The coiled coil domain of DEF6 facilitates formation of large vesicle-like, cytoplasmic aggregates that trap the P-body marker DCP1 and exhibit prion-like features

ABSTRACTDEF6, also known as SLAT and IBP, is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse. However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. DEF6 can adopt multiple conformations that result in different cellular localisations and functions. Post translational modifications such as phosphorylation result in conformational change liberating functional domains that are masked in the native stage of DEF6. ITK phosphorylation of Try210/222 liberates the N-terminal end and to a certain extend also the C-terminal coiled coil domain of DEF6 resulting in P-body colocalisation. In fact, the N-terminal 45 amino acids of DEF6 that encode a Ca2+-binding EF hand are sufficient to target P-bodies. Mutant proteins that unleashed the C-terminal coiled coil domain of DEF6 spontaneously aggregated forming large vesicle-like, cytoplasmic structures. These aggregates trapped proteins such as the P-body component DCP1 altering its cytoplasmic localisation. However, cellular stress reversed aggregate formation in mutant DEF6 proteins that contained ITAM and PH domain in conjunction with the coiled coil domain resulting in colocalisation with DCP1. Furthermore, coiled coil-mediated aggregates appeared to function like prions enforcing conformational change onto wild type DEF6 protein.

Ultrastructural and cytochemical studies on vitellogenesis in trypanorhynch cestode Dollfusiella spinulifera Beveridge, Neifar et Euzet, 2004 (Eutetrarhynchidae)

The first description of vitellogenesis in the Trypanorhyncha is presented in this paper. Though the type of vitellogenesis and mature vitellocyte in Dollfusiella spinulifera appear to be unique among the Eucestoda, to some extent they resemble that observed in the lower cestodes, namely the Tetraphyllidea and Pseudophyllidea. Maturation is characterized by: (1) an increase in cell volume; (2) extensive development of large, parallel, frequently concentric cisternae of GER that produce proteinaceous granules; (3) development of Golgi complexes engaged in packaging this material; (4) continuous enlargement of proteinaceous granules within vesicles and their transformation into shell globule clusters; and (5) progressive fusion of all vesicles, with flocculent material containing the proteinaceous granules and shell globule clusters, into a single very large vesicle that characterises mature vitellocytes of this tapeworm. Cell inclusions in and around the large vesicle consist of flocculent material of a very low density, a few shell globule clusters, moderately dense proteinaceous granules and numerous large droplets of unsaturated lipids. A new previously unreported mode of transformation of proteinaceous granules into shell globule clusters, that evidently differs from that of pseudophyllideans and tetraphyllideans, is described. Cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for polysaccharides indicates a strongly positive reaction for membrane-bound glycoproteins in all membranous structures such as GER, mitochondria, Golgi complexes, nuclear and cell plasma membranes. Similar staining revealed β-glycogen particles scattered in the cytoplasm of maturing vitellocytes. Typical cytoplasmic β-glycogen particles appear mainly during early vitellocyte maturation but it is characteristic for this species that they are only seldom visible in mature cells. Some working hypotheses concerning the interrelationship between this particular pattern of vitellogensis, possible mode of egg formation in D. spinulifera, its embryonic development and trypanorhynchean life cycle, are drawn and discussed.

Spatial variability in release at the frog neuromuscular junction measured with FM1-43

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.Key words: synaptic transmission, exocytosis, synaptic vesicles, neuromuscular junction.