Biology subjects

Lin, K.-Y.

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

6 recordsLinked to original sources

bioRxiv2024

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating suicide exon and Cre recombinase

Chemical-inducible gene expression systems have been frequently used to regulate gene expression for functional genomics in various plant species. However, a convenient chemical-inducible system that can tightly regulate transgene expression in Nicotiana benthamiana is still missing. In this study, we developed a tightly regulated copper-inducible system that can be used to regulate transgene expression and perform cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system showed the least concerns of leakiness issues. Using the MoClo-based synthetic biology approach, we optimized the design of the copper-inducible system and incorporated the use of the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested NLRs and their matching AVRs, and it can also be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings provide new approaches for both fundamental and translational studies in plant functional genomics.

plant biology↗

bioRxiv2024

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein q subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

developmental biology↗

bioRxiv2024

The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is great interest in using lytic bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of intravenously administered bacteriophage in uninfected mice. A single dose of LPS-5, an antipseudomonal bacteriophage recently used in human clinical trials, was administered intravenously to both wild-type BALB/c and neutropenic ICR mice. Phage concentrations were assessed in peripheral blood and spleen at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance is only minimally affected by neutropenia. Indeed, the half-life of phages in blood in BALB/c and ICR mice is 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that substantial functional inactivation of circulating phages occurs over time. These data indicate that circulating factors, but not neutrophils, inactivate intravenously administered phages.

pharmacology and toxicology↗

bioRxiv2024

Arl2 Associates with Cdk5rap2 to Regulate Cortical Development via Microtubule Organization

ADP ribosylation factor-like GTPase 2 (Arl2) is crucial for controlling mitochondrial fusion and microtubule assembly in various organisms. Arl2 regulates the asymmetric division of neural stem cells in Drosophila via microtubule growth. However, the function of mammalian Arl2 during cortical development was unknown. Here, we demonstrate that mouse Arl2 plays a new role in corticogenesis via regulating microtubule growth, but not mitochondria functions. Arl2 knockdown leads to impaired proliferation of neural progenitor cells (NPCs) and neuronal migration. Arl2 knockdown in mouse NPCs significantly diminishes centrosomal microtubule growth and delocalization of centrosomal proteins Cdk5rap2 and {gamma}-tubulin. Moreover, Arl2 physically associates with Cdk5rap2 by in silico prediction using AlphaFold Multimer and in vitro binding assays. Remarkably, Cdk5rap2 overexpression significantly rescues the neurogenesis defects caused by Arl2 knockdown. Therefore, Arl2 plays an important role in mouse cortical development through microtubule growth via the centrosomal protein Cdk5rap2.

neuroscience↗

bioRxiv2023

Functional divergence shaped the network architecture of plant immune receptors

In solanaceous plants, several sensor NLRs and their helper NLRs, known as NRC, form a complex network to confer immunity against pathogens. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, the evolution and molecular basis of the complex network structure remained elusive. Here we demonstrated that functional divergence of NRC3 variants has shaped the genetic architecture of the NLR network. Natural NRC3 variants form three allelic groups displaying distinct compatibilities with sensor NLRs. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility, with two residues critical for subfunctionalization. Co-functioning Rpi-blb2 and NRC3 variants showed stronger transient interactions upon effector detection, with NRC3 membrane-associated complexes forming subsequently. Our findings reveal how mutations in helper NLRs, particularly NRC3, have driven the evolution of their transient interactions with sensor NLRs, leading to subfunctionalization and contributing significantly to the complexity of the NRC network in plant immunity. TeaserHelper NLR subfunctionalization alters transient interactions with sensor NLRs, enhancing plant immune system complexity.

plant biology↗

bioRxiv2022

Canonical Wnt signaling promotes formation of somatic permeability barrier for proper germ cell differentiation

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

developmental biology↗