Ubiquitination of the protocadherin-γA3 variable cytoplasmic domain modulates cell-cell interaction.

The family of ∼60 clustered protocadherins (Pcdhs) are cell adhesion molecules encoded by a genomic locus that regulates expression of distinct combinations of isoforms in individual neurons resulting in what is thought to be a neural surface "barcode" which mediates same-cell interactions of dendrites, as well as interactions with other cells in the environment. Pcdh mediated same-cell dendrite interactions were shown to result in avoidance while interactions between different cells through Pcdhs, such as between neurons and astrocytes, appear to be stable. The cell biological mechanism of the consequences of Pcdh based adhesion is not well understood although various signaling pathways have been recently uncovered. A still unidentified cytoplasmic regulatory mechanism might contribute to a "switch" between avoidance and adhesion. We have proposed that endocytosis and intracellular trafficking could be part of such a switch. Here we use "stub" constructs consisting of the proximal cytoplasmic domain (lacking the constant carboxy-terminal domain spliced to all Pcdh-γs) of one Pcdh, Pcdh-γA3, to study trafficking. We found that the stub construct traffics primarily to Rab7 positive endosomes very similarly to the full length molecule and deletion of a substantial portion of the carboxy-terminus of the stub eliminates this trafficking. The intact stub was found to be ubiquitinated while the deletion was not and this ubiquitination was found to be at non-lysine sites. Further deletion mapping of the residues required for ubiquitination identified potential serine phosphorylation sites, conserved among Pcdh-γAs, that can reduce ubiquitination when pseudophosphorylated and increase surface expression. These results suggest Pcdh-γA ubiquitination can influence surface expression which may modulate adhesive activity during neural development.

Gamma-protocadherin localization at the synapse is associated with parameters of synaptic maturation.

Clustered protocadherins (Pcdhs) are a family of ~60 cadherin-like proteins (divided into subclasses α, ß, and γ) that regulate dendrite morphology and neural connectivity. Their expression is controlled through epigenetic regulation at a gene cluster encoding the molecules. During neural development, Pcdhs mediate dendrite self-avoidance in some neuronal types through an uncharacterized anti-adhesive mechanism. Pcdhs are also important for dendritic complexity in cortical neurons likely through a pro-adhesive mechanism. Pcdhs have also been postulated to participate in synaptogenesis and connectivity. Some synaptic defects were noted in knockout animals, including synaptic number and physiology, but the role of these molecules in synaptic development is not understood. The effect of Pcdh knockout on dendritic patterning may present a confound to studying synaptogenesis. We showed previously that Pcdh-γs are highly enriched in intracellular compartments in dendrites and spines with localization at only a few synaptic clefts. To gain insight into how Pcdh-γs might affect synapses, we compared synapses that harbored Pcdh-γs versus those that did not for parameters of synaptic maturation including pre- and postsynaptic size, postsynaptic perforations, and spine morphology by light microscopy in cultured hippocampal neurons and by serial section immuno-electron microscopy in hippocampal CA1. In mature neurons, synapses immunopositive for Pcdh-γs were larger in diameter with more frequent perforations. Analysis of spines in cultured neurons revealed that mushroom spines were more frequently immunopositive for Pcdh-γs at their tips than thin spines. These results suggest that Pcdh-γ function at the synapse may be related to promotion of synaptic maturation and stabilization.

Electron microscopic characterization of nuclear egress in the sea urchin gastrula.

Nuclear egress, also referred to as nuclear envelope (NE) budding, is a process of transport in which vesicles containing molecular complexes or viral particles leave the nucleus through budding from the inner nuclear membrane (INM) to enter the perinuclear space. Following this event, the perinuclear vesicles (PNVs) fuse with the outer nuclear membrane (ONM), where they release their contents into the cytoplasm. Nuclear egress is thought to participate in many functions such as viral replication, cellular differentiation, and synaptic development. The molecular basis for nuclear egress is now beginning to be elucidated. Here, we observe in the sea urchin gastrula, using serial section transmission electron microscopy, strikingly abundant PNVs containing as yet unidentified granules that resemble the ribonucleoprotein complexes (RNPs) previously observed in similar types of PNVs. Some PNVs were observed in the process of fusion with the ONM where they appeared to release their contents into the cytoplasm. These vesicles were abundantly observed in all three presumptive germ layers. These findings indicate that nuclear egress is likely to be an important mechanism for nucleocytoplasmic transfer during sea urchin development. The sea urchin may be a useful model to characterize further and gain a better understanding of the process of nuclear egress.

