Biology subjects

Gu, A.

Publications and source records attributed to Gu, A..

6 recordsLinked to original sources

bioRxiv2021

Sensory cilia act as a specialized venue for regulated EV biogenesis and signaling

Extracellular vesicles play major roles in intercellular signaling, yet fundamental aspects of their biology remain poorly understood. Ciliary EV shedding is evolutionary conserved. Here we use super resolution, real time imaging of fluorescent-protein tagged EV cargo combined with in vivo bioassays to study signaling EVs in C. elegans. We find that neuronal sensory cilia shed the TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. Blocking ciliary tip shedding results in excessive EV shedding from the base. Finally, we demonstrate that C. elegans male ciliated neurons modulate EV cargo composition in response to sensory stimulation by hermaphrodite mating partners. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

cell biology↗

bioRxiv2020

Bedshift: perturbation of genomic interval sets

Functional genomics experiments, like ChIP-Seq or ATAC-Seq, produce results that are summarized as a region set. Many tools have been developed to analyze region sets, including computing similarity metrics to compare them. However, there is no way to objectively evaluate the effectiveness of region set similarity metrics. In this paper we present Bedshift, a command-line tool and Python API to generate new BED files by making random perturbations to an original BED file. Perturbed files have known similarity to the original file and are therefore useful to benchmark similarity metrics. To demonstrate, we used Bedshift to create an evaluation dataset of hundreds of perturbed files generated by shifting, adding, and dropping regions from a reference BED file. Then, we compared four similarity metrics: Jaccard score, coverage score, Euclidean distance, and cosine similarity. Our results highlight differences in behavior among these metrics, such as that Jaccard score is most sensitive to added or dropped regions, while coverage score is most sensitive to shifted regions. Together, we show that Bedshift is a useful tool for creating randomized region sets for a variety of uses. AvailabilityBSD2-licensed source code and documentation can be found at https://bedshift.databio.org.

bioinformatics↗

bioRxiv2020

CCP1, a tubulin deglutamylase, increases survival of rodent spinal cord neurons following glutamate-induced excitotoxicity

Microtubules (MTs) are cytoskeletal elements that provide structural support, establish morphology, and act as roadways for intracellular transport in cells. Neurons extend and must maintain long axons and dendrites to transmit information through the nervous system. Therefore, in neurons, the ability to independently regulate cytoskeletal stability and MT-based transport in different cellular compartments is essential. Post-translational modification of MTs is one mechanism by which neurons can regulate the cytoskeleton. The carboxypeptidase CCP1 negatively regulates post-translational glutamylation of MTs. We previously demonstrated that the CCP1 homolog in C. elegans is important for maintenance of cilia. In mammals, loss of CCP1, and the resulting hyperglutamylation of MTs, causes neurodegeneration. It has long been known that CCP1 expression is activated by neuronal injury; however, whether CCP1 plays a neuroprotective role after injury is unknown. Furthermore, it not yet clear whether CCP1 acts on ciliary MTs in spinal cord neurons. Using an in vitro model of excitotoxic neuronal injury coupled with shRNA-mediated knockdown of CCP1, we demonstrate that CCP1 protects neurons from excitotoxic death. Unexpectedly, excitotoxic injury reduced CCP1 expression in our system, and knockdown of CCP1 did not result in loss or shortening of cilia in cultured spinal cord neurons. Our results suggest that CCP1 acts on axonal and dendritic MTs to promote cytoskeletal rearrangements that support neuroregeneration and that enzymes responsible for glutamylation of MTs might be therapeutically targeted to prevent excitotoxic death after spinal cord injuries.

cell biology↗

bioRxiv2020

Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

genetics↗

bioRxiv2020

Sensory-evoked extracellular vesicle release and targeting

Extracellular vesicles (EVs) are emerging as a universal means of cell-to-cell communication and hold great potential in diagnostics and regenerative therapies [1]. An urgent need in the field is a fundamental understanding of physiological mechanisms driving EV generation and function. Ciliary EVs act as signaling devices in Chlamydomonas and C. elegans [2-4]. Mammalian cilia shed EVs to eliminate unwanted receptors [5] or to retract cilia before entering the cell cycle [6]. Here we used our established C. elegans model to study sensory-evoked ciliary EV release and targeting using a fluorescently labeled EV cargo polycystin-2 (PKD-2). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 and polycystin-2 localize to cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation [7]. We find that males deposit PKD-2-carrying EVs onto the vulva of the hermaphrodite during mating. We also show that mechanical stimulation triggers release of PKD-2-carrying EVs from cilia. To our knowledge this is the first report of mechanoresponsive nature of the ciliary EV release and of ciliary EV directional transfer from one animal to another animal. Since the polycystins are evolutionarily conserved ciliary EV cargoes, our findings suggest that similar mechanisms for EV release and targeting may occur in other systems and biological contexts.

cell biology↗

bioRxiv2019

Impact of Influent Carbon to Phosphorus Ratio on Performance and Phenotypic Dynamics in Enhanced Biological Phosphorus Removal (EBPR) System - Insights into Carbon Distribution, Intracellular Polymer Stoichiometry and Pathways Shifts

This study investigated the impact of influent carbon to phosphorus (P) ratio on the variation in P-removal performance and associated intracellular polymers dynamics in key functionally relevant microbial populations, namely, PAOs and GAOs, at both individual and populations levels, in laboratory scale sequencing batch reactor-EBPR systems. Significant variations and dynamics were evidenced for the formation, utilization and stoichiometry of intracellular polymers, namely polyphosphate, glycogen and Polyhydroxyalkanoates in PAOs and GAOs in the EBPR systems that were operated with influent C/P ranged from 20 to 50, presumably as results of phylogenetic diversity changes and, or metabolic functions shifts in these two populations at different influent C/P ratios. Single cell Raman micro-spectroscopy enabled quantification of differentiated polymer inclusion levels in PAOs and GAOs and, showed that as the influent rbCOD/P ratio increases, the excessive carbon beyond stoichiometric requirement for PAOs would be diverted into GAOs. Our results also evidenced that when condition becomes more P limiting at higher rbCOD/P ratios, both energy and reducing power generation required for acetate uptake and PHB formation might shift from relying on both polyP hydrolysis and glycolysis pathway, to more enhancement and dependence on glycolysis in addition to partial/reverse TCA cycle. These findings provided new insights into the metabolic elasticity of PAOs and GAOs and their population-level parameters for mechanistic EBPR modeling. This study also demonstrated the potential of application of single cell Raman micro-spectroscopy method as a powerful tool for studying phenotypic dynamics in ecological systems such as EBPR.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC=\"FIGDIR/small/671081v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (37K):\norg.highwire.dtl.DTLVardef@13227d5org.highwire.dtl.DTLVardef@1d52d42org.highwire.dtl.DTLVardef@8e67c4org.highwire.dtl.DTLVardef@13eb1aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