Nuclear import of an intact preassembled proteasome particle

The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated "+" factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development.     

A simplified method for the generation of human osteoclasts in vitro

Osteoclasts are large, multinucleated cells responsible for the resorption of mineralized bone matrix. These cells are critical players in the bone turnover involved in bone homeostasis. Osteoclast activity is connected to the establishment and expansion of skeletal metastases from a number of primary neoplasms. Thus, the formation and activation of osteoclasts is an area of research with many potential avenues for clinical translation. Past studies of osteoclast biology have utilized primary murine cells cultured in vitro. Recently, techniques have been described that involve the generation of osteoclasts from human precursor cells. However, these protocols are often time-consuming and insufficient for generating large numbers of osteoclasts. We therefore developed a simplified protocol by which human osteoclasts may be easily and reliably generated in large numbers in vitro. In this study, osteoclasts were differentiated from bone marrow cells that had been aliquotted and frozen. Cells were generated by culture with recombinant macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Both human and murine RANKL were shown to efficiently generate osteoclasts, although higher concentrations of murine RANKL were required. Formation of osteoclasts was demonstrated qualitatively by tartrate-resistant acid phosphatase (TRAP) staining. These cells were fully functional, as confirmed by their ability to form resorption pits on cortical bone slices. Functional human osteoclasts can be difficult to generate in vitro by current protocols. We have demonstrated a simplified system for the generation of human osteoclasts in vitro that allows for large numbers of osteoclasts to be obtained from a single donor.     

Introduction, rescue and expression of plasmid genes in mammalian cells and Escherichia coli

A shuttle-vector system is described for the study of mutational specificity in mammalian cells. Using a plasmid (pGKTK) carrying the E. coli galactokinase gene (gk) and the herpes simplex virus thymidine kinase gene (tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK- mouse fibroblasts) and its efficient rescue back into E. coli. This system makes use of two genes, each of which can expressed in both E. coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host. In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing. In the case of a putative single-copy transformant (T8), about half of the rescued plasmids are identical in size and restriction pattern to the original plasmid. Each of these expressed the tk gene, indicating the fidelity of the rescue system.     

A reliable method for assessing rotational power

Rotational core training is said to be beneficial for rotational power athletes. Currently, there has been no method proposed for the reliable assessment of rotational power. Therefore, our purpose was to determine the test-retest reliability of kinetic and kinematic rotational characteristics of a pulley system when performing a rotational exercise of the axial skeleton in the transverse plane to find out if this would be a reliable tool for evaluating rotational power. Healthy, college-aged men (n = 8) and women (n = 15) reported for 3 testing sessions. The participants were seated on a box, and they held the handle with both arms extended in front of their body, starting their motion with their torso rotated toward the machine. All the participants rotated their torso forcefully until they reached 180° of rotation, and they then slowly returned to the starting position, 3 times per trial, with 3 loads: 9% body weight (BW), 12% BW, and 15% BW. The repetition with the greatest power for each trial for each load was analyzed. The mean peak power repetition (watts) for all the subjects was 20.09 ± 7.16 (9% BW), 26.17 ± 8.6 (12% BW), and 30.74 ± 11.022 (15% BW) in the first training session and 22.3 ± 8.087 (9% BW), 28.7 ± 11.295 (12% BW), and 33.52 ± 12.965 (15% BW) in the second training session with intraclass correlation coefficients of 0.97 (9%BW), 0.94 (12%BW), and 0.95 (15%BW). When the participants were separated by sex, there were no significant differences between groups. Based on these results, it was found that a pulley system and an external dynamometer can be used together as a reliable research tool to assess rotational power.     

Healing while parasitized: impact of a naturally-occurring nematode during energy-intensive wound-healing in a beetle

The rigours of the daily lives of insects sometimes lead to minor injuries and wounds, which must be healed to avoid entry of pathogens and to resume normal function. Such healing requires energy, which must be diverted from other bodily reserves. What happens if energy reserves are already low, as would occur in individuals coping with internal parasites? This question is addressed in the presemt study, using horned passalus beetles (Odontotaenius disjunctus) and their naturally-occurring nematode Chondronema passali. Oxygen consumption rates are tested at rest, as well as after an experimental wound is applied, to evaluate energy requirements of wound-healing in parasitized and nonparasitized hosts. Furthermore, wound-healing rates are visually tracked with a numerical scoring system to directly measure the cost of parasitism on healing. At rest, parasitized beetles show no elevation in respiration (oxygen consumption). After wounding, the oxygen consumption of parasitized beetles is 10% higher than that in nonparasitized beetles. Beetles with moderate- to heavy worm burdens have slower healing than those with few or no nematodes. These results show that this parasite carries little cost to the host during day-to-day activities, whereas, during times of immediate energy demand, there is a cost; hosts require more energy to repair wounds, and the wounds take longer to close. This conclusion leads to the question of whether this parasite is truly benign, and how many other apparently benign parasites, in insects or other animals, have similar ‘hidden’ effects.     

Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2–interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.     

Coordinating morphogenesis: epithelial integrity during heart tube assembly

Formation of the embryonic heart tube requires the medial migration and merger of bilateral precursor populations. A new study of zebrafish cardiogenesis published in this issue of Developmental Cell indicates that precursor migration involves formation of a coherent epithelium and that fibronectin plays an important role in maintaining cardiac epithelial integrity.