Glucose Homeostasis and Retinal Histopathology in CSAD KO Mice.

In this study we examined glucose homeostasis and retinal histology in homozygous knockout mice lacking CSAD (CSAD-KO). Two-month-old male mice were used including wild type (WT), homozygotes with without supplementation of taurine in the drinking water (1% w/v). Mice were sacrificed and the eyes processed for histology and immunohistochemistry. Additional mice were subjected to a glucose tolerance test (7.5 mg/kg BW) after 12 h fasting. We found that CSAD-KO and CSAD-KO treated with taurine were slightly hypoglycemic prior to glucose injection and showed a significantly reduced plasma glucose at 30, 60 and 120 min post-glucose injection, compared to WT. While glucose homeostasis in CSAD-KO was significantly different compared to WT, CSAD-KO supplemented with taurine was without effect. Analysis of retinas by electron microscopy showed that CSAD-KO without taurine supplementation exhibited substantial retinal degeneration. Remaining photoreceptor outer and inner segments were disorganized. Retinal nuclear and synaptic layers were largely absent and there was apparent reorganization of the pigmented epithelial cells. The choroid and sclera were intact. These histological aberrations were largely rectified by taurine supplementation in the drinking water.These data indicate that taurine deficiency alters glucose homeostasis and retinal structure and taurine supplementation improves these retinal abnormalities, but not in hypoglycemia.

Pah1p negatively regulates the expression of V-ATPase genes as well as vacuolar acidification.

In yeast, PAH1 plays an important role in cell homeostasis and lipid biosynthesis. PAH1 encodes for the PA phosphatase, Pah1p, which is responsible for de novo TAG and phospholipid synthesis. It has been suggested that the lack of Pah1p causes irregular vacuolar morphology and dysfunctional V-ATPase pump activity. However, the molecular connection between Pah1p and V-ATPase activity has remained unclear. Through real-time PCR, we have shown that PAH1 is maximally induced at the stationary stage in the presence of inositol. We also found that vacuoles were less fragmented when PAH1 is maximally expressed. Subsequently, we observed that vacuoles from pah1Δ cells were more acidic than those in WT cells. Furthermore, V-ATPase genes were upregulated in the absence of Pah1p. These results suggest that Pah1p plays an important role in vacuolar activity by negatively regulating the expression of V-ATPase genes. As such, we provide evidence to show the role of Pah1p in vacuolar acidification and fragmentation.

Clustered protocadherin trafficking.

The cluster of almost 60 protocadherin genes, divided into the α, ß and γ subgroups, is a hallmark of vertebrate nervous system evolution. These clustered protocadherins (Pcdhs) are of interest for several reasons, one being the arrangement of the genes, which allows epigenetic regulation at the cluster and single-cell identity. Another reason is the still ambiguous effect of Pcdhs on cell-cell interaction. Unlike the case for classical cadherins, which typically mediate strong cell adhesion and formation of adherens junctions, it has been challenging to ascertain exactly how Pcdhs affect interacting cells. In some instances, Pcdhs appear to promote the association of membranes, while in other cases the Pcdhs are anti-adhesive and cause avoidance of interacting membranes. It is clear that Pcdh extracellular domains bind homophillically in an antiparallel conformation, typical of adhesive interactions. How can molecules that would seemingly bind cells together be able to promote the avoidance of membranes? It is possible that Pcdh trafficking will eventually provide insights into the role of these molecules at the cell surface. We have found that endogenous and expressed Pcdhs are generally less efficient at targeting to cell junctions and synapses than are classical cadherins. Instead, Pcdhs are prominently sequestered in the endolysosome system or other intracellular compartments. What role this trafficking plays in the unique mode of cell-cell interaction is a current topic of investigation. It is tempting to speculate that modulation of endocytosis and endolysosomal trafficking may be a part of the mechanism by which Pcdhs convert from adhesive to avoidance molecules.