TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. IntroductionPolymorphonuclear leukocytes (PMNs, neutrophils) are highly specialized cellular effectors in host defense and immune surveillance. Mature human PMNs from healthy adults have a unique repertoire of activities, including phagocytosis, degranulation of antimicrobial enzymes and peptides, and generation of oxygen radicals with antimicrobial properties. 1-6 Synthesis of inflammatory and regulatory lipids and proteins complements these innate mechanisms. 1,4,5 PMNs have evolved for capture, containment, and destruction of bacteria and fungi and also have activity against intracellular pathogens and viruses. 2,3 PMNs have additional important roles in tissue repair and integration of innate and adaptive immune responses. 6 If, however, these specialized defensive mechanisms become dysregulated or unregulated, PMNs can paradoxically be mediators of inflammatory tissue injury. 1,6 Consistent with their requisite activities in host defense, defects in PMN functions cause immune deficiency syndromes. 2,7 Neutrophil defects can be hereditary, developmental, or acquired in nature. Specific genetic deficiencies in PMN function cause significant morbidity in subsets of children and adults and, in parallel, provide unique insights into molecular mechanisms that regulate leukocyte activities. 7,8 Nevertheless, these disorders are rare and arcane. In contrast, the developmental syndrome of neonatal neutrophil dysfunction, which is particularly important in premature infants, is common and contributes to infections in infants worldwide. As an example, neonatal PMN dysfunction is thought to be a pivotal feature of sepsis in the newborn. 9-11 The incidence of neonatal sepsis is estimated to be 1 to 5 cases per 1000 live births in the United States and to be even higher after very low birth weight premature deliveries (15-19/1000); in contrast, the incidence of sepsis is much lower in children older than 1 year of age and in young adults. 12-15 Furthermore, the incidence of neonatal sepsis is as high as 25% in some areas of the developing world. 16,17 Thus, neonatal PMN dysfunction is a contributor to a public health problem of significant proportions, and also may pr...

We wish to report that we have discovered an error in the AD-ROCK1 yeast two hybrid construct used in the experiment displayed in Figure 1D. This error introduced a stop codon after ROCK1 amino acid 123, so that the two hybrid interactions that we reported as being between TSG101 and full-length ROCK1 were actually between TSG101 and a truncated construct that expressed only the first 123 residues of ROCK1. This error does not affect the corresponding ROCK1-TSG101 co-immunoprecipitation experiment displayed in Figure 1B of the original paper. Based on the co-immunoprecipitation experiment, the conclusion that ROCK1 can interact with TSG101 stands.The authors apologize for any inconvenience caused.

The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSG101, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links. Here, we have systematically analyzed the requirements for late-acting mammalian ESCRT-III and VPS4 proteins at different stages of mitosis and cell division. We found that depletion of VPS4A, VPS4B, or any of the 11 different human ESCRT-III (CHMP) proteins inhibited abscission. Remarkably, depletion of individual ESCRT-III and VPS4 proteins also altered centrosome and spindle pole numbers, producing multipolar spindles (most ESCRT-III/VPS4 proteins) or monopolar spindles (CHMP2A or CHMP5) and causing defects in chromosome segregation and nuclear morphology. VPS4 proteins concentrated at spindle poles during mitosis and then at midbodies during cytokinesis, implying that these proteins function directly at both sites. We conclude that ESCRT-III/VPS4 proteins function at centrosomes to help regulate their maintenance or proliferation and then at midbodies during abscission, thereby helping ensure the ordered progression through the different stages of cell division.T he ESCRT pathway functions across eukaryotes and many archaeal species, where it helps mediate (i) vesicle formation at multivesicular bodies (MVB) (1), (ii) enveloped virus budding (2), and (iii) the abscission stage of cytokinesis (3-7). These seemingly disparate biological processes all involve the resolution of thin, cytoplasm-filled membrane tubules, implying that ESCRT machinery can be recruited to different biological membranes to mediate topologically similar membrane fission events. Most ESCRT pathway proteins function as subunits of five multiprotein complexes, termed the ESCRT-0, -I, -II, -III, and VPS4 complexes. Other ESCRT factors, such as ALIX, function as discrete proteins. Classic studies in yeast have established that the ESCRT components are recruited sequentially to endosomal membranes where they assemble into higher order complexes that mediate protein sorting, membrane remodeling, and fission (8). Initially, early-acting factors such as ESCRT-I, ESCRT-II, and ALIX interact with upstream recruiting factors, concentrate protein cargoes, and help deform membranes (9). These early-acting factors recruit subunits of the ESCRT-III complex, which form filaments within the necks of membrane tubules and mediate membrane fission (10-16). ESCRT-III assemblies, in turn, recruit VPS4 ATPases, which use the energy of ATP hydrolysis to disassemble the ESCRT complexes (10-14, 17).ESCRT-III and VPS4 homologs mediate abscission in hyperthermophilic crenarchaeal species that diverged from eukaryotes several billion years ago, suggesting that cell division may have been the primordial function of the E...

