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Biology subjects

Lin, Y.-C.

Publications and source records attributed to Lin, Y.-C..

At least 73 records · Page 4Linked to original sources

Traction Force Microscopy by Deep Learning

Cells interact mechanically with their surrounding by exerting forces and sensing forces or force-induced displacements. Traction force microscopy (TFM), purported to map cell-generated forces or stresses, represents an important tool that has powered the rapid advances in mechanobiology. However, to solve the ill-posted mathematical problem, its implementation has involved regularization and the associated compromises in accuracy and resolution. Here we applied neural network-based deep learning as a novel approach for TFM. We modified a network for processing images to process vector fields of stress and strain. Furthermore, we adapted a mathematical model for cell migration to generate large sets of simulated stresses and strains for training the network. We found that deep learning-based TFM yielded results qualitatively similar to those from conventional methods but at a higher accuracy and resolution. The speed and performance of deep learning TFM make it an appealing alternative to conventional methods for characterizing mechanical interactions between cells and the environment. Statement of SignificanceTraction Force Microscopy has served as a fundamental driving force for mechanobiology. However, its nature as an ill-posed inverse problem has posed serious challenges for conventional mathematical approaches. The present study, facilitated by large sets of simulated stresses and strains, describes a novel approach using deep learning for the calculation of traction stress distribution. By adapting the UNet neural network for handling vector fields, we show that deep learning is able to minimize much of the limitations of conventional approaches to generate results with speed, accuracy, and resolution.

biophysics↗

Altered Expression of Cadherin-8 and Cadherin-11 in Neural Circuit Development: Implications for Autism

Autism spectrum disorder (ASD) is a neurological condition characterized by alterations in social interaction and communication, and restricted and/or repetitive behaviors. The classical type II cadherins cadherin-8 (Cdh8, CDH8) and cadherin-11 (Cdh11, CDH11) have been implicated as autism risk gene candidates. To explore the role of cadherins in the etiology of autism, we investigated their expression patterns during mouse brain development and in autism-specific human tissue. In mice, expression of cadherin-8 and cadherin-11 was developmentally regulated and enriched in the cortex, hippocampus, and thalamus/striatum during the peak of dendrite formation and synaptogenesis. Both cadherins were expressed in synaptic compartments but only cadherin-8 associated with the excitatory synaptic marker neuroligin-1. Induced pluripotent stem cell (iPSC)-derived cortical neural precursor cells (NPCs) and cortical organoids generated from individuals with autism showed upregulated CDH8 expression levels while CDH11 expression levels were downregulated. We used Cdh11 knockout mice of both sexes to analyze the function of cadherin-11, which could help explain phenotypes observed in autism. Cdh11-/- hippocampal neurons exhibited increased dendritic complexity along with altered neuronal and synaptic activity. Similar to the expression profiles in human tissue, levels of cadherin-8 were significantly elevated in Cdh11 knockout brains. Additionally, excitatory synaptic markers neuroligin-1 and PSD-95 were both increased. Together, these results strongly suggest that cadherin-11 is involved in regulating the development of neuronal circuitry and that alterations in the expression levels of cadherin-11 may contribute to the etiology of autism. Significance StatementAutism is a neurodevelopmental condition with high genetic and phenotypic heterogeneity. Multiple genes have been implicated in autism, including the cadherin superfamily of adhesion molecules, cadherin-8 and cadherin-11. This study first characterizes the expression profiles of cadherin-8 and cadherin-11 to understand the potential roles they play in the development of neurons. The study further describes novel contributions of cadherin-11 in neural circuit formation. Loss of cadherin-11 in mice results in altered levels of several synaptic proteins, including PSD-95, neuroligin-1, and cadherin-8, and changes the morphology and activity of excitatory neurons. The levels of cadherin-8 and cadherin-11 in human cells of autistic individuals are both altered, strengthening the hypothesis that these two cadherins may involve in aspects of autism etiology.

