A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.     

Extracellular generation of hydrogen peroxide is responsible for activation of EGF receptor by ultraviolet A radiation

Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) have been proposed to be activated in cells exposed to ultraviolet A (UVA) radiation (320-400 nm) and to be involved in photocarcinogenesis. Singlet oxygen and hydrogen peroxide are being discussed as mediators of the activation of signal transduction pathways by UVA. It is demonstrated here that EGFR is not activated in cells exposed to UVA in the absence of extracellular photosensitizers. Rather, UVA was capable of activating the EGFR and the related ErbB2 receptor tyrosine kinase in HeLa cells and human keratinocytes only under conditions that allowed for the extracellular photochemical generation of H(2)O(2), such as when cells were covered with cell culture medium during exposure to UVA. Pretreatment of cells with vanadate was required for UVA-induced EGFR activation, pointing to the involvement of protein tyrosine phosphatases. Unlike H(2)O(2), photochemically generated singlet oxygen did not activate EGFR but instead impaired the activation of EGFR by its ligand, EGF. In summary, extracellularly generated H(2)O(2) mediates UVA-induced activation of the EGFR and of ErbB2, whereas intracellular generation of reactive oxygen species upon exposure of cells to UVA is not sufficient for activation of the receptor.     

Microtubule motors at the intersection of trafficking and transport

Molecular motors drive the transport of vesicles and organelles within the cell. Traditionally, these transport processes have been considered separately from membrane trafficking events, such as regulated budding and fusion. However, recent progress has revealed mechanistic links that integrate these processes within the cell. Rab proteins, which function as key regulators of intracellular trafficking, have now been shown to recruit specific motors to organelle membranes. Rab-independent recruitment of motors by adaptor or scaffolding proteins is also a key mechanism. Once recruited to vesicles and organelles, these motors can then drive directed transport; this directed transport could in turn affect the efficiency of trafficking events. Here, we discuss this coordinated regulation of trafficking and transport, which provides a powerful mechanism for temporal and spatial control of cellular dynamics.     

Haplotype divergence in Beta vulgaris and microsynteny with sequenced plant genomes

We characterized two overlapping sugar beet ( Beta vulgaris) bacterial artificial chromosome (BAC) clones representing different haplotypes. A total of 254 kbp of the genomic sequence was determined, of which the two BACs share 92 kbp. Eleven of 15 genes discovered in the sequenced interval locate to the overlap region. The haplotypes differ in exons by 1% (nucleotide level) and in non-coding regions by 9% (6% mismatches, 3% gaps; alignable regions only). Large indels or high sequence divergence comprised 11% of either sequence. Of such indels, 68 and 45%, respectively, could be attributed to haplotype-specific integration of transposable elements. We identified novel repeat candidates by comparing the two BAC sequences to a set of genomic sugar beet sequences. Synteny was found with Arabidopsis chromosome 1 (At1), At2 and At4, Medicago chromosome 7, Vitis chromosome 15 and paralogous regions on poplar chromosomes II and XIV.     

Targeting human langerin promotes HIV-1 specific humoral immune responses

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4⁺ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.     

Functionally Relevant Domains of the Prion Protein Identified In Vivo

The prion consists essentially of PrP^Sc, a misfolded and aggregated conformer of the cellular protein PrP^C. Whereas PrP^C deficient mice are clinically healthy, expression of PrP^C variants lacking its central domain (PrP_ΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrP^C (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrP_ΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrP_ΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrP_ΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants.     

Tooth wear in captive wild ruminant species differs from that of free-ranging conspecifics

The mesowear method evaluates the wear patterns of herbivore cheek teeth by visually evaluating the facet development of the occlusal surfaces. It thus allows classification of most herbivorous ungulates into browsers, grazers or intermediate feeders, due to the fact that in grazers, tooth wear is characterized by a comparatively high degree of abrasion, most probably due to the presence of silicacious phytoliths in grasses, a higher amount of dust and grit adhering to their forage, or both. It has been suggested that excessive tooth wear could be a particularly limiting factor in the husbandry of captive large browsing species, and major tooth wear was demonstrated in captive as compared to free-ranging giraffe. If this increased tooth wear in captivity was an effect of feeding type and diets fed, then it would be expected that other browsing species are affected in a similar manner. In order to test this hypothesis, we investigated the dental mesowear pattern in captive individuals of 19 ruminant species and compared the results to data on free-ranging animals. Compared to free-ranging populations, captive browsers show a significantly more abrasion-dominated tooth wear signal. The reverse applies to captive grazers, which tend to show a less abrasion-dominated wear in captivity. Captive ruminants were generally more homogenous in their wear signature than free-ranging ruminants. If grit contamination in the natural habitat is a major cause of dental wear in grazers, then diets in captivity, although similar in botanical composition, most likely contain less abrasives due to feeding hygiene. If dental wear is one of the major factors limiting longevity, then captive grazers should achieve longer lifespans than both captive browsers and free-ranging grazers. In particular with respect to browsers, the results suggest that captive feeding regimes could be improved.     

Reversible Phosphorylation as a Molecular Switch to Regulate Plasma Membrane Targeting of Acylated SH4 Domain Proteins

Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes. Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated down-regulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.     

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.