Spatio-Temporal Cellular Dynamics of the Arabidopsis Flagellin Receptor Reveal Activation Status-Dependent Endosomal Sorting

The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor FLAGELLIN SENSING2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)–sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b– and ARA6/Rab F1–positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.     

BRCA1-directed,enhanced and aberrant homologous recombination: Mechanism and potential treatment strategies

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.Key words: BRCT, DNA repair, peptide, radiation, RING, ubiquitylation     

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.     

Fish behavioural responses to river discharge trends and the importance of habitat connectivity

Population behaviours associated with the migrations of coarse (non‐salmonid) fishes within river basins are amongst the most poorly understood dispersion mechanisms of temperate freshwater organisms, which in rivers are expected to be influenced by river discharge. We examined the timing and intensity of fish movements (via trapping) between the River Avon (Hampshire, England) and a small floodplain tributary, Ibsley Brook, and tested for correlations with trends in river discharge (i.e. mean cm of change in stage during trapping), water temperature and brook water velocity over twelve months in 1999–2000. 0‐group fishes dominated the catches. Intensities of movement between the brook and the river were similar in most months, but seasonal patterns were observed overall and for individual species. Few significant differences in overall numbers of fish were observed between the discharge trends, but many individual species demonstrated differences, mostly as more intense movement under fast rising discharge. Fish numbers in five species were correlated with river discharge trend, and movements in some species were correlated with the rate of temperature change (Δ° C 10 h sampling), and with changes in brook water velocity. Our results suggest daily movements between the river and small tributary brooks are triggered by changes in light intensity and water velocity, whereas seasonal movements of species between the river and brook are driven by changes in river discharge and water temperature, in particular associated with flood events. This study emphasizes the importance of connectivity in river systems, as fish movement between the Avon and its annexes occur under all flow regimes, but especially with rapidly rising discharge.     

The effects of heparin cofactor II-derived chemotaxins on neutrophil actin conformation and cyclic AMP levels

The serine proteinase inhibitor heparin cofactor II (HC) can be cleaved by polymorphonuclear leukocyte (PMN) elastase (LE) to yield potent chemotactic activity for PMN and monocytes. In contrast to the bacterially-derived chemotaxin formyl-Met-Leu-Phe (fMLP), the HC-derived chemotaxin does not stimulate PMN degranulation or oxidative burst activity. We compared the effects of HC-derived chemotaxins to the effects of fMLP on PMN actin conformation and on the cAMP levels. Both the HC chemotaxins and fMLP rapidly induced an increase in F-actin which was similar in magnitude and time-course. However, in contrast to fMLP, HC-derived chemotaxins did not elevate cAMP levels. HC-derived chemotaxins may be useful probes of chemotactic responses, since they do not have the mixed biological activities of fMLP.     

Regeneration of new fibers in muscles of old rats reduces contraction-induced injury.

Skeletal muscles are injured by their own contractions. Compared with muscles in young animals, those in old animals are injured more easily and more severely and regenerate less well afterward. Injection of a myotoxin (bupivacaine) causes complete degeneration of fibers in extensor digitorum longus (EDL) muscles of rats, followed by full regeneration within 60 days. We tested the specific hypothesis that, 3 days after a protocol of pliometric (lengthening) contractions, the newly regenerated muscle fibers in bupivacaine-treated EDL muscles in both young and old rats would show a lesser deficit in maximum force and fewer damaged fibers than muscles in nontreated EDL muscles. The treated and nontreated EDL muscles of young and old male Wistar rats were administered a protocol of 225 pliometric contractions and were evaluated 3 days afterward, when morphological damage to muscle fibers is most severe. In treated compared with nontreated EDL muscles of both young and old rats, the force deficit and the number of damaged fibers were each reduced by approximately 75%. We conclude that newly regenerated fibers in muscles of young and old animals are resistant to injury and that maintenance of newly regenerated fibers by conditioning may prevent inadvertent damage, particularly in muscles of elderly people.     

Muscle grafts overloaded by ablation of synergistic muscles

We hypothesized that the mass and maximum tetanic tension (Po) of nerve-intact grafts overloaded by ablation of synergistic muscles would be greater than that of standard nerve-intact grafts or of control soleus muscles. Soleus muscles were grafted orthotopically and bilaterally in 35 female rats. Control soleus muscles were obtained from 30 age-matched cohorts. Twenty-eight days following grafting, gastrocnemius muscles were ablated bilaterally in half of the animals. Comparisons were made between 28 and 112 days following grafting. By 112 days the wet mass of the overload nerve-intact grafts was 138% of the standard grafts and 152% of the control soleus muscles, whereas the Po was 161% and 107%, respectively. Specific tension stabilized at approximately 19 +/- 1 N/cm2 for both types of grafts, significantly lower than the value of 24 +/- 1 N/cm2 for control soleus muscles. Ablation of synergistic muscles resulted in a significant and sustained increase in mass and Po in regenerating skeletal muscle autografts. We conclude that provided the appropriate conditioning stimulus small grafts (100-200 mg) are capable of achieving the values for the mass and Po of control muscles.     

Mass Culturing of Ditylenchus dipsaci to Yield Large Quantities of Inoculum

Methods are described for rearing large quantities of Ditylenchus dipsaci on alfalfa tissues. Nematodes and alfalfa seed were disinfected and nematodes were reared in quantities sufficient to provide a continuous supply of inoculum for our alfalfa-breeding program. Nematodes reproduced best in darkness at 20-25 C. Cultures reached maximum numbers in 3-6 wk.