A screening procedure was used to identify cell fusion (hyphal anastomosis) mutants in the Neurospora crassa single gene deletion library. Mutants with alterations in 24 cell fusion genes required for cell fusion between conidial anastomosis tubes (CATs) were identified and characterized. The cell fusion genes identified included 14 genes that are likely to function in signal transduction pathways needed for cell fusion to occur (mik-1, mek-1, mak-1, nrc-1, mek-2, mak-2, rac-1, pp2A, so/ham-1, ham-2, ham-3, ham-5, ham-9, and mob3). The screening experiments also identified four transcription factors that are required for cell fusion (adv-1, ada-3, rco-1, and snf5). Three genes encoding proteins likely to be involved in the process of vesicular trafficking were also identified as needed for cell fusion during the screening (amph-1, ham-10, pkr1). Three of the genes identified by the screening procedure, ham-6, ham-7, and ham-8, encode proteins that might function in mediating the plasma membrane fusion event. Three of the putative signal transduction proteins, three of the transcription factors, the three putative vesicular trafficking proteins, and the three proteins that might function in mediating cell fusion had not been identified previously as required for cell fusion.The process of cell-to-cell fusion plays a vital role in the life cycles of almost all multicellular organisms. Fertilization, the fusion of an egg and sperm, is a required cell-to-cell fusion event for sexually reproducing organisms. For vertebrates, cell fusion is a critical step in the development of muscle, placenta, and bone and plays an essential role in the formation of multinucleated giant cells in the immune system. Hyphal cell fusion plays an important role in the life cycle of Neurospora crassa and other filamentous fungi (1,6,12,24,38). During the life cycle of the filamentous fungus Neurospora crassa, cell fusions occur during at least three stages (12, 38). During sexual development, fertilization occurs as a protoperithecium (immature female mating structure) generates a trichogyne (long specialized hyphae) that chemotrophically grows toward a conidium (asexual spore) or hypha of the opposite mating type and undergoes cell fusion with it. During the germination of N. crassa conidia, the cells produce short, specialized thin hyphae called conidial anastomosis tubes (CATs), which mediate cell fusion between the germlings and generate an interconnected hyphal network (38,41). This process of cell fusion between conidia allows the cells to share resources and may be critical to the establishment of a colony under some environmental conditions. Cell fusions also occur during the growth of a vegetative N. crassa colony. A few millimeters behind the growing edge of the colony, specialized fusion hyphae are formed as branches from the vegetative hyphae. The fusion hyphae grow toward each other in a directed manner and undergo cell fusion to generate an interconnected hyphal network within the colony (18). Cytoplasm and organelles flow f...

Human immunodeficiency virus-associated neurological disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy. The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, "reversible" DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging human immunodeficiency virus (HIV)-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson's disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously, we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here, we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT V max , without changes in K m or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by total internal reflection fluorescence microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3␤. We conclude that Tat activates RyRs via a calcium-and calpain-mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3␤ inhibition as clinical therapeutic approaches for HAND. Finally, we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients and by demonstrating similar findings in Tat-expressing transgenic mice.

Mutants of Neurospora crassa unable to participate in vegetative hyphal fusion (anastomosis) were isolated and characterized. From this analysis, three genes, rcm-1, rco-1 and ham-5, were identified and shown to be required for hyphal fusion. The rcm-1 and rco-1 genes are homologues of the Saccharomyces cerevisiae SSN6 and TUP1 genes, which encode a dimeric transcription factor in yeast. We demonstrate that in N. crassa the rcm-1 and rco-1 genes are required for hyphal fusion and normal hyphal morphology, and influence both asexual and sexual development. The ham-5 gene encodes a 1686 amino acid protein with two putative WD40 domains, which might participate in protein–protein interactions. ham-5 deletion mutants had a reduced rate of hyphal extension and altered hyphal morphology, and were unable to produce the conidial anastomosis tubes that are required for hyphal fusion during colony initiation.

Tetrahymena telomeres usually consist of ϳ250 base pairs of T 2 G 4 repeats, but they can grow to reach a new length set point of up to 900 base pairs when kept in log culture at 30°C. We have examined the growth profile of individual macronuclear telomeres and have found that the rate and extent of telomere growth are affected by the subtelomeric region. When the sequence of the rDNA subtelomeric region was altered, we observed a decrease in telomere growth regardless of whether the GC content was increased or decreased. In both cases, the ordered structure of the subtelomeric chromatin was disrupted, but the effect on the telomeric complex was relatively minor. Examination of the telomeres from non-rDNA chromosomes showed that each telomere exhibited a unique and characteristic growth profile. The subtelomeric regions from individual chromosome ends did not share common sequence elements, and they each had a different chromatin structure. Thus, telomere growth is likely to be regulated by the organization of the subtelomeric chromatin rather than by a specific DNA element. Our findings suggest that at each telomere the telomeric complex and subtelomeric chromatin cooperate to form a unique higher order chromatin structure that controls telomere length. INTRODUCTIONTelomeres from most organisms exhibit a characteristic mean length that results from a balance between addition of telomeric DNA by telomerase or recombination and loss of DNA due to incomplete replication or nuclease activity (Greider, 1996;McEachern et al., 2000). If this balance is perturbed, telomeres grow or shrink until a new length set point is reached. Factors that control the balance, and hence regulate telomere length, include telomere and telomerase components, replication and repair proteins, and environmental conditions. In Saccharomyces cerevisiae, more than a dozen different proteins have been shown to affect telomere length (Bourns et al., 1998;Blackburn, 2001), whereas rapid proliferation and increased culture temperature induce telomere growth in Tetrahymena, trypanosomes, and Candida albicans (Bernards et al., 1983;Larson et al., 1987;McEachern and Hicks, 1993).In organisms such as Tetrahymena and S. cerevisiae that have relatively short telomeres (250 -350 nt), the entire telomeric tract can be packaged into a nonnucleosomal complex (Blackburn and Chiou, 1981;Wright et al., 1992;Cohen and Blackburn, 1998), but in organisms with longer telomeres, the telomeric DNA is bound by a combination of nucleosomes and specialized telomere proteins (Tommerup et al., 1994). Both short and long telomeres seem to be subject to a second level of packaging that involves folding or looping of the telomeric tract to form a more compact higher order structure (Grunstein, 1997;Griffith et al., 1999). In S. cerevisiae, this folding is mediated by protein-protein interactions with the telomere protein Rap1 interacting with SIR proteins bound to nucleosomes along the subtelomeric region (Grunstein, 1997). In vertebrate and plant cells, the folding seems ...

Activation of nuclear factor-kappaB (NF-kappaB), a key feature of the neurotrophin signaling, has been shown to be critical for neuronal survival under pathologic settings. However, the precise mechanism by which neurotrophins activate NF-kappaB is not well understood. Here we report that the Ankyrin-rich Membrane Spanning (ARMS/Kidins220) protein, a novel transmembrane substrate of tropomyosin receptor kinase B (TrkB), plays an important role in NF-kappaB signaling elicited by brain-derived neurotrophic factor (BDNF). Accordingly, depletion of ARMS by specific RNA interference, or disruption of ARMS-TrkB interaction with expression of dominant-negative ARMS mutant, abolished BDNF-induced signaling to NF-kappaB. Our data further suggests that ARMS may promote NF-kappaB signaling via activation of mitogen-activated kinase (MAPK) and IkappaB kinase (IKK), thereby facilitating phosphorylation of RelA (major NF-kappaB subunit) at an IKK-sensitive site. The results shown here identify ARMS as a major factor that links neurotrophin signaling to NF-kappaB.