Biology subjects

Hall, D. H.

Publications and source records attributed to Hall, D. H..

34 records · Page 2Linked to original sources

bioRxiv2020

A genetic screen identifies processes that couple oocyte maturation to proteostasis in the immortal C. elegans germ lineage

Somatic cells age and die, but the germ-cell lineage is immortal. In C. elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte-maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in ER activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.

cell biology↗

bioRxiv2020

Beyond the connectome: A map of a brain architecture derived from whole-brain volumetric reconstructions

Animal nervous system organization is crucial for all body functions and its disruption can manifest in severe cognitive and behavioral impairment. This organization relies on features across scales, from nano-level localization of synapses, through multiplicities of neuronal morphologies and their contribution to circuit organization, to the high level stereotyped connections between different regions of the brain. The sheer complexity of this organ means that to date, we have yet to reconstruct and model the structure of a complete nervous system that is integrated across all these scales. Here, we present a complete structure-function model of the nematode C. elegans main neuropil, the nerve ring, which we derive by integrating the volumetric reconstruction from two animals with corresponding synaptic and gap junctional connectomes. Whereas previously the nerve ring was considered a densely packed tract of axons, we uncover internal organization into 5 functional bundles and show how they spatially constrain and support the synaptic connectome. We find that the C. elegans connectome is not invariant, but that a precisely wired core circuit is embedded in a background of variable connectivity, and propose a corresponding reference connectome for the core circuit. Using this reference, we show that the architecture of the C. elegans brain can be viewed as a modular Residual Network that supports sensory computation and integration, sensory-motor convergence, and brain-wide coordination. These findings point to scalable and robust features of brain organization that are likely universal across phyla.

neuroscience↗

bioRxiv2020

A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans

Biological tubes must develop and maintain their proper diameter in order to transport materials efficiently. These tubes are molded and protected in part by apical extracellular matrices (aECMs) that line their lumens. Despite their importance, aECMs are difficult to image in vivo and therefore poorly understood. The C. elegans vulva has been a paradigm for understanding many aspects of organogenesis. Here we describe the vulva luminal matrix, which contains chondroitin proteoglycans, Zona Pellucida (ZP) domain proteins, and other glycoproteins and lipid transporters related to those in mammals. Confocal and transmission electron microscopy revealed, with unprecedented detail, a complex and dynamic aECM. Different matrix factors assemble on the apical surfaces of each vulva cell type, with clear distinctions seen between Ras-dependent (1{degrees}) and Notch-dependent (2{degrees}) cell types. Genetic perturbations suggest that chondroitin and other aECM factors together generate a structured scaffold that both expands and constricts lumen shape.

developmental biology↗

bioRxiv2020

F-box protein MEC-15 promotes microtubule stability and neurite growth by antagonizing the HSP90 chaperone network in Caenorhabditis elegans

Molecular chaperones often work collaboratively with the ubiquitination-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development, and neuronal functions, and these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and cochaperones. MEC-15 likely functions in a SCF complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and chaperones; this antagonism regulates TRN development as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between UPS and chaperones tightly controls neuronal differentiation. Summary statementMolecular chaperones are known to protect cells from stress. However, in this study the authors showed that the Hsp90 chaperone negatively regulates neuronal differentiation when the ubiquitination-proteasome system is compromised.

developmental biology↗

bioRxiv2019

The connectome of the Caenorhabditis elegans pharynx

Detailed anatomical maps of individual organs and entire animals have served as invaluable entry points for ensuing dissection of their evolution, development, and function. The pharynx of the nematode Caenorhabditis elegans is a simple neuromuscular organ with a self-contained, autonomously acting nervous system, composed of 20 neurons that fall into 14 anatomically distinct types. Using serial EM reconstruction, we re-evaluate here the connectome of the pharyngeal nervous system, providing a novel and more detailed view of its structure and predicted function. Contrasting the previous classification of pharyngeal neurons into distinct inter- and motorneuron classes, we provide evidence that most pharyngeal neurons are also likely sensory neurons and most, if not all, pharyngeal neurons also classify as motorneurons. Together with the extensive cross-connectivity among pharyngeal neurons, which is more widespread than previously realized, the sensory-motor characteristics of most neurons define a shallow network architecture of the pharyngeal connectome. Network analysis reveals that the patterns of neuronal connections are organized into putative computational modules that reflect the known functional domains of the pharynx. Compared to the somatic nervous system, pharyngeal neurons both physically associate with a larger fraction of their neighbors and create synapses with a greater proportion of their neighbors. We speculate that the overall architecture of the pharyngeal nervous system may be reminiscent of the architecture of ancestral, primitive nervous systems.