neuroscience↗

Genomic, geographic and temporal distributions of SARS-CoV-2 mutations

The COVID-19 pandemic is the most significant public health issue in recent history. Its causal agent, SARS-CoV-2, has evolved rapidly since its first emergence in December 2019. Mutations in the viral genome have critical impacts on the adaptation of viral strains to the local environment, and may alter the characteristics of viral transmission, disease manifestation, and the efficacy of treatment and vaccination. Using the complete sequences of 1,932 SARS-CoV-2 genomes, we examined the genomic, geographic and temporal distributions of aged, new, and frequent mutations of SARS-CoV-2, and identified six phylogenetic clusters of the strains, which also exhibit a geographic preference in different continents. Mutations in the form of single nucleotide variations (SNVs) provide a direct interpretation for the six phylogenetic clusters. Linkage disequilibrium, haplotype structure, evolutionary process, global distribution of mutations unveiled a sketch of the mutational history. Additionally, we found a positive correlation between the average mutation count and case fatality, and this correlation had strengthened with time, suggesting an important role of SNVs on disease outcomes. This study suggests that SNVs may become an important consideration in virus detection, clinical treatment, drug design, and vaccine development to avoid target shifting, and that continued isolation and sequencing is a crucial component in the fight against this pandemic. Significance StatementMutation is the driving force of evolution for viruses like SARS-CoV-2, the causal agent of COVID-19. In this study, we discovered that the genome of SARS-CoV-2 is changing rapidly from the originally isolated form. These mutations have been spreading around the world and caused more than 2.5 million of infected cases and 170 thousands of deaths. We found that fourteen frequent mutations identified in this study can characterize the six main clusters of SARS-CoV-2 strains. In addition, we found the mutation burden is positively correlated with the fatality of COVID-19 patients. Understanding mutations in the SARS-CoV-2 genome will provide useful insight for the design of treatment and vaccination.

microbiology↗

A Piezo1 open state reveals a multi-fenestrated ion permeation pathway

Force-sensing Piezo channels are essential to many aspects of vertebrate physiology. Activation of Piezo1 is facilitated by the presence of negative membrane lipids in the inner leaflet, such as phosphatidylinositol-4,5-bisphosphate (PIP2). Here, to study how Piezo1 opens, we performed molecular dynamics simulations of Piezo1 in membranes flattened by the periodic boundary effect and with or without PIP2 lipids. The Piezo1 pore spontaneously opens in the asymmetrical bilayer but not in the symmetric membrane or when PIP2 lipids are neutralized. Electrophysiological characterization of putative PIP2-interacting Piezo1 residues suggests the contribution of multiple PIP2 binding sites. Our Piezo1 open state recapitulates ionic selectivity, unitary conductance and mutant phenotypes obtained from numerous experimental studies. Tracking ion diffusion through the open pore reveals the presence of intracellular and extracellular fenestrations, delineating a multi-fenestrated permeation pathway. This open state sheds light on the mechanisms of lipid modulation, permeation, and selectivity in a Piezo channel.

biophysics↗

PIP3 depletion rescues myoblast fusion defects in human rhabdomyosarcoma cells

Myoblast fusion is required for myotube formation during myogenesis, and defects in myoblast differentiation and fusion have been implicated in a number of diseases, including human rhabdomyosarcoma. While the transcriptional regulation of the myogenic program has been studied extensively, the mechanisms controlling myoblast fusion remain largely unknown. This study identified and characterized the dynamics of a distinct class of blebs, termed bubbling blebs, which are smaller than those that participate in migration. The formation of these bubbling blebs occurred during differentiation and decreased alongside a decline in phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) at the plasma membrane before myoblast fusion. In a human rhabdomyosarcoma-derived (RD) cell line that exhibits strong blebbing dynamics and myoblast fusion defects, PIP3 was constitutively abundant on the membrane during myogenesis. Targeting phosphatase and tensin homolog (PTEN) to the plasma membrane reduced PIP3 levels, inhibited bubbling blebs, and rescued myoblast fusion defects in RD cells. These findings highlight the differential distribution and crucial role of PIP3 during myoblast fusion and reveal a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma. Summary StatementThis study reveals a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma cells involving PIP3, whose depletion with PTEN rescues myoblast fusion defects.

cell biology↗

Relationship between maximal incremental and high-intensity interval exercise performance in elite athletes

