SUMMARYAtrophy of neurons in the prefrontal cortex (PFC) plays a key role in the pathophysiology of depression and related disorders. The ability to promote both structural and functional plasticity in the PFC has been hypothesized to underlie the fast-acting antidepressant properties of the dissociative anesthetic ketamine. Here, we report that, like ketamine, serotonergic psychedelics are capable of robustly increasing neuritogenesis and/or spinogenesis both in vitro and in vivo. These changes in neuronal structure are accompanied by increased synapse number and function, as measured by fluorescence microscopy and electrophysiology. The structural changes induced by psychedelics appear to result from stimulation of the TrkB, mTOR, and 5-HT2A signaling pathways and could possibly explain the clinical effectiveness of these compounds. Our results underscore the therapeutic potential of psychedelics and, importantly, identify several lead scaffolds for medicinal chemistry efforts focused on developing plasticity-promoting compounds as safe, effective, and fast-acting treatments for depression and related disorders.

The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC–dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC–dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post–SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1−/− implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes centromere and chiasma function.

Abstract. To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nablp, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A) + RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nablp is identical to the Npl3p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and prerRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TP//mRNAs, a concomitant accumulation of unspliced ACTI pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing. HETEROGENEOUS nuclear RNAs (hnRNAs) t are the products of RNA polymerase II transcription, and include polyadenylated and nonpolyadenylated premRNAs and mRNAs as well as several uncharacterized RNAs (Dreyfuss et al., 1993). An array of nuclear factors bind to hnRNAs during transcription, including heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear RNP (snRNP) particles (Amero et al., 1992;Matunis et al., 1993). Numerous studies have established the roles of snRNP particles in a variety of nuclear processes such as pre-mRNA splicing (Green, 1991;Guthrie, 1991; Ruby and Abelson, 1991;Baserga and Steitz, 1993). In contrast, the functions of hnRNPs have remained unclear. HnRNPs are nuclear RNA-binding proteins whose primary and stable RNA-binding site is hnRNA. Current ideas about the functional roles of hnRNPs in pre-mRNA processing have Address all correspondence to M.

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH 2 -terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH 2 -terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

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Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm. The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1. Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not. These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm.

The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. We find that, between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of alphaPS integrin, inhibitors of Armadillo/beta-catenin nuclear activity and baculovirus caspase inhibitor p35. We conclude that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution.

Cytokines such as interferon-gamma (IFN-␥), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-␥ or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-␥ is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-␥(95-132) but not the peptide IFN-␥(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-␥(95-132). This observation suggests that the NLS in IFN-␥ may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-␥, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-␥(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-␥ was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-␥ in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-␥ is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-␥.The interferons (IFNs) 1 are cytokines that use the well studied JAK/STAT pathway for signal transduction to the nucleus (1). This pathway is initiated by the binding of the ligand to the extracellular domain of the appropriate receptor complex followed by the activation of select members of the JAK family of tyrosine kinases at the intracellular cytoplasmic domain of the receptor subunits. These tyrosine kinases in turn phosphorylate appropriate members of the STAT family of transcription factors present in the cytoplasm, thereby targeting these factors, through unknown mechanisms, for translocation to the nucleus to activate transcription. Transcription factors the size of STATs must be taken into the cell nucleus by an active transport process through the nuclear pore complex.Active nuclear import of a large number of nuclear proteins occurs through the Ran/importin pathway (for review, see Ref.2). In this pathway, the nuclear protein initially binds to a heterodimeric nuclear transport protein called importin which contains a ␣ and ␤ subunit. Nuclear proteins interact with the ␣ subunit, importin ␣, of the transporter via a specific nuclear localization sequence (NLS). These NLSs generally consi...