neuroscience↗

bioRxiv2019

A ciliary BBSome-ARL-6-PDE6D pathway trafficks RAB-28, a negative regulator of extracellular vesicle biogenesis

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that cells shed to influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis and environment release are almost entirely unknown. Here, we show that the ciliary G-protein RAB28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans. We also find that sequential targeting of lipidated RAB28 to periciliary and ciliary membranes is highly dependent on the BBSome and PDE6D, respectively, and that BBSome loss causes excessive and ectopic EV production. Our data indicate that RAB28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane. Our findings also suggest that EVs control sensory organ homeostasis by mediating communication between ciliated neurons and glia, and that defects in ciliary EV biogenesis may contribute to human ciliopathies.

cell biology↗

bioRxiv2019

The Marginal Cells of the Caenorhabditis elegans Pharynx Scavenge Cholesterol and Other Hydrophobic Small Molecules

The nematode worm Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worms pharynx, a bacterial suspension is sucked into the pharynxs lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels that are made from specialized pharyngeal cells called marginal cells. Through the characterization of our library of worm-bioactive small molecules (a.k.a. wactives), we found that more than one third of wactives visibly accumulate inside of the marginal cells as crystals or globular spheres. Wactives that visibly accumulate are typically more hydrophobic than those that do not. To understand why wactives accumulate specifically in marginal cells, we performed a forward genetic screen for mutants that resist the lethality associated with one crystallizing wactive. We identified a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. Sphingomyelin is a phospholipid that is enriched in the outer leaflet of the plasma membranes of most metazoans. We find that the predicted terminal enzyme of this pathway, sphingomyelin synthase 5 (SMS-5), is expressed in the pharynx, contributes to sphingomyelin abundance, and that its expression in marginal cells is sufficient for wactive accumulation. We also find that the expression of SMS-5 in the marginal cells is necessary for the proper absorption of exogenous cholesterol, without which C. elegans cannot develop. We conclude that the sphingomyelin-rich plasma membrane of the marginal cells acts as a sink to scavenge important hydrophobic nutrients from the filtered liquid that might otherwise be shunted back into the environment. One sentence summaryThe anterior pharynx of C. elegans is a Sink for Hydrophobic Small Molecules

developmental biology↗

bioRxiv2018

Volumetric reconstruction of main Caenorhabditis elegans neuropil at two different time points

Detailed knowledge of both synaptic connectivity and the spatial proximity of neurons is crucial for understanding wiring specificity in the nervous system. Here, we volumetrically reconstructed the C. elegans nerve ring from legacy serial-sectioned electron micrographs at two distinct time points: the L4 and young adult. The new volumetric reconstructions provide detailed spatial and morphological information of neural processes in the nerve ring. Our analysis suggests that the nerve ring exhibits three levels of wiring specificity: spatial, synaptic and subcellular. Neuron classes innervate well defined neighborhoods and aggregate functionally similar synapses to support distinct computational pathways. Connectivity fractions vary based on neuron class and synapse type. We find that the variability in process placement accounts for less than 20% of the variability in synaptic connectivity and models based only on spatial information cannot account for the reproducibility of synaptic connections among homologous neurons. This suggests that additional, non-spatial factors also contribute to synaptic and subcellular specificity. With this in mind, we conjecture that a spatially constrained, genetic model could provide sufficient synaptic specificity. Using a model of cell-specific combinatorial genetic expression, we show that additional specificity, such as sub-cellular domains or alternative splicing, would be required to reproduce the wiring specificity in the nerve ring.

developmental biology↗

bioRxiv2018

MET-2, a SETDB1 family methyltransferase, coordinates embryo events through distinct histone H3 methylation states