It remains unclear whether the number of total bouts to limitation (Blim) in high-intensity interval testing (HIIT) differs among individuals, no matter if performed at the same relative intensity. This study aimed to explore the physiologic factors determining tolerance to effort during a HIIT. Forty-seven female participants (15-28 years old) were included: 23 athletes from Taiwan national or national reserve teams, and 24 moderately-active female. Each participant underwent maximal incremental (INC; modified-Bruce protocol) cardiopulmonary exercise testing and HIIT on treadmill, on separate days. HIIT protocol alternated a 1-min effort at 120% of the maximal speed and the same slope reached at the end of INC, with a 1-min rest, until volitional exhaustion. Gas-exchanges, and muscle oxygenation at right vastus lateralis by near-infrared spectroscopy, were continuously recorded. Additionally, bioelectrical impedance was utilized for body composition analysis. The result showed that Blim differed greatly (range: 2.6 to 12) among participants. Stepwise regression revealed that Blim was determined primarily by oxygen consumption (VO2) and heart rate (HR) at second-minute recovery; and, muscle tissue saturation index at peak of INC (R=0.644). Also, age and percent body fat were linearly correlated with Blim (adjusted R=-0.475, -0.371, p<0.05). Therefore, HIIT performance is determined by fast recovery of VO2 and HR, rather than maximal VO2 or muscle oxygenation recovery. Moreover, capacity to sustain a HIIT declines with age since as early as late adolescent. Further investigations on which factors should be manipulated to further improve athletes performance are warrant.

physiology↗

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Clathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle. Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis.

cell biology↗

Altering microtubule dynamics is synergistically toxic with inhibition of the spindle checkpoint

Chromosome instability (CIN) and aneuploidy are hallmarks of cancer. As the majority of cancers are aneuploid, targeting aneuploidy or CIN may be an effective way to target a broad spectrum of cancers. Here, we perform two small molecule compound screens to identify drugs that selectively target cells that are aneuploid or exhibit a CIN phenotype. We find that aneuploid cells are much more sensitive to the energy metabolism regulating drug ZLN005 than their euploid counterparts. Furthermore, cells with an ongoing CIN phenotype, induced by spindle assembly checkpoint (SAC) alleviation, are significantly more sensitive to the Src kinase inhibitor SKI606. We show that inhibiting Src kinase increases microtubule polymerization rates and, more generally, that deregulating microtubule polymerization rates is particularly toxic to cells with a defective SAC. Our findings therefore suggest that tumors with a dysfunctional SAC are particularly sensitive to microtubule poisons and, vice versa, that compounds alleviating the SAC provide a powerful means to treat tumors with deregulated microtubule dynamics.

cancer biology↗

High-content interrogation of human induced pluripotent stem cell-derived cortical organoid platforms.

The need for scalable and high-throughput approaches to screening using 3D human stem cell models remains a central challenge in using stem cell disease models for drug discovery. It is imperative to develop standardized systems for phenotypic screening, yet most researchers screen cells across different platforms using a multitude of assays. In this study, we have developed a workflow centered on a small array of assays that can be employed to screen 3D stem cell cultures across a set of platforms. This workflow can be used as a starting point for a standardized approach to phenotypic screening. In this manuscript we hope to provide a roadmap for groups looking to start high-content screening using 3D organoid systems. To do this, we employ serum-free embryoid bodies (SFEBs) created from human induced pluripotent stem cells (hiPSCs). SFEBs are used in this study because they do not display the same level of heterogeneity observed in other neural organoid systems and they are amenable to high content imaging without cryosectioning. They contain populations of excitatory and inhibitory neurons that form synaptically active networks1 and medium- to high-throughput electrophysiology can be performed using SFEBs via the multielectrode array (MEA). The assays outlined in this study allow SFEBs to be scanned for neurite outgrowth, cell number and electrophysiological activity. SFEBs derived from control and disease hiPSCs can be used in combination with high-throughput screening assays to generate sufficient statistical power to compensate for the biological and experimental variability common in 3D cultures, while significantly decreasing processing speed, thus making this an efficient starting point for phenotypic drug screening.

neuroscience↗

STXBP5/tomosyn regulates both the small Rho GTPase and the surface expression of AMPA receptors to control the dendritic stability of neurons

Tomosyn, a protein encoded by syntaxin-1-binding protein 5 (STXBP5) gene, has a well-established presynaptic role in the inhibition of neurotransmitter release and the reduction of synaptic transmission by its conical interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. The postsynaptic role of tomosyn in dendritic arborization, spine stability, and trafficking of ionotropic glutamate receptors remains to be elucidated. We used short hairpin RNA (shRNA) to knock down tomosyn in mouse primary neurons to evaluate the postsynaptic cellular function and molecular signaling regulated by tomosyn. Knockdown of tomosyn led to an increase of RhoA GTPase activity accompanied by compromised dendritic arborization, loss of dendritic spines, decreased surface expression of AMPA receptors, and reduced miniature excitatory postsynaptic current (mEPSC) frequency. Inhibiting RhoA signaling was sufficient to rescue the abnormal dendritic morphology and the surface expression of AMPA receptors. The function of tomosyn regulating RhoA is mediated through the N-terminal WD40 motif, where two variants each carrying a single nucleotide mutation in this region, were found in individuals with autism spectrum disorder (ASD). We demonstrated that these variants displayed loss-of-function phenotypes. Unlike the wild-type tomosyn, these two variants failed to restore the reduced dendritic complexity, spine density, as well as decreased surface expression of AMPA receptors in tomosyn knockdown neurons. This study uncovers a critical role of tomosyn, independent of its interaction with the SNARE machinery, in maintaining neuronal function by inhibiting RhoA activity. Further analysis of tomosyn variants also provides a potential mechanism for explaining cellular pathology in ASD.\n\nSignificance StatementThis study unveils a vital role of tomosyn in the maintenance of neuronal morphology, basal synaptic transmission, and AMPA receptor surface expression that is distinct from its presynaptic role. Tomosyn affects dendritic stability and glutamate receptor trafficking via the regulation of the Rho signaling pathway and this interaction is likely independent of the interaction with the dendritic SNARE complex, such as syntaxin-4. The WD40 domain of tomosyn is necessary to conduct the Rho regulation and two autism-associated variants localized at the WD40 domain perturb this function. The current study reveals a novel molecular link between dendritic stability and synaptic function, which could advance a greater understanding of the cellular pathologies involved in neurodevelopmental disorders, such as ASD.

neuroscience↗

Reference genome for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

Mycobacterium bovis bacillus Calmette-Guerin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). This study reports on an integrated genome analysis workflow for BCG, resulting in the completely assembled genome sequence of BCG Danish 1331 (07/270), one of the WHO reference strains for BCG vaccines. We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of this vaccine strain.

microbiology↗

Nifedipine Modulated Renal Lipogenesis via AMPK-SREBP Transcriptional Pathways

Lipid accumulation in renal cells has been implicated in the pathogenesis of obesity-related kidney disease, and lipotoxicity occurring in the kidney can be a surrogate marker for renal failure or renal fibrosis. Nifedipine-induced renal lipotoxicity has never been cited, although a few studies have shown that nifedipine inhibits lipogenesis via activation of the LKB1-AMPK pathway. Therefore, we utilized NRK52E cell models to examine this further. We pre-treated cells with varying concentrations of nifedipine (7.5, 15, or 30 M) for 24 or 48 h prior to examining the activity of lipogenesis enzymes and lipotoxicity.\n\nNifedipine was found to activate acetyl CoA synthetase, acetyl CoA carboxylase, long chain fatty acyl CoA elongase, ATP-citrate lyase, and HMG CoA reductase, suggesting elevated production of cholesterol, triacylglycerides, and phospholipids. Nifedipine exposure induced a vast accumulation of cytosolic free fatty acids (FFA) and stimulated the production of reactive oxygen species, upregulated CD36 and KIM-1 (kidney injury molecule-1) expression, inhibited p-AMPK activity, and triggered the transcription of SREBP-1/2 and lipin-1, underscoring the potential of nifedipine to induce lipotoxicity with renal damage.\n\nIn the present study, we elucidated the mechanism of action of nifedipine that leads to renal lipotoxicity via the AMPK-SREBP-1/2 pathway. To our knowledge, this is the first report demonstrating nifedipine-induced lipid accumulation in the kidney.

biochemistry↗

Red blood cell tension controls Plasmodium falciparum invasion and protects against severe malaria in the Dantu blood group

