"Ménage à trois": the presence/absence of thyme shapes the mutualistic interaction between the host plant Medicago truncatula (Fabaceae) and its symbiotic bacterium Sinorhizobium meliloti

The long-term maintenance of specialized mutualisms remains an evolutionary puzzle. Recent focus has been on factors governing the stability of these mutualisms, including sanctions by the host, partner choice, and coevolutionary constraint, that is, the genetic correlation (r(G)) between fitness of both partners. So far these studies have been typically carried out in a single environment. Here, we ask if the genetic correlation between fitness of the host plant Medicago truncatula (Fabaceae) and its bacterial symbiont Sinorhizobium meliloti is affected by the presence/absence of a monoterpene (carvacrol) leached into the soil by Thymus vulgaris-a common plant of the Mediterranean vegetation, often co-occuring with Medicago. We show that the presence of carvacrol in the soil dramatically affects fitness of the rhizobial partner and increases the magnitude of r(G) between plant and rhizobia fitness (r(G) = 0.02 ± 0.05 vs. r(G) = 0.57 ± 0.02). This finding emphasizes the importance of heterogeneity in the biotic environment for understanding the evolution of species interactions.     

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.     

RIP2 regulates growth and differentiation of normal myoblasts and of rhabdomyosarcoma cells

RIP2 is an important regulator of myoblast proliferation and differentiation. We have previously demonstrated that in the myoblast cell line C2C12 and in primary myoblasts, downregulation of rip2 gene expression is a prerequisite for differentiation. To further study the role of rip genes in myogenesis, we compared expression patterns of rip1–4 in two myoblast cell lines, C2C12 and C2F3, after the induction of differentiation. These two cell lines are derived from the same clonal origin, but differ with respect to their differentiation behaviour: specifically, the differentiation process is slower and more incomplete in C2F3 cells. When analyzing cells up to 4 days after the induction of differentiation, we found no downregulation of rip2 gene expression in C2F3 cells, which might be linked to the low differentiation potential of these cells. In addition, in contrast to C2C12 cells, the rip3 gene was not expressed in C2F3 cells. To further study the role of rip genes in the regulation of myoblast growth and differentiation, we analyzed expression patterns of rip1–4 in rhabdomyosarcoma cell lines. We found that in these cells, rip2 expression was not downregulated after the induction of differentiation. Furthermore, in contrast to normal myoblasts, they did not express the rip3 and rip4 genes. Thus, we focused on the functional role of RIP2 in rhabdomyosarcoma cells. Inhibition of rip2 gene expression in C2C12 and in rhabdomyosarcoma cells using specific siRNAs led to decreased proliferation and promoted the differentiation process of these cells. These data indicate that differential expression of rip genes can be associated with abnormal growth and differentiation behaviour of skeletal myoblasts.     

Isolation and characterization of phosphatases from vascular smooth muscle

Actomyosin preparations of the carotid arteries of cattle contain a soluble phosphatase activity, which can be removed from the contractile proteins by repeated washings. This enzyme activity is lowered by high ionic strength, potassium fluoride, zinc acetate, ammonium molybdate, and vanadate. Magnesium ions enhance the enzyme activity. The phosphatase activity shows a maximum between pH 5.5 and 6.0 and a plateau of pH 7-9. By means of gel filtration on Sepharose 6B the phosphate activity is separated into three peaks, which are characterized with respect to their inhibition by potassium fluoride, ammonium molybate, and vanadate and their dependence on pH.     

High-density genetic mapping for coffee leaf rust resistance

Coffee leaf rust caused by the fungus Hemileia vastatrix causes considerable economic losses for coffee producers. Although agrochemical products can provide sufficient disease control, the use of resistant cultivars is a safer alternative. This resistance may be constrained by one or a few genetic factors, mainly those found in material originating from interspecific hybrids. In this study, the genetic analysis of an F ₂ population consisting of 224 plants derived from a crossing of Híbrido de Timor UFV 427-15 (resistant) with Catuaí Amarelo IAC 30 (susceptible) showed that a dominant gene confers the resistance of coffee to race II of H. vastatrix. From a genetic map saturated with 25 amplified fragment length polymorphism (AFLP) markers linked to the resistance gene, we developed a high-density genetic map with six sequence-characterized amplified region (SCAR) markers delimiting a chromosomal region of 9.45 cM and flanking the dominant gene at 0.7 and 0.9 cM. This is the first saturated and high-density genetic map obtained from this region containing the resistance gene. The results of this study are of great importance for the introduction of molecular markers for marker-assisted selection; they will also facilitate studies related to the cloning, structure, and function of race-specific genes involved in the resistance of coffee trees to H. vastatrix.     

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].     

Coherent Neutron Scattering and Collective Dynamics in the Protein,GFP

Collective dynamics are considered to be one of the major properties of soft materials, including biological macromolecules. We present coherent neutron scattering studies of the low-frequency vibrations, the so-called boson peak, in fully deuterated green fluorescent protein (GFP). Our analysis revealed unexpectedly low coherence of the atomic motions in GFP. This result implies a low amount of in-phase collective motion of the secondary structural units contributing to the boson peak vibrations and fast conformational fluctuations on the picosecond timescale. These observations are in contrast to earlier studies of polymers and glass-forming systems, and suggest that random or out-of-phase motions of the β-strands contribute greater than two-thirds of the intensity to the low-frequency vibrational spectra of GFP.