Yansong Miao - Academia.edu (original) (raw)

Papers by Yansong Miao

Research paper thumbnail of Faculty Opinions recommendation of Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2022

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space... more Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases. Editor's evaluation In elegant quantitative live-cell imaging and biochemical experiments, the authors show how activity of the plant immune signaling complex FLS2-BAK1 is affected by nanoscale mobility behaviors mediated through peptide signaling and the receptor kinase FERONIA (FER). Additionally, they are able to define separable roles for FER domains in different biological activities. The details of this work advance our understanding of plant immunity, but also provide generalizable concepts about the roles of nanoscale organization in signaling.

Research paper thumbnail of Plant bioreactors for pharmaceuticals 363

Plant bioreactors are attractive expression systems for economic production of pharmaceuticals. V... more Plant bioreactors are attractive expression systems for economic production of pharmaceuticals. Various plant expression systems or platforms have been tested with certain degrees of success over the past years. However, further development and improvement are needed for more effective plant bioreactors. In this review we first summarize recent progress in various plant bioreactor expression systems and then focus on discussing protein compartmentation to unique organelles and various strategies for developing better plant bioreactors.

Research paper thumbnail of A teamwork promotion of formin-mediated actin nucleation by Bud6 and Aip5 in Saccharomyces cerevisiae

Molecular Biology of the Cell

Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucle... more Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucleation-promoting factors (NPFs) into functional protein complexes. Selective inter- and intramolecular interactions between the nucleation complex constituents enable diverse modes of complex assembly in initiating actin polymerization upon demand. Budding yeast has two formins, Bni1 and Bnr1, which are teamed up with different NPFs. However, the selective pairing between formin NFs and NPFs into the nucleation core for actin polymerization is not completely understood. By examining the functions and interactions of NPFs and NFs via biochemistry, genetics, and mathematical modeling approaches, we found that two NPFs, Aip5 and Bud6, showed joint teamwork effort with Bni1 and Bnr1, respectively, by interacting with the C-terminal intrinsically disordered region (IDR) of formin, in which two NPFs work together to promote formin-mediated actin nucleation. Although the C-terminal IDRs of Bni1 ...

Research paper thumbnail of Intrinsically Disordered Region of Actin Binding Protein Regulates Dynamic Actin Assembly

Research paper thumbnail of Quantitative Analysis of Clathrin-Mediated Endocytosis in Yeast by Live Cell Fluorescence Microscopy

Methods in molecular biology, 2018

The budding yeast Saccharomyces cerevisiae has provided a useful model for studying clathrin-medi... more The budding yeast Saccharomyces cerevisiae has provided a useful model for studying clathrin-mediated endocytosis due to ease of genetic manipulation and crosssectional imaging of individual endocytic sites. This protocol describes a method for using live cell fluorescence microscopy to analyze clathrin-mediated endocytosis and the contributions of actin to the process.

Research paper thumbnail of Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth

Journal of Cell Science

Dynamic assembly and remodeling of actin is critical for many cellular processes during developme... more Dynamic assembly and remodeling of actin is critical for many cellular processes during development and stress adaptation. In filamentous fungi and budding yeast, actin cables align in a polarized manner along the mother-to-daughter cell axis, and are essential for the establishment and maintenance of polarity; moreover, they rapidly remodel in response to environmental cues to achieve an optimal system response. A formin at the tip region within a macromolecular complex, called the polarisome, is responsible for driving actin cable polymerization during polarity establishment. This polarisome undergoes dynamic assembly through spatial and temporally regulated interactions between its components. Understanding this process is important to comprehend the tuneable activities of the formin-centered nucleation core, which are regulated through divergent molecular interactions and assembly modes within the polarisome. In this Review, we focus on how intrinsically disordered regions (IDRs...

Research paper thumbnail of Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Proceedings of the National Academy of Sciences

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family.... more Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutate...

