Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation.

T cells coordinate intercellular communication through the meticulous regulation of cytokine secretion. Direct visualization of vesicular transport and intracellular distribution of cytokines provides valuable insights into the temporal and spatial mechanisms involved in regulation. Employing Jurkat E6-1 T cells and interleukin-2 (IL-2) as a model system, we investigated vesicular dynamics using single-particle tracking and the nanoscale distribution of intracellular IL-2 in fixed T cells using superresolution microscopy. Live-cell imaging revealed that in vitro activation resulted in increased vesicular dynamics. Direct stochastic optical reconstruction microscopy and 3D structured illumination microscopy revealed nanoscale clustering of IL-2. In vitro activation correlated with spatial accumulation of IL-2 nanoclusters into more pronounced and elongated clusters. These observations provide visual evidence that accelerated vesicular transport and spatial concatenation of IL-2 clusters at the nanoscale may constitute a potential mechanism for modulating cytokine release by Jurkat T cells.

Cell cycle-related kinase regulates mammalian eye development through positive and negative regulation of the Hedgehog pathway.

Cell cycle-related kinase (CCRK) is a conserved regulator of ciliogenesis whose loss in mice leads to a wide range of developmental defects, including exencephaly, preaxial polydactyly, skeletal abnormalities, and microphthalmia. Here, we investigate the role of CCRK in mouse eye development. Ccrk mutants show dramatic patterning defects, with an expansion of the optic stalk domain into the optic cup, as well as an expansion of the retinal pigment epithelium (RPE) into neural retina (NR) territory. In addition, Ccrk mutants display a shortened optic stalk. These defects are associated with bimodal changes in Hedgehog (Hh) pathway activity within the eye, including the loss of proximal, high level responses but a gain in distal, low level responses. We simultaneously removed the Hh activator GLI2 in Ccrk mutants (Ccrk-/-;Gli2-/-), which resulted in rescue of optic cup patterning and exacerbation of optic stalk length defects. Next, we disrupted the Hh pathway antagonist GLI3 in mutants lacking CCRK (Ccrk-/-;Gli3-/-), which lead to even greater expansion of the RPE markers into the NR domain and a complete loss of NR specification within the optic cup. These results indicate that CCRK functions in eye development by both positively and negatively regulating the Hh pathway, and they reveal distinct requirements for Hh signaling in patterning and morphogenesis of the eyes.

Proper ciliary assembly is critical for restricting Hedgehog signaling during early eye development in mice.

Patterning of the vertebrate eye into optic stalk, retinal pigment epithelium (RPE) and neural retina (NR) territories relies on a number of signaling pathways, but how these signals are interpreted by optic progenitors is not well understood. The primary cilium is a microtubule-based organelle that is essential for Hedgehog (Hh) signaling, but it has also been implicated in the regulation of other signaling pathways. Here, we show that the optic primordium is ciliated during early eye development and that ciliogenesis is essential for proper patterning and morphogenesis of the mouse eye. Ift172 mutants fail to generate primary cilia and exhibit patterning defects that resemble those of Gli3 mutants, suggesting that cilia are required to restrict Hh activity during eye formation. Ift122 mutants, which produce cilia with abnormal morphology, generate optic vesicles that fail to invaginate to produce the optic cup. These mutants also lack formation of the lens, RPE and NR. Such phenotypic features are accompanied by strong, ectopic Hh pathway activity, evidenced by altered gene expression patterns. Removal of GLI2 from Ift122 mutants rescued several aspects of optic cup and lens morphogenesis as well as RPE and NR specification. Collectively, our data suggest that proper assembly of primary cilia is critical for restricting the Hedgehog pathway during eye formation in the mouse.

Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

Spata7 is required for maintenance of the retinal connecting cilium.

SPATA7, an early onset LCA3 retinal disease gene, encodes a putative scaffold protein that is essential for the proper assembly of the connecting cilium (CC) complex in photoreceptors. Previous studies have shown that SPATA7 interacts with other photoreceptor-specific ciliary proteins, such as RPGR and RPGRIP1, and maintains the integrity of CC integrity. However, although it is known that Spata7 is required for early formation of the CC, it is unclear if Spata7 is also required for the maintenance of the CC. To investigate Spata7 function in the retina at the adult stage, loss of function was induced in the adult retina upon tamoxifen induction of an inducible Spata7 knockout allele (Spata7flox/-; UbcCreERT2/+). The phenotype of mutant retina was characterized by a combination of histology, immunobiochemistry, and electroretinography (ERG). Our results demonstrated that Spata7 is also essential for maintaining the integrity of the mature retinal CC. Loss of Spata7 in adults caused phenotypes similar to those seen in germline mutant mice, including photoreceptor cell degeneration and defective ERG responses. Close examination of the CC revealed significantly shortened NPHP1 length as a result of Spata7 deletion. Furthermore, mislocalization of rhodopsin, leading to ER stress-mediated apoptosis, was observed in the retinal layers. Our results indicate that Spata7 is required not only for the establishment but also for the maintenance of the CC of photoreceptors.