Two-way signalling through the LFA-1 lymphocyte adhesion receptor

T lymphocyte recognition of foreign antigens and migration throughout the body require the regulated adhesion of lymphocytes to diverse types of cells and to the extracellular matrix. The lymphocyte adhesion 'receptor' LFA-1, a member of the integrin family, interacts with ICAM-1 and other counter-receptors to mediate adhesion. The LFA-1/ICAM-1 interaction is regulated by signals transmitted from the cytoplasm to the extracellular space. Conversely, LFA-1 transmits signals from the extracellular space to the cytoplasm to regulate T lymphocyte activation. The observed properties of LFA-1 and related adhesion 'receptors' are incorporated into a general model for adhesion during immune surveillance and recognition of foreign antigens.     

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.     

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Rewiring coral: Anthropogenic nutrients shift diverse coral–symbiont nutrient and carbon interactions toward symbiotic algal dominance

Improving coral reef conservation requires heightened understanding of the mechanisms by which coral cope with changing environmental conditions to maintain optimal health. We used a long‐term (10 month) in situ experiment with two phylogenetically diverse scleractinians (Acropora palmata and Porites porites) to test how coral–symbiotic algal interactions changed under real‐world conditions that were a priori expected to be beneficial (fish‐mediated nutrients) and to be harmful, but non‐lethal, for coral (fish + anthropogenic nutrients). Analyzing nine response variables of nutrient stoichiometry and stable isotopes per coral fragment, we found that nutrients from fish positively affected coral growth, and moderate doses of anthropogenic nutrients had no additional effects. While growing, coral maintained homeostasis in their nutrient pools, showing tolerance to the different nutrient regimes. Nonetheless, structural equation models revealed more nuanced relationships, showing that anthropogenic nutrients reduced the diversity of coral–symbiotic algal interactions and caused nutrient and carbon flow to be dominated by the symbiont. Our findings show that nutrient and carbon pathways are fundamentally “rewired” under anthropogenic nutrient regimes in ways that could increase corals’ susceptibility to further stressors. We hypothesize that our experiment captured coral in a previously unrecognized transition state between mutualism and antagonism. These findings highlight a notable parallel between how anthropogenic nutrients promote symbiont dominance with the holobiont, and how they promote macroalgal dominance at the coral reef scale. Our findings suggest more realistic experimental conditions, including studies across gradients of anthropogenic nutrient enrichment as well as the incorporation of varied nutrient and energy pathways, may facilitate conservation efforts to mitigate coral loss.     

A chemically triggered transition from conflict to cooperation in burying beetles

Although interspecific competition has long been recognised as a major driver of trait divergence and adaptive evolution, relatively little effort has focused on how it influences the evolution of intraspecific cooperation. Here we identify the mechanism by which the perceived pressure of interspecific competition influences the transition from intraspecific conflict to cooperation in a facultative cooperatively breeding species, the Asian burying beetle Nicrophorus nepalensis. We not only found that beetles are more cooperative at carcasses when blowfly maggots have begun to digest the tissue, but that this social cooperation appears to be triggered by a single chemical cue – dimethyl disulfide (DMDS) – emitted from carcasses consumed by blowflies, but not from control carcasses lacking blowflies. Our results provide experimental evidence that interspecific competition promotes the transition from intraspecific conflict to cooperation in N. nepalensis via a surprisingly simple social chemical cue that is a reliable indicator of resource competition between species.     

Physiological dormancy in seeds of tropical montane woody species in Hawai`i

Worldwide, there is relatively little information on seed dormancy and germination of tropical montane species. Our aim was to help fill this knowledge gap by conducting seed dormancy/germination studies on woody species from this vegetation zone in Hawai`i. All species had water-permeable seeds with a fully developed embryo. Seeds of 29 species (23 genera) were incubated in light/dark at 15/6, 20/10 and 25/15°C and germination monitored at 2-week intervals for 16–128 weeks. Seeds of Chenopodium oahuense, Dubautia menziesii and Silene lanceolata were non-dormant (ND) and those of 26 other species had physiological dormancy (PD); 10 of the 26 species had conditional PD. The optimum germination temperature regime(s) was (were) 25/15°C, 17 species; 25/10 and 20/10°C, 2; 20/10°C, 6; 20/10 and 15/6°C, 2; and 15/6°C, 2. Worldwide, PD in the woody genera included in our study is more common than ND. In addition to its contribution to the world biogeography of seed dormancy/germination, this study will be useful to conservation biologists who need to germinate seeds of tropical montane species.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.