Dendritically localized transcripts are sorted into distinct ribonucleoprotein particles that display fast directional motility along dendrites of hippocampal neurons.

Localization of mRNAs to postsynaptic sites and their subsequent translation is thought to contribute to synapse-specific plasticity. However, the direct visualization of dendritic RNA transport in living neurons remains a major challenge. Here, we analyze the transport of Alexa-labeled RNAs microinjected into mature hippocampal neurons. We show that microinjected MAP2 and CaMKIIalpha RNAs form particles that localize into dendrites as their endogenous counterparts. In contrast, nonlocalizing RNAs or truncated CaMKIIalpha, lacking the dendritic targeting element, remain in the cell body. Furthermore, our microinjection approach allowed us to identify a novel dendritically localized RNA, Septin7. Time-lapse videomicroscopy of neurons injected with CaMKIIalpha and Septin7 RNAs demonstrates fast directional movement along the dendrites of hippocampal neurons, with similar kinetics to Staufen1 ribonucleoprotein particles (RNPs). Coinjection and simultaneous visualization of two RNAs, as well as double detection of the corresponding endogenous RNAs, reveal that neuronal transcripts are differentially sorted in dendritic RNPs.

Proteasome subunit Rpn1 binds ubiquitin-like protein domains.

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.

A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis.

The dsRNA-binding protein Staufen was the first RNA-binding protein proven to play a role in RNA localization in Drosophila. A mammalian homolog, Staufen1 (Stau1), has been implicated in dendritic RNA localization in neurons, translational control, and mRNA decay. However, the precise mechanisms by which it fulfills these specific roles are only partially understood. To determine its physiological functions, the murine Stau1 gene was disrupted by homologous recombination. Homozygous stau1(tm1Apa) mutant mice express a truncated Stau1 protein lacking the functional RNA-binding domain 3. The level of the truncated protein is significantly reduced. Cultured hippocampal neurons derived from stau1(tm1Apa) homozygous mice display deficits in dendritic delivery of Stau1-EYFP and beta-actin mRNA-containing ribonucleoprotein particles (RNPs). Furthermore, these neurons have a significantly reduced dendritic tree and develop fewer synapses. Homozygous stau1(tm1Apa) mutant mice are viable and show no obvious deficits in development, fertility, health, overall brain morphology, and a variety of behavioral assays, e.g., hippocampus-dependent learning. However, we did detect deficits in locomotor activity. Our data suggest that Stau1 is crucial for synapse development in vitro but not critical for normal behavioral function.

High-efficiency transfection of mammalian neurons via nucleofection.

Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.

Control of receptor-induced signaling complex formation by the kinetics of ligand/receptor interaction.

Tumor necrosis factor (TNF) exists both as a membrane-integrated type II precursor protein and a soluble cytokine that have different bioactivities on TNFR2 (CD120b) but not on TNFR1 (CD120a). To identify the molecular basis of this disparity, we have investigated receptor chimeras comprising the cytoplasmic part of Fas (CD95) and the extracellular domains of the two TNF receptors. The membrane form of TNF, but not its soluble form, was capable of inducing apoptosis as well as activation of c-Jun N-terminal kinase and NF-kappaB via the TNFR2-derived chimera. In contrast, the TNFR1-Fas chimera displayed strong responsiveness to both TNF forms. This pattern of responsiveness is identical to that of wild type TNF receptors, demonstrating that the underlying mechanisms are independent of the particular type of the intracellular signaling machinery and rather are controlled upstream of the intracellular domain. We further demonstrate that the signaling strength induced by a given ligand/receptor interaction is regulated at the level of adaptor protein recruitment, as shown for FADD, caspase-8, and TRAF2. Since both incidents, strong signaling and robust adapter protein recruitment, are paralleled by a high stability of individual ligand-receptor complexes, we propose that half-lives of individual ligand-receptor complexes control signaling at the level of adaptor protein recruitment.