The rockefeller university

CLC proteins transport chloride (Cl-) ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl- ion channels, whereas others are secondary active transporters that exchange Cl- ions and protons (H+) with a 2: 1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2: 1 Cl-/H+ exchange and a simple mechanistic connection between CLC channels and transporters.

Approximately 2% of the worldwide population is infected with hepatitis C virus (HCV), the major causative agent of non-A, non-B hepatitis. Although substantial progress has been made in developing tools to dissect the viral life cycle, most in vitro studies rely on hepatoma cell lines, which are functionally disparate from the natural in vivo target of the virus - hepatocytes. To gain insights into virus-host interactions, there is a need for HCV-model systems that more closely mimic the physiological environment of the liver. Here, we discuss recent advances in culture and detection systems that facilitate the study of HCV in primary cells. Use of these new models may help bridge the gap between in vitro studies and clinical research.

The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e. g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field.

Tetherin/BST2 is a type-II membrane protein that inhibits the release of a range of enveloped viruses, including HIV-1. Here we report three crystal structures of human tetherin, including the full-length ectodomain, a triple cysteine mutant and an ectodomain truncation. These structures show that tetherin forms a continuous alpha helix encompassing almost the entire ectodomain. Tetherin helices dimerize into parallel coiled coils via interactions throughout the C-terminal portion of the ectodomain. A comparison of the multiple structures of the tetherin dimer reveals inherent constrained flexibility at two hinges positioned at residues A88 and G109. In the crystals, two tetherin ectodomain dimers associate into a tetramer by forming an antiparallel four-helix bundle at their N termini. However, mutagenesis studies suggest that the tetrametric form of tetherin, although potentially contributing to, is not essential for its antiviral activity. Nonetheless, the structural and chemical properties of the N terminus of the ectodomain are important for optimal tethering function. This study provides detailed insight into the mechanisms by which this broad-spectrum antiviral restriction factor can function.

Handed asymmetry in organ shape and positioning is a common feature among bilateria, yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. We utilize the directional 360 degrees clockwise rotation of genitalia in Drosophila to study LA-dependent organ looping. Using time-lapse imaging, we show that rotation of genitalia by 360 degrees results from an additive process involving two ring-shaped domains, each undergoing 180 degrees rotation. Our results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. We further reveal a specific pattern of apoptosis at the ring boundaries and show that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180 degrees LR module.

Objective: To evaluate the benefit of Y microdeletion testing. Design: Retrospective analysis. Setting: University-based male fertility clinic and genetics laboratory. Patient(s): A total of 1,591 men with sperm concentrations less than 5 million sperm/mL. Intervention(s): Semen analysis, Y microdeletion testing, microdissection testicular sperm extraction (TESE). Main Outcome Measure(s): Sperm concentration, incidence and nature of Y microdeletions, microdissection TESE outcome. Result(s): We identified 149 microdeletions (9.4%). 10.4% of azoospermic men and 10.1% of men with sperm concentrations >0-1 million sperm/mL harbored microdeletions. Two-thirds of microdeletions in azoospermic men were AZFa, AZFb, AZFb+c, or complete Yq deletions. Virtually all microdeletions in oligozoospermic patients were AZFc deletions. Seven hundred eighteen patients underwent microdissection TESE, including 41 with microdeletions. Microdissection TESE failed in all patients with AZFa, AZFb, AZFb+c, and complete Yq deletions. Sperm were retrieved in 15/21 AZFc deleted patients (71.4%). The presence of an AZFc deletion was associated with increased likelihood of sperm retrieval when compared with the 48.8% retrieval rate in 385 idiopathically azoospermic men who consecutively underwent microdissection TESE at our institution during the study period. Clinical pregnancy was achieved in 10/15 azoospermic AZFc deleted patients for whom sperm were successfully retrieved. Conclusion(s): Of azoospermic and severely oligozoospermic American men, 10% harbor Y microdeletions that alter prognosis for surgical sperm retrieval and are vertically transmissible. Y microdeletion testing is essential for genetic and preoperative counseling in these patients. (Fertil Steril (R) 2010; 94: 1753-6. (C)2010 by American Society for Reproductive Medicine.)

A hallmark of mitosis is the appearance of high levels of histone phosphorylation, yet the roles of these modifications remain largely unknown. Here, we demonstrate that histone H3 phosphorylated at threonine 3 is directly recognized by an evolutionarily conserved binding pocket in the BIR domain of Survivin, which is a member of the chromosomal passenger complex (CPC). This binding mediates recruitment of the CPC to chromosomes and the resulting activation of its kinase subunit Aurora B. Consistently, modulation of the kinase activity of Haspin, which phosphorylates H3T3, leads to defects in the Aurora B-dependent processes of spindle assembly and inhibition of nuclear reformation. These findings establish a direct cellular role for mitotic histone H3T3 phosphorylation, which is read and translated by the CPC to ensure accurate cell division.

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.