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here we examine the relationship between these two processes, and we probe the determinants that contribute to their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, likely through mono- and di- methylation of H3K9 (me1/me2), and promotes heterochromatin formation, likely through H3K9me3. We examine how MET-2 is regulated and find that methylated H3K9 appears gradually and depends on the accumulated time of embryogenesis. H3K9me is independent of zygotic genome activation or cell counting. These data reveal how central events are synchronized during embryogenesis and distinguish distinct roles for different H3K9 methylation states.\n\nSummary StatementDuring early embryogenesis, heterochromatin formation and loss of developmental plasticity are coordinately regulated by distinct Histone H3 Lysine 9 (H3K9) methylation states, by the methyltransferase MET-2.

developmental biology↗

bioRxiv2018

Cell type-specific structural plasticity of the ciliary transition zone in C. elegans

Background informationThe current consensus on cilia development posits that the ciliary transition zone (TZ) is formed via extension of nine centrosomal microtubules. In this model, TZ structure remains unchanged in microtubule number throughout the cilium life cycle. This model does not however explain structural variations of TZ structure seen in nature, and could also lend itself to the misinterpretation that deviations from nine-doublet microtubule ultrastructure represent an abnormal phenotype. Thus, a better understanding of events that occur at the TZ in vivo during metazoan development is required.\n\nResultsTo address this issue, we characterized ultrastructure of two types of sensory cilia in developing Caenorhabditis elegans. We discovered that, in cephalic male (CEM) and inner labial quadrant (IL2Q) sensory neurons, ciliary TZs are structurally plastic and remodel from one structure to another during animal larval development. The number of microtubules doublets forming the TZ can be increased or decreased over time, depending on cilia type. Both cases result in structural TZ intermediates different from TZ in adult cilia. In CEM cilia, axonemal extension and maturation occurs concurrently with TZ structural maturation.\n\nConclusions and SignificanceOur work extends the current model to include the structural plasticity of metazoan transition zone, which can be structurally delayed, maintained or remodeled in cell type-specific manner.

cell biology↗

bioRxiv2018

Extracellular matrix regulates morphogenesis and function of ciliated sensory organs in Caenorhabditis elegans

Cilia and extracellular vesicles (EVs) are signaling organelles that play important roles in human health and disease. In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. Hence, the nematode offers an excellent system in which to address central questions regarding the biology of cilia, EVs, and the polycystins. We discovered an unexpected role of the mec-1, mec-5, and mec-9 genes encoding extracellular matrix (ECM) components. We determined that these ECM encoding genes regulate polycystin localization and function, ciliary EV release, cilia length, dendritic morphology, and neuron-glia interactions. Abnormal ECM and fibrosis are observed in ciliopathies such as ADPKD, nephronophthisis, and Bardet-Biedl Syndrome. Our studies reveal multifaceted roles for ECM proteins in the ciliated nervous system of the worm and provide a powerful new in vivo model to study the relationship between ECM, the polycystins, and ciliopathies.

cell biology↗

bioRxiv2018

Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos

Heterochromatin formation during early embryogenesis is timed precisely, but it has been elusive how this process is regulated. Here we report the discovery of a histone methyltransferase complex whose nuclear accumulation and activation establishes the onset of heterochromatin formation in C. elegans embryos. We find that the inception of heterochromatin generation coincides with the accumulation of the Histone H3 Lysine 9 (H3K9) methyltransferase MET-2 (SETDB) into nuclear hubs. The absence of MET-2 results in delayed and disturbed heterochromatin formation, whereas accelerated nuclear localization of the methyltransferase leads to precocious H3K9 methylation. We identify two factors that bind to and function with MET-2: LIN-65, which resembles ATF7IP, localizes MET-2 into nuclear hubs, and ARLE-14, orthologous to ARL14EP, promotes stable association of MET-2 with chromatin. These data reveal that nuclear accumulation of MET-2 in conjunction with LIN-65 and ARLE-14 regulates timing of heterochromatin domains during embryogenesis.\n\nONE SENTENCE SUMMARYMET-2/SETDB1 and interactors LIN-65/ATF7IP and ARLE-14/ARL14EP initiate heterochromatin formation during embryogenesis.

developmental biology↗

bioRxiv2018

A direct glia-to-neuron natural transdifferentiation ensures nimble sensory-motor coordination of male mating behaviour

