Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for "surveying" mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing β-globin mRNA in intact single cells after the induction of β-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing β-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free β-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.

A complex nervous system requires precise numbers of various neuronal types produced with exquisite spatiotemporal control. This striking diversity is generated by a limited number of neural stem cells (NSC), where spatial and temporal patterning intersect. Drosophila is a genetically tractable model system that has significant advantages for studying stem cell biology and neuronal fate specification. Here we review the latest findings in the rich literature of temporal patterning of neuronal identity in the Drosophila central nervous system. Rapidly changing consecutive transcription factors expressed in NSCs specify short series of neurons with considerable differences. More slowly progressing changes are orchestrated by NSC intrinsic temporal factor gradients which integrate extrinsic signals to coordinate nervous system and organismal development.

Driven either by external landmarks or by internal dynamics, hippocampal neurons form sequences of cell assemblies. The coordinated firing of these active cells is organized by the prominent "theta" oscillations in the local field potential (LFP): place cells discharge at progressively earlier theta phases as the rat crosses the respective place field ("phase precession"). The faster oscillation frequency of active neurons and the slower theta LFP, underlying phase precession, creates a paradox. How can faster oscillating neurons comprise a slower population oscillation, as reflected by the LFP? We built a mathematical model that allowed us to calculate the population activity analytically from experimentally derived parameters of the single neuron oscillation frequency, firing field size (duration), and the relationship between within-theta delays of place cell pairs and their distance representations ("compression"). The appropriate combination of these parameters generated a constant frequency population rhythm along the septo-temporal axis of the hippocampus, while allowing individual neurons to vary their oscillation frequency and field size. Our results suggest that the faster-than-theta oscillations of pyramidal cells are inherent and that phase precession is a result of the coordinated activity of temporally shifted cell assemblies, relative to the population activity, reflected by the LFP.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

During the development of the central nervous system (CNS) of Drosophila, neuronal stem cells, the neuroblasts (NBs), first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, ’secondary’ neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ’bi-oriented’ kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

Theoretical neuroscientists often try to understand how the structure of a neural network relates to its function by focusing on structural features that would either follow from optimization or occur consistently across possible implementations. Both optimization theories and ensemble modeling approaches have repeatedly proven their worth, and it would simplify theory building considerably if predictions from both theory types could be derived and tested simultaneously. Here we show how tensor formalism from theoretical physics can be used to unify and solve many optimization and ensemble modeling approaches to predicting synaptic connectivity from neuronal responses. We specifically focus on analyzing the solution space of synaptic weights that allow a thresholdlinear neural network to respond in a prescribed way to a limited number of input conditions. For optimization purposes, we compute the synaptic weight vector that minimizes an arbitrary quadratic loss function. For ensemble modeling, we identify synaptic weight features that occur consistently across all solutions bounded by an arbitrary quadratic function. We derive a common solution to this suite of nonlinear problems by showing how each of them reduces to an equivalent linear problem that can be solved analytically. Although identifying the equivalent linear problem is nontrivial, our tensor formalism provides an elegant geometrical perspective that allows us to solve the problem numerically. The final algorithm is applicable to a wide range of interesting neuroscience problems, and the associated geometric insights may carry over to other scientific problems that require constrained optimization.

BACKGROUND: Conditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation. RESULTS: To investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons. CONCLUSIONS: The finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.