Research paper thumbnail of Salicylic acid regulates PIN2 auxin transporter hyperclustering and root gravitropic growth via Remorin ‐dependent lipid nanodomain organisation in Arabidopsis thaliana

Research paper thumbnail of Review: F-Actin Remodelling during Plant Signal Transduction via Biomolecular Assembly

Plant Science

During signal transduction, multivalent interactions establish dynamic molecular connectivities t... more During signal transduction, multivalent interactions establish dynamic molecular connectivities that propagate molecular cascades throughout the entire signaling pathway. Such multivalent interactions include the initial activation, cascade signal transduction, and the amplification and assembly of structural machinery. For example, plants rapidly remodel the actin cytoskeleton during signal transduction by perceiving a wide range of mechanical and chemical cues from developmental and defense pathways. Actin treadmilling is stepwise-regulated by interactions between actin and actin-binding proteins (ABPs). Emerging evidence shows that intrinsically disordered regions (IDRs) enable flexible and promiscuous interactions that serve as the functional hub for generating cellular interactomes underlying various signaling events. Though IDRs are present in a majority of ABPs, few of the functional roles of IDR in the interaction and functions of ABPs have been defined. The distinct features of IDRs create diverse and dynamic molecular interactions that introduce a new paradigm to our understanding of the structure-function relationships for actin assembly. In this review, we will create a snapshot of recent advances in IDR-mediated plant actin remodeling and discuss future research directions in studying the complexity of actin assembly via multifaceted biomolecular assembly during signal transduction.

Research paper thumbnail of The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related ... more Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely under-studied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of oligomerization states and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis, with a particular increase of the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol remo...

Research paper thumbnail of Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro

BIO-PROTOCOL

Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capp... more Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin purification and polymerization assays in this protocol, the readers could quickly get the affordable and straightforward assays to study actin dynamics.

Research paper thumbnail of An Effective Recombinant Protein Expression and Purification System in Saccharomyces cerevisiae

Current protocols in molecular biology, 2018

The expression and purification of recombinant proteins using bacterial vectors is a mature and p... more The expression and purification of recombinant proteins using bacterial vectors is a mature and preferred system to obtain folded and stable proteins. However, functional post-translational protein modifications, such as glycosylation or phosphorylation, can only be achieved using eukaryotic expression systems. In addition, insolubility is another challenge when using proteins expressed in Escherichia coli, such as certain intrinsically disordered proteins, which are more prone to aggregation than folded proteins. Eukaryotic protein expression systems, including human cells, baculovirus/insect cells, and yeast, have become indispensable for the production of functional eukaryotic proteins. This article describes a detailed protocol for performing cytosolic protein expression, protein purification, and protein characterization using the budding yeast Saccharomyces cerevisiae. The introduced protein expression and purification system in yeast are advantageous due to the low cost, high...

Research paper thumbnail of Molecular characterization of plant vacuolar sorting receptor (VSR) proteins

... Yansong Miao1 • Minghui Yang1 • Kwun Yee Li1 • Pui Kit Suen1 • Junli ... University of Hong K... more ... Yansong Miao1 • Minghui Yang1 • Kwun Yee Li1 • Pui Kit Suen1 • Junli ... University of Hong Kong, Shatin, NT, Hong Kong, China 2 State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen ...

Research paper thumbnail of Orchestrated actin nucleation by the Candida albicans polarisome complex enables filamentous growth

Journal of Biological Chemistry

Candida albicans is a dimorphic fungus that converts from a yeast form to a hyphae form during in... more Candida albicans is a dimorphic fungus that converts from a yeast form to a hyphae form during infection. This switch requires the formation of actin cable to coordinate polarized cell growth. It's known that nucleation of this cable requires a multiprotein complex localized at the tip called the polarisome, but the mechanisms underpinning this process were unclear. Here, we found that C. albicans Aip5, a homolog of polarisome component ScAip5 in Saccharomyces cerevisiae that nucleates actin polymerization and synergizes with the formin ScBni1, regulates actin assembly and hyphae growth synergistically with other polarisome proteins Bni1, Bud6, and Spa2. The C terminus of Aip5 binds directly to G-actin, Bni1, and the C-terminal of Bud6, which form the core of the nucleation complex to polymerize F-actin. Based on insights from structural biology and molecular dynamic simulations, we propose a possible complex conformation of the actin nucleation core, which provides cooperative ...

