Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Human and Mouse Type I Natural Killer T Cell Antigen Receptors Exhibit Different Fine Specificities for CD1d-Antigen Complex

Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4′,4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4′-OH position in comparison with the 3′-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4′-OH > 3′-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.     

Distinguishing Closely Related Amyloid Precursors Using an RNA Aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β₂-microglobulin (hβ₂m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ₂m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ₂m with an EC₅₀ of ∼200 nm. NMR spectroscopy was used to assign the ¹H-¹⁵N HSQC spectrum of the B6-hβ₂m complex, revealing that the aptamer binds to the face of hβ₂m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ₂m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.     

ARFGAP1 Is Dynamically Associated with Lipid Droplets in Hepatocytes

The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.     

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.     

Assessing Nitrogen-Saturation in a Seasonally Dry Chaparral Watershed: Limitations of Traditional Indicators of N-Saturation

To evaluate nitrogen (N) saturation in xeric environments, we measured hydrologic N losses, soil N pools, and microbial processes, and developed an N-budget for a chaparral catchment (Sierra Nevada, California) exposed to atmospheric N inputs of approximately 8.5 kg N ha^?1 y^?1. Dual-isotopic techniques were used to trace the sources and processes controlling nitrate (NO₃ ^?) losses. The majority of N inputs occurred as ammonium. At the onset of the wet season (November to April), we observed elevated streamwater NO₃ ^? concentrations (up to 520 µmol l^?1), concomitant with the period of highest gaseous N-loss (up to 500 ng N m^?2 s^?1) and suggesting N-saturation. Stream NO₃ ^? δ¹⁵N and δ¹⁸O and soil N measurements indicate that nitrification controlled NO₃ ^? losses and that less than 1% of the loss was of atmospheric origin. During the late wet season, stream NO₃ ^? concentrations decreased (to <2 µmol l^?1) as did gaseous N emissions, together suggesting conditions no longer indicative of N-saturation. We propose that chaparral catchments are temporarily N-saturated at ≤8.5 kg N ha^?1 y^?1, but that N-saturation may be difficult to reach in ecosystems that inherently leak N, thereby confounding the application of N-saturation indicators and annual N-budgets. We propose that activation of N sinks during the typically rainy winter growing season should be incorporated into the assessment of ecosystem response to N deposition. Specifically, the N-saturation status of chaparral may be better assessed by how rapidly catchments transition from N-loss to N-retention.     

ABC transporters: how small machines do a big job

Transporters from the ATP-binding cassette (ABC) superfamily operate in all organisms, from bacteria to humans, to pump substances across biological membranes. Recent high-resolution views of ABC transporters in different conformational states provide clues as to how ATP might be used to drive the structural reorganizations that accompany membrane transport. Importantly, it now appears that a putative translocation pathway running through the center of the transporter might be gated alternately, either at the inside or the outside of the cytoplasmic membrane, coupling substrate translocation to a cycle of ATP-dependent conformational changes. ATP binding and ATP hydrolysis have distinct roles in this cycle: binding favors the outward-facing orientation, whereas hydrolysis returns the transporter to an inward-facing conformation.     

Identifying conservation hotspots in non-breeding areas: a case study of Lake Sturgeon (<Emphasis Type="Italic">Acipenser fulvescens</Emphasis>) in the Great Lakes

Limited conservation resources necessitate the prioritization of management efforts and one of the mechanisms for prioritization is the identification of hotspots. For some species of conservation concern, hotspots have been delineated, often using individual abundance as the metric. However, areas that attract individuals from multiple breeding populations may be a higher priority for conservation. These areas may have more abundant resources or habitat quality, and multiple breeding populations simultaneously benefit from conservation actions in these areas. We identified non-spawning aggregations of Lake Sturgeon Acipenser fulvescens in lakes Huron and Erie of the Great Lakes. The spawning period in Lake Sturgeon only represents a brief portion of their life and conservation goals need to include non-spawning areas. Based on 12 microsatellite loci, we determined the population of origin of individuals in the aggregations and computed a modified population-level Simpson’s diversity index (pD). Using this criterion, two hotspots were identified. The North Channel of Lake Huron had the highest diversity of represented populations (pD = 3.14). The second identified hotspot was Saginaw Bay in Lake Huron (pD = 1.78). Both hotspots had evidence of long-distance movement into the area. Monitoring of aggregations should continue to assess temporal variability in hotspot delineation.