Differences in protein mobility between pioneer versus follower growth cones

Navigating growth cones need to integrate, process and respond to guidance signals, requiring dynamic information transfer within and between different compartments. Studies have shown that, faced with different navigation challenges, growth cones display dynamic changes in growth kinetics and morphologies. However, it remains unknown whether these are paralleled by differences in their internal molecular dynamics. To examine whether there are protein mobility differences during guidance, we developed multiphoton fluorescence recovery after photobleaching methods to determine molecular diffusion rates in pathfinding growth cones in vivo. Actively navigating growth cones (leaders) have consistently longer recovery times than growth cones that are fasciculated and less actively navigating (followers). Pharmacological perturbations of the cytoskeleton point to actin as the primary modulator of diffusion in differently behaving growth cones. This approach provides a powerful means to quantify mobility of specific proteins in neurons in vivo and reveals that diffusion is important during axon navigation

Ascending midbrain dopaminergic axons require descending GAD65 axon fascicles for normal pathfinding

The Nigrostriatal pathway (NSP) is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold

The Effect of Group Size on Individual Roles and the Potential for Cooperation in Group Bubble-net Feeding Humpback Whales (Megaptera novaeangliae)

Group foraging is observed in many species as a means to increase the ability of members of the group to find and exploit patchy prey. Group foraging can be exhibited in a number of different contexts based on the relationships between the participants, including by-product mutualism. One variant of by-product mutualism is cooperation, in which individuals achieve a greater energetic gain by feeding together than they would alone. In cooperation, individuals adopt a role in the group, and in the most complex interactions there may be multiple roles, resulting in a division of labor that occasionally includes role specialization. Humpback whales (Megaptera novaeangliae) are one of the few baleen whale species that have been observed feeding in groups, utilizing behaviors that are hypothesized to be cooperative. One of these behaviors is group bubble-net feeding, which has been observed in the Northeastern Pacific, Northwestern Atlantic, and Southern Oceans. This study utilized multi-sensor archival tag data from 26 humpback whales from the southern Gulf of Maine, 4 from Southeast Alaska, and 1 from the Western Antarctic Peninsula to analyze individual bubble-net feeding behaviors and compare these across populations. Linear mixed effects models were used to determine if dive behaviors varied with group sizes to test the hypothesis that group size influences individual behavior. The results indicate that individuals in the southern Gulf of Maine, for which sufficient data were available, were consistent in their bubble-net feeding behaviors across group sizes, which suggests that individuals utilize set roles in group feeding events. There was evidence for a division of labor and role specialization among whales utilizing certain bubble-net feeding tactics in the southern Gulf of Maine. The three populations performed different variations of bubble-net feeding that are likely based on the speed and schooling patterns of the prey. The results are consistent with the hypothesis that bubble-net feeding is an example of by-product mutualism in these populations, though was not enough data to suggest that group bubble-net feeding in Southeast Alaska was a form of by-product mutualism. The prevalence of herding dives in feeding groups suggest that each individual takes on a role to herd the prey to the surface, and provide evidence against a producer-scrounger relationship in the southern Gulf of Maine, and potentially in the Western Antarctic Peninsula

Frizzled-3a and Wnt-8b genetically interact during forebrain commissural formation in embryonic zebrafish

The commissural plate forms the rostral surface of the embryonic vertebrate forebrain and provides a cellular substrate for forebrain commissural axons. We have previously reported that the Wnt receptor frizzled-3a (fzd3a) restricts the expression of the chemorepulsive guidance ligand slit2 to a discrete domain of neuroepithelial cells in the commissural plate of embryonic zebrafish. Loss of Fzd3a function perturbed slit2 expression and disrupted the formation of glial bridges which guide the formation of forebrain commissures. We now show that Wnt8b is also necessary for anterior commissural formation as well as for patterning of slit2 expression at the midline. Knock down of Wnt8b produced the same phenotype as loss of Fzd3a which suggested that these genes were acting together to regulate axon guidance. Simultaneous sub-threshold knock down of both Fzd3a and Wnt8b led to a greater than additive increase in the penetrance of the mutant phenotype which indicated that these two genes were indeed interacting. We have shown here that Fzd3a/Wnt8b signaling is essential for normal patterning of the commissural plate and that loss-of-function in either receptor or ligand causes Slit2-dependent defects in glial bridge morphology which indirectly attenuated axon midline crossing in the embryonic vertebrate forebrain. (C) 2013 Elsevier B.V. All rights reserved

Glucose transporter (GLUT-4) is targeted to secretory granules in rat atrial cardiomyocytes

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the trans-Golgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulin-dependent movement to the cell surface. To examine the relationship between these GLUT-4-containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT-4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Caveolin-1 (Cav1) is an essential component of caveolae whose Src kinase-dependent phosphorylation on tyrosine 14 (Y14) is associated with regulation of focal adhesion dynamics. However, the relationship between these disparate functions remains to be elucidated. Caveola biogenesis requires expression of both Cav1 and cavin-1, but Cav1Y14 phosphorylation is dispensable. In this paper, we show that Cav1 tyrosine phosphorylation induces caveola biogenesis via actin-dependent mechanotransduction and inactivation of the Egr1 (early growth response-1) transcription factor, relieving inhibition of endogenous Cav1 and cavin-1 genes. Cav1 phosphorylation reduces Egr1 binding to Cav1 and cavin-1 promoters and stimulates their activity. In MDA-231 breast carcinoma cells that express elevated levels of Cav1 and caveolae, Egr1 regulated Cav1, and cavin-1 promoter activity was dependent on actin, Cav1, Src, and Rho-associated kinase as well as downstream protein kinase C (PKC) signaling. pCav1 is therefore a mechanotransducer that acts via PKC to relieve Egr1 transcriptional inhibition of Cav1 and cavin-1, defining a novel feedback regulatory loop to regulate caveola biogenesis

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice