southern Tibeto-Burman (STB) populations were primarily formed by two parental groups: northern immigrants and native southerners. Furthermore, the admixture has a bias between male and female lineages, with a stronger influence of northern immigrants on the male lineages (∼62%) and with the southern natives contributing more extensively to the female lineages (∼56%) in the extant STBs. This is the first genetic evidence revealing sex-biased admixture in STB populations, which has genetic, historical, and anthropological implications

Female germline cells do not require <i>auxilin</i> to send Delta signals to follicle cells.

<p>(A) A diagram of an oocyte/nurse cell complex (stage 6–7) is shown. The fifteen nurse cells are diploid, and the cytoplasms of the nurse cells and the oocyte are interconnected by cytoplasmic bridges. (B–D’) Confocal microscope images of oocyte/nurse cell complexes are shown. The complexes were immunolabeled to reveal Notch activation in the follicle cells (anti-Hnt) and F-actin (phalloidin). Homozygous mutant cell nuclei are marked by the absence of GFP. (B,B’) Wild-type (WT) complexes are shown. Notch is activated in the follicle cells. (C,C’) A mosaic complex with <i>aux-</i> germ-line cells and <i>aux+</i> follicle cells is shown. Notch was activated in the follicle cells. The clone was generated in females of the genotype <i>hs-flp/+; ubi-gfp tub-aux FRT40A/FRT40A; aux¹³⁶/aux⁷²⁷</i>. (D,D’) As in (C,C’), except the genotype was <i>hs-flp/+; FRT^5-5Z3515, aux^F956*/FRT^5-5Z3515, ubi-ngfp</i>. Reduced levels of Hnt were seen at the poles of the <i>aux+/aux-</i> mosaic oocyte/nurse cell complexes, as was also observed in <i>Chc+/Chc-</i> mosaics [SLW and DB, unpublished observation]. This is quite distinct, however, from the absence of Hnt throughout the follicle epithelium observed with <i>lqf-</i> or <i>Dl-</i> germ line clones <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018259#pone.0018259-Windler1" target="_blank">[40]</a>. Scale bar 20 µm.</p

Overexpression of clathrin heavy chain and/or epsin suppresses the adult eye defects in <i>aux</i> loss-of-function mutants.

<p>(A–E) Light micrographs of adult external eyes of the genotypes indicated beneath are shown. (F) A diagram of an apical tangential section of a single ommatidium is shown. The numbers are photoreceptor cells R1 – R7. The black circular projections from each cells are the light-gathering organelles called rhabdomeres. The hexagonal shape is formed by pigment cells. (G–K) Small fields of apical tangential sections of adult eyes are shown. (H) Ommatidia of <i>aux</i> hypomorphs are usually disorganized, and often have extra photoreceptors. (I–K) Addition of genomic DNA transgenes that express <i>Chc+</i> or <i>lqf+</i> suppresses the eye morphology defects of <i>aux</i> hypomorphs. The fraction of phenotypically wild-type (wt) ommatidia was determined by observing 300–500 ommatidia in 4–5 eyes of each genotype. The error is one standard deviation. Scale bar 10 µm (G–K) and 60 µm (A–E).</p