Malaria has had a major effect on the human genome, with many protective polymorphisms such as sickle cell trait having been selected to high frequencies in malaria endemic regions1, 2. Recently, we showed that a novel blood group variant, Dantu, provides 74% protection against all forms of severe malaria in homozygous individuals3-5. This is a similar degree of protection to sickle cell trait and considerably greater than the most advanced malaria vaccine, but until now the mechanism of protection has been unknown. In the current study, we demonstrate a significant impact of Dantu on the ability of Plasmodium falciparum merozoites to invade RBCs. The Dantu variant was associated with extensive changes to the RBC surface protein repertoire, but unexpectedly the malaria protective effect did not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we found a strong link between RBC tension and parasite invasion and, even in non-Dantu RBCs, identified a tension threshold above which RBC invasion did not occur. Dantu RBCs had higher average tension, meaning that a higher proportion of Dantu RBCs could not be invaded. These findings not only provide an explanation for the protective effect of Dantu against severe malaria, but also provide fresh insights into the essential process of P. falciparum parasite invasion, and how invasion efficiency varies across the heterogenous populations of RBCs that are present both within and between individuals.

genetics↗

A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO2 and ABA responses

The identification of homologous genes with functional overlap in forward genetic screens is severely limited. Here we report the generation of over 14,000 amiRNA-expressing plants that enable screens of the functionally redundant gene space in Arabidopsis. A protocol is developed here for isolating robust and reproducible amiRNA-mutants. Examples of validation approaches and essential controls are presented for two new amiRNA mutants that exhibit genetically redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a strong proof of principle for this approach. A new ABA insensitive amiRNA line is isolated, which targets three genes encoding avirulence-induced gene2-like (AIG2) genes. A thermal imaging screen for plants with impaired stomatal opening in response to low CO2 exposure led here to isolation of a new amiRNA targeting two essential proteasomal subunits, PAB1 and PAB2. The seed library of 14,000 T2 amiRNA lines generated here provides a new platform for forward genetic screens and is being made available to the Arabidopsis Biological Resource Center (ABRC) and optimized procedures for amiRNA screening and controls are described.\n\nHighlightThe generation of over 14,000 amiRNA-expressing plants is reported that are being made publicly available enabling screens of redundant genes in Arabidopsis. Identification of known and new genes is reported.

plant biology↗

FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for Gria2 mRNA processing

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function and pathology have not been explored. Here, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity include TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS). FUS translocation occurs through a calcium-dependent mechanism and coincides with striking alterations in nucleocytoplasmic transport. Further, glutamate-induced upregulation of Gria2 in neurons was dependent on FUS expression, consistent with a functional role for FUS under excitotoxic stress. These findings reveal a link between prominent factors in neurodegenerative disease, namely excitotoxicity, disease-associated RBPs and nucleocytoplasmic transport.

neuroscience↗

The Pseudomonas aeruginosa complement of lactate dehydrogenases enables use of D- and L-lactate and metabolic crossfeeding

Pseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to D-lactate during anaerobic survival, and LldE and LldD, which oxidize D-lactate and L-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant L-lactate oxidation during growth in aerobic cultures in both a defined MOPS-based medium and synthetic CF sputum medium. However, LldA differs from LldD in that its expression is induced specifically by the L-enantiomer of lactate. We also show that all four enzymes perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of D-lactate in the anaerobic zone that is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection.\n\nIMPORTANCELactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the L-form, generally produced by the host, and the D-form, which is usually produced by bacteria including the pulmonary pathogen Pseudomonas aeruginosa. Here, we characterize P. aeruginosas set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic D-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways could contribute to P. aeruginosa growth and survival in the lung.

microbiology↗

Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here we develop a method to rapidly deplete tubulin glutamlyation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

cell biology↗

Drug discovery to counteract antinociceptive tolerance with mu-opioid receptor endocytosis

Morphine antinociceptive tolerance is highly correlated with its poor ability to promote mu-opioid- receptor (MOR) endocytosis. Our objective was to discover a novel positive allosteric modulator of MOR to enhance morphine-induced MOR endocytosis. We used high-throughput screening to identify several cardiotonic steroids as positive allosteric modulators of morphine-induced MOR endocytosis having high potency and efficacy, independently of Na+/K+-ATPase inhibition. Convallatoxin was found to enhance morphine-induced MOR endocytosis through an adaptor protein 2/clathrin-dependent mechanism without regulating G protein- or {beta}-arrestin-mediated pathways. Both F243 and I292 residues of MOR were essential to the effect of convallatoxin on MOR endocytosis. Co-treatment with chronic morphine and convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance in morphine-tolerant mice. These findings suggest that cardiotonic steroids are potentially therapeutic for morphine side effects and open a new avenue for the study of MOR trafficking.

neuroscience↗