The coordinated execution of innate, stereotyped sexual behaviours, such as courtship and mating, requires sexually dimorphic sensory-motor circuits that are genetically specified during development (reviewed in [1-3]). Studies in the nematode Caenorhabditis elegans, in which the development and function of neural circuits can be interrogated with single cell resolution, have revealed two general developmental mechanisms underlying sexual dimorphism in the nervous system. The first involves the acquisition of sexually dimorphic features in sex-shared neurons during sexual maturation, which include changes in terminal gene expression, such as odorant receptors, neurotransmitters and synaptic regulators [4-10]. The second mechanism involves the generation of sex-specific neurons [11-13]. This requires sex-specific cell death [14]or neurogenesis events resulting from extensive sex differences in the cell division patterns and neurodevelopmental programmes of post-embryonic cell lineages (reviewed in [3]). Here we identify a third, novel way to generate sexual dimorphism in the nervous system. We find that during sexual maturation (L4 stage), a class of sex-shared glial cells acquires sexually dimorphic function by undergoing a direct glia-to-neuron transdifferentiation that results in the production of male-specific neurons. This plasticity is regulated cell-intrinsically by the sex-determination pathway. These previously unnoticed neurons, which we term PHDs, are putative proprioceptors that regulate male locomotion during specific steps of mating. One of these steps is a novel readjustment movement performed when intromission becomes difficult to achieve. Our results reveal sex-specific direct transdifferentiation as a novel mechanism for generating sex-specific neurons and also show the importance of proprioceptive feedback during the complex steps of mating for successful reproduction.

neuroscience↗

bioRxiv2018

Intermediate filaments EXC-2 and IFA-4 Maintain Luminal Structure of the Tubular Excretory Canals in Caenorhabditis elegans

The excretory canals of Caenorhabditis elegans are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts that lack a complete terminal web at the apical surface, and accumulate filamentous material in the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes intermediate filament protein IFC-2. EXC-2/IFC-2 protein, fluorescently tagged via CRISPR/Cas9, is located at the apical surface of the canals independently of other intermediate filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either protein results in narrow but shortened canals. These results are consistent with a model whereby three intermediate filaments in the canals, EXC-2, IFA-4, and IFB-1, restrain swelling of narrow tubules in concert with actin filaments that guide the extension and direction of tubule outgrowth, while allowing the tube to bend as the animal moves.\n\nArticle SummaryThe C. elegans excretory canals form a useful model for understanding formation of narrow tubes. exc-2 mutants start to form normal canals that then swell into fluid-filled cysts. We show that exc-2 encodes a large intermediate filament (IF) protein previously not thought to be located in the canals. EXC-2 is located at the apical (luminal) membrane, binds to another IF protein, and appears to be one of three IF proteins that form a flexible meshwork to maintain the thin canal diameter. This work provides a genetically useful model for understanding the interactions of IF proteins with other cytoskeletal elements to regulate tube size and growth.

developmental biology↗

bioRxiv2017

C. elegans DBL-1/BMP Regulates Lipid Accumulation via Interaction with Insulin Signaling

Metabolic homeostasis is coordinately controlled by diverse inputs, which must be understood to combat metabolic disorders. Here we introduce DBL-1, the C. elegans BMP2/4 homolog, as a significant regulator of lipid homeostasis. We used neutral lipid staining and a lipid droplet marker to demonstrate that both increases and decreases in DBL-1/BMP signaling result in reduced lipid stores and lipid droplet count. We find that lipid droplet size, however, correlates positively with the level of DBL 1/BMP signaling. Regulation of lipid accumulation in the intestine occurs through non-cell-autonomous signaling, since expression of SMA-3, a Smad signal transducer, in the epidermis (hypodermis) is sufficient to rescue the loss of lipid accumulation. Finally, genetic evidence indicates that DBL-1/BMP functions upstream of Insulin/IGF-1 Signaling (IIS) in lipid metabolism. We conclude that BMP signaling regulates lipid metabolism in C. elegans through inter-organ signaling to IIS, shedding light on a less well-studied regulatory mechanism for metabolic homeostasis.

cell biology↗

bioRxiv2017

Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an -tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.\n\nTable of Content (TOC) Highlight SummaryDifferent tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One -tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.

cell biology↗