Synapses are the site of chemical communication between neurons and between neurons and muscles. The synaptic vesicle (SV) is a prominent presynaptic organelle which contains chemical neurotransmitters and fuses with the plasma membrane to mediate neurotransmission. There are about 50 or so synaptic proteins which are either integral vesicle membrane proteins (e.g., synaptotagmin, syt; and synaptobrevin, syb) or vesicle-associated proteins (e.g., cysteine string protein, CSP; Fernandez-Chacon and Sudhof, 1999). We have transformed Drosophila with a novel syt-eGFP (enhanced GFP) fusion protein, the fluorescence pattern of which colocalizes with native SV proteins at synapses, suggesting that the syt-eGFP fusion protein is correctly localized as an integral SV protein and therefore a good SV marker in living synapses. We demonstrate that the syt-eGFP line can be used to study SV dynamics in vivo by fluorescence recovery after photobleach (FRAP).The syt-eGFP fusion was constructed as shown in Figure 1. The eGFP carries double substitution of Phe 64 to Leu and Ser 65 to Thr and fluoresces 35-fold more intensely than wild-type GFP when excited at 488 nm, based on spectral analysis of equal amounts of soluble protein (Cormack et al., 1996). Four syt-eGFP transgenic lines were generated; one with insertion on the X chromosome, two on the second chromosome, and one on the third chromosome. All of these lines produced clear fluorescence when crossed to a pan-neuronal GAL4 driver (elav-GAL4, see Fig. 2) or a subset neuronal GAL4 driver 4G-GAL4 (data not shown). 4G-GAL4 is identified from an enhancer trap screen for neuronal-specific genes; it starts expression at late embryogenesis panneuronally, but in a subset of motor neurons in the third instar larvae, and enriched in mushroom body in adult brain. The eGFP-positive animals, from embryos to adults, can be readily recognizable under a fluorescence dissecting scope. Stocks with expression of syt-eGFP in all neurons (recombinant chromosome carrying both elav-GAL4 and syt-eGFP on the X chromosome) or subset of neurons (recombinant chromosome carrying both 4G-GAL4 and syt-eGFP on the second chromosome) were established.Multiple lines of evidence indicate that syt-eGFP is present in SVs, with expression similar to the native syt (Fig. 2). First, syt-eGFP is highly enriched in the neuropil region of the ventral nerve cord (VNC) of the embryo (data not shown) and larva ( Fig. 2A, left), as well as in the axonal lobes of the larval mushroom body ( Fig. 2A, right). These neuropil regions are densely packed with neuronal synapses. Second, at neuromuscular junction (NMJ) synapses, where we have higher resolution of single synaptic boutons, the syt-eGFP pattern perfectly matches the staining pattern seen with antibodies against SV-associated proteins (Fig. 2B) FIG. 1.Map of syt-eGFP and syb-eGFP fusion constructs. Syt (accession number M55048) or syb (neuronal synaptobrevin, accession number S66686) coding region was fused to the N-terminal of eGFP (enhanced GFP, catalog number 6...