Daniel A. Colón-Ramos

Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition.

Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis of apoptotic substrates. IAPs occlude the active sites of caspases to which they are bound and can function as ubiquitin ligases. IAPs are also reported to ubiquitinate themselves and caspases. Several proteins induce apoptosis, at least in part, by binding and inhibiting IAPs. Among these are the Drosophila melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal IAP-binding motif. We report here that Rpr not only inhibits IAP function, but also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibited protein translation. As the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis.

Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.

Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy

Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.

Functional Organization of a Neural Network for Aversive Olfactory Learning in Caenorhabditis elegans

Many animals use their olfactory systems to learn to avoid dangers, but how neural circuits encode naive and learned olfactory preferences, and switch between those preferences, is poorly understood. Here, we map an olfactory network, from sensory input to motor output, which regulates the learned olfactory aversion of Caenorhabditis elegans for the smell of pathogenic bacteria. Naive animals prefer smells of pathogens but animals trained with pathogens lose this attraction. We find that two different neural circuits subserve these preferences, with one required for the naive preference and the other specifically for the learned preference. Calcium imaging and behavioral analysis reveal that the naive preference reflects the direct transduction of the activity of olfactory sensory neurons into motor response, whereas the learned preference involves modulations to signal transduction to downstream neurons to alter motor response. Thus, two different neural circuits regulate a behavioral switch between naive and learned olfactory preferences.

Netrin (UNC-6) mediates dendritic self-avoidance

Dendrites from a single neuron may be highly branched but typically do not overlap. Self-avoidance behavior has been shown to depend on cell-specific membrane proteins that trigger mutual repulsion. Here we report the unexpected discovery that a diffusible cue, the axon guidance protein UNC-6 (Netrin), is required for self-avoidance of sister dendrites from the PVD nociceptive neuron in Caenorhabditis elegans. We used time-lapse imaging to show that dendrites fail to withdraw upon mutual contact in the absence of UNC-6 signaling. We propose a model in which the UNC-40 (Deleted in Colorectal Cancer; DCC) receptor captures UNC-6 at the tips of growing dendrites for interaction with UNC-5 on the apposing branch to induce mutual repulsion. UNC-40 also responds to dendritic contact through another pathway that is independent of UNC-6. Our findings offer a new model for how an evolutionarily conserved morphogenic cue and its cognate receptors can pattern a fundamental feature of dendritic architecture.

Chapter 2 Synapse Formation in Developing Neural Circuits

The nervous system consists of hundreds of billions of neurons interconnected into the functional neural networks that underlie behaviors. The capacity of a neuron to innervate and function within a network is mediated via specialized cell junctions known as synapses. Synapses are macromolecular structures that regulate intercellular communication in the nervous system, and are the main gatekeepers of information flow within neural networks. Where and when synapses form determines the connectivity and functionality of neural networks. Therefore, our knowledge of how synapse formation is regulated is critical to our understanding of the nervous system and how it goes awry in neurological disorders. Synapse formation involves pairing of the pre- and postsynaptic partners at a specific neurospatial coordinate. The specificity of synapse formation requires the precise execution of multiple developmental events, including cell fate specification, cell migration, axon guidance, dendritic growth, synaptic target selection, and synaptogenesis (Juttner and Rathjen in Cell. Mol. Life Sci. 62:2811, 2005; Salie et al., in Neuron 45:189, 2005; Waites et al., in Annu. Rev. Neurosci. 28:251, 2005). Remarkably, during the development of the vertebrate nervous system, these developmental processes occur almost simultaneously in billions of neurons, resulting in the formation of trillions of synapses. How this remarkable specificity is orchestrated during development is one of the outstanding questions in the field of neurobiology, and the focus of discussion of this chapter. We center the discussion of this chapter on the early developmental events that orchestrate the process of synaptogenesis prior to activity-dependent mechanisms. We have therefore limited the discussion of important activity-dependent synaptogenic events, which are discussed in other chapters of this book. Moreover, our discussion is biased toward lessons we have learned from invertebrate systems, in particular from C. elegans and Drosophila. We did so to complement the discussions from other chapters in this book, which focus on the important findings that have recently emerged from the vertebrate literature. The chapter begins with a brief history of the field of synaptic biology. This serves as a backdrop to introduce some of the historically outstanding questions of synaptic development that have eluded us during the past century, and which are the focus of this review. We then discuss some general features of synaptic structure as it relates to its function. In particular, we will highlight evolutionarily conserved traits shared by all synaptic structures, and how these features have helped optimize these ancient cellular junctions for interneural communication. We then discuss the regulatory signals that orchestrate the precise assembly of these conserved macromolecular structures. This discussion will be framed in the context of the neurodevelopmental process. Specifically, much of our discussion will focus on how the seemingly disparate developmental processes are intimately linked at a molecular level, and how this relationship might be crucial in the developmental orchestration of circuit assembly. We hope that the discussion of the multifunctional cues that direct circuit development provides a conceptual framework into understanding how, with a limited set of signaling molecules, precise neural wiring can be coordinated between synaptic partners.

Dynamic Encoding of Perception, Memory, and Movement in a C. elegans Chemotaxis Circuit

Brain circuits endow behavioral flexibility. Here, we study circuits encoding flexible chemotaxis in C. elegans, where the animal navigates up or down NaCl gradients (positive or negative chemotaxis) to reach the salt concentration of previous growth (the set point). The ASER sensory neuron mediates positive and negative chemotaxis by regulating the frequency and direction of reorientation movements in response to salt gradients. Both salt gradients and set point memory are encoded in ASER temporal activity patterns. Distinct temporal activity patterns in interneurons immediately downstream of ASER encode chemotactic movement decisions. Different interneuron combinations regulate positive versus negative chemotaxis. We conclude that sensorimotor pathways are segregated immediately after the primary sensory neuron in the chemotaxis circuit, and sensory representation is rapidly transformed to motor representation at the first interneuron layer. Our study reveals compact encoding of perception, memory, and locomotion in an experience-dependent navigational behavior in C. elegans.

Dual-view plane illumination microscopy for rapid and spatially isotropic imaging

We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3–8 h, depending on the size of the data.

A GH3-like Domain in Reaper Is Required for Mitochondrial Localization and Induction of IAP Degradation*

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.

Netrin instructs synaptic vesicle clustering through Rac GTPase, MIG-10, and the actin cytoskeleton

A signaling module known to organize the actin cytoskeleton during neuronal guidance also instructs synaptic vesicle clustering during synapse assembly.