The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced "truncated receptors" with identical extracellular ligandbinding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1-and trkB.T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.

At fertilization, signals at the site of sperm-egg interaction cause a rise in cytosolic Ca 2ϩ (1,2). This opens ion channels and stimulates exocytosis of cortical granules, resulting in blocks to polyspermy, and also stimulates the resumption of the cell cycle (3-5). The Ca 2ϩ rise results, at least in large part, from Ca 2ϩ release from the endoplasmic reticulum, mediated by inositol 1,4,5-trisphosphate (IP 3 ) 1 (6 -10). Much recent work on fertilization has focused on the signal transduction pathways that lead to IP 3 production.The phospholipase C family of enzymes produces IP 3 and diacylglycerol from the membrane lipid phosphatidylinositol 4,5-bisphosphate (11). In echinoderm eggs, it is the ␥ isoform of phospholipase C (PLC␥) that functions at fertilization. PLC␥ enzyme activity increases by 30 s post-fertilization in sea urchin eggs (12), and inhibition of PLC␥ activation inhibits Ca 2ϩ release at fertilization in both sea urchin and starfish eggs (13)(14)(15). In these experiments, PLC␥ activity was inhibited by injection of eggs with excess Src homology 2 (SH2) domains of PLC␥. SH2 domains are found in many signaling proteins, and provide a site for specific interaction of a particular protein with a particular phosphorylated tyrosine on another protein (16). Excess SH2 domains, introduced into cells by microinjection, function as specific dominant negative inhibitors of such interactions.PLC␥ can be activated by phosphorylation of a regulatory tyrosine, although other factors may also be significant (17)(18)(19)(20). In sea urchin eggs, attempts to determine if PLC␥ is tyrosine phosphorylated at fertilization have been inconclusive, since the phosphotyrosine in PLC␥ immunoprecipitates was barely detectable either before or after fertilization (12, 21). As discussed by these authors, a local increase at the site of spermegg interaction might have been too small to detect by the methods used. Nevertheless, tyrosine kinase activity increases within 15 s after fertilization (22), and the tyrosine kinase inhibitor genistein delays Ca 2ϩ release at fertilization (23). One group of tyrosine kinases that participates, directly or indirectly, in activation of PLC␥ is the Src family (20, 24 -26), and in vitro experiments with starfish eggs have shown a fertilization-dependent association of a Src-like kinase with the SH2 domains of PLC␥ (27). Further evidence that a Src family kinase functions to activate PLC␥ at fertilization comes from findings that in both starfish and sea urchin eggs, injection of excess SH2 domains of Src family kinases inhibits Ca 2ϩ release at fertilization (28,29). In addition, in sea urchin eggs, the Src family kinase inhibitor PP1 delays Ca 2ϩ release at fertilization, and the activity of a Src-like kinase increases by 30 s postinsemination (29).These findings indicate that a Src-like kinase may, directly or through intermediate molecules, activate PLC␥ at fertilization, leading to Ca 2ϩ release and egg activation. In this report, we examine four questions related to this model. 1) I...