Research paper thumbnail of Localization of Green Fluorescent Protein Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY2 Cells1(W)(OA)

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.

Research paper thumbnail of Overexpression of Arabidopsis AGD7 Causes Relocation of Golgi-Localized Proteins to the Endoplasmic Reticulum and Inhibits Protein Trafficking in Plant Cells1(C)(OA)

ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bou... more ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bound to Arfs to GDP, which plays a pivotal role in regulating Arfs by converting the active GTP-bound forms of these proteins into their inactive GDP- bound forms. Here, we investigated the biological role of AGD7, an Arf GAP homolog, in Arabidopsis (Arabidopsis thaliana). We show that AGD7

Research paper thumbnail of Molecular Characterization of Plant Prevacuolar and Endosomal Compartments

Journal of Integrative Plant Biology, 2007

Prevacuolar compartments (PVCs) and endosomal compartments are membrane-bound organelles mediatin... more Prevacuolar compartments (PVCs) and endosomal compartments are membrane-bound organelles mediating protein traffic to vacuoles in the secretory and endocytic pathways of plant cells. Over the years, great progress has been made towards our understanding in these two compartments in plant cells. In this review, we will summarize our contributions toward the identification and characterization of plant prevacuolar and endosomal compartments. Our studies will serve as important steps in future molecular characterization of PVC biogenesis and PVC-mediated protein trafficking in plant cells.

Research paper thumbnail of Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

Plant physiology, 2006

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT s...

Research paper thumbnail of Localization of GFP Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY2 Cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis thaliana contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on previously proved hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to GFP (green fluorescent protein) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types including Arabidopsis, rice, pea, and mungbean. Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells and wortmannin-induced vacuolation of PVCs is a general response in plants. www.plant.org on July 10, 2015 -Published by www.plantphysiol.org Downloaded from

Research paper thumbnail of Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

Plant Physiology, 2006

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.

Research paper thumbnail of Faculty Opinions recommendation of Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2022

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space... more Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases. Editor's evaluation In elegant quantitative live-cell imaging and biochemical experiments, the authors show how activity of the plant immune signaling complex FLS2-BAK1 is affected by nanoscale mobility behaviors mediated through peptide signaling and the receptor kinase FERONIA (FER). Additionally, they are able to define separable roles for FER domains in different biological activities. The details of this work advance our understanding of plant immunity, but also provide generalizable concepts about the roles of nanoscale organization in signaling.

Research paper thumbnail of Plant bioreactors for pharmaceuticals 363

Plant bioreactors are attractive expression systems for economic production of pharmaceuticals. V... more Plant bioreactors are attractive expression systems for economic production of pharmaceuticals. Various plant expression systems or platforms have been tested with certain degrees of success over the past years. However, further development and improvement are needed for more effective plant bioreactors. In this review we first summarize recent progress in various plant bioreactor expression systems and then focus on discussing protein compartmentation to unique organelles and various strategies for developing better plant bioreactors.

Research paper thumbnail of A teamwork promotion of formin-mediated actin nucleation by Bud6 and Aip5 in Saccharomyces cerevisiae

Molecular Biology of the Cell

Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucle... more Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucleation-promoting factors (NPFs) into functional protein complexes. Selective inter- and intramolecular interactions between the nucleation complex constituents enable diverse modes of complex assembly in initiating actin polymerization upon demand. Budding yeast has two formins, Bni1 and Bnr1, which are teamed up with different NPFs. However, the selective pairing between formin NFs and NPFs into the nucleation core for actin polymerization is not completely understood. By examining the functions and interactions of NPFs and NFs via biochemistry, genetics, and mathematical modeling approaches, we found that two NPFs, Aip5 and Bud6, showed joