Russell S. Hamilton

Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei

The metaphase–anaphase transition is orchestrated through proteolysis of numerous proteins by a ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C). A crucial aspect of this process is sister chromatid separation, which is thought to be mediated by separase, a thiol protease activated by the APC/C. Separase cleaves cohesin, a ring-shaped complex that entraps sister DNAs. It is a matter of debate whether cohesin-independent forces also contribute to sister chromatid cohesion. Using 4D live-cell imaging of Drosophila melanogaster syncytial embryos blocked in metaphase (via APC/C inhibition), we show that artificial cohesin cleavage is sufficient to trigger chromosome disjunction. This is nevertheless insufficient for correct chromosome segregation. Kinetochore–microtubule attachments are rapidly destabilized by the loss of tension caused by cohesin cleavage in the presence of high Cdk1 (cyclin-dependent kinase 1) activity, as occurs when the APC/C cannot destroy mitotic cyclins. Metaphase chromosomes undergo a bona fide anaphase when cohesin cleavage is combined with Cdk1 inhibition. We conclude that only two key events, opening of cohesin rings and downregulation of Cdk1, are sufficient to drive proper segregation of chromosomes in anaphase.

A PAR-1–dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte

A PAR-1–mediated bias in microtubule organization in the Drosophila oocyte underlies posterior-directed mRNA transport.

A common variant in the 3′UTR of the GRIK4 glutamate receptor gene affects transcript abundance and protects against bipolar disorder

Underactivity of the glutamatergic system is an attractive model for the pathophysiology of several major mental illnesses. We previously described a chromosome abnormality disrupting the kainate class ionotropic glutamate receptor gene, GRIK4/KA1, in an individual with schizophrenia and learning disability (mental retardation). We also demonstrated in a case-control study that two physically separated haplotypes within this gene were significantly associated with increased risk of schizophrenia and decreased risk of bipolar disorder, respectively. The latter protective haplotype was located at the 3′ end of the gene. We now report the identification from carriers of the protective haplotype of a deletion variant within the 3′ untranslated region of the gene. The deletion allele also was found to be negatively associated with bipolar disorder in both initial (P = 0.00000019) and replication (P = 0.0107) case-control studies. Expression studies indicated that deletion-carrying mRNA transcripts were relatively more abundant. We postulate that this may be a direct consequence of the differences in the RNA secondary structures predicted for the insertion and deletion alleles. These data suggest a mechanism whereby the genetic protective effect is mediated through increased kainate receptor expression.

Prediction of contacts from correlated sequence substitutions.

Recent work has led to a substantial improvement in the accuracy of predictions of contacts between amino acids using evolutionary information derived from multiple sequence alignments. Where large numbers of diverse sequence relatives are available and can be aligned to the sequence of a protein of unknown structure it is now possible to generate high-resolution models without recourse to the structure of a template. In this review we describe these exciting new techniques and critically assess the state-of-the-art in contact prediction in the light of these. While concentrating on methods, we also discuss applications to protein and RNA structure prediction as well as potential future developments.

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution

Background: NDE1 and NDEL1 are neurodevelopmental and mitotic proteins with extended coiled-coil N termini, but unknown C-terminal structure. Results: Recombinant NDE1/NDEL1 form dimers and tetramers in which their C termini interact with their N-terminal domains. Conclusion: NDE1/NDEL1 each adopt a sharply bent back structure. Significance: This explains the existence of two distinct dynein-binding domains on NDE1/NDEL1 and instability of disease-associated mutants lacking C termini. Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.

Identifying and searching for conserved RNA localisation signals.

RNA localisation is an important mode of delivering proteins to their site of function. Cis-acting signals within the RNAs, which can be thought of as zip-codes, determine the site of localisation. There are few examples of fully characterised RNA signals, but the signals are thought to be defined through a combination of primary, secondary, and tertiary structures. In this chapter, we describe a selection of computational methods for predicting RNA secondary structure, identifying localisation signals, and searching for similar localisation signals on a genome-wide scale. The chapter is aimed at the biologist rather than presenting the details of each of the individual methods.

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.

Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse.

ABSTRACT Synaptic plasticity involves the modulation of synaptic connections in response to neuronal activity via multiple pathways. One mechanism modulates synaptic transmission by retrograde signals from the post-synapse that influence the probability of vesicle release in the pre-synapse. Despite its importance, very few factors required for the expression of retrograde signals, and proper synaptic transmission, have been identified. Here, we identify the conserved RNA binding protein Syncrip as a new factor that modulates the efficiency of vesicle release from the motoneuron and is required for correct synapse structure. We show that syncrip is required genetically and its protein product is detected only in the muscle and not in the motoneuron itself. This unexpected non-autonomy is at least partly explained by the fact that Syncrip modulates retrograde BMP signals from the muscle back to the motoneuron. We show that Syncrip influences the levels of the Bone Morphogenic Protein ligand Glass Bottom Boat from the post-synapse and regulates the pre-synapse. Our results highlight the RNA-binding protein Syncrip as a novel regulator of synaptic output. Given its known role in regulating translation, we propose that Syncrip is important for maintaining a balance between the strength of presynaptic vesicle release and postsynaptic translation.

Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon

Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and β chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the ‘MHCII RNA operon’ in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5′- and 3′-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

ParticleStats: open source software for the analysis of particle motility and cytoskeletal polarity

The study of dynamic cellular processes in living cells is central to biology and is particularly powerful when the motility characteristics of individual objects within cells can be determined and analysed statistically. However, commercial programs only offer a limited range of inflexible analysis modules and there are currently no open source programs for extensive analysis of particle motility. Here, we describe ParticleStats (http://www.ParticleStats.com), a web server and open source programs, which input the X,Y coordinate positions of objects in time, and output novel analyses, graphical plots and statistics for motile objects. ParticleStats comprises three separate analysis programs. First, ParticleStats:Directionality for the global analysis of polarity, for example microtubule plus end growth in Drosophila oocytes. Second, ParticleStats:Compare for the analysis of saltatory movement in terms of runs and pauses. This can be applied to chromosome segregation and molecular motor-based movements. Thirdly ParticleStats:Kymographs for the analysis of kymograph images, for example as applied to separation of chromosomes in mitosis. These analyses have provided key insights into molecular mechanisms that are not possible from qualitative analysis alone and are widely applicable to many other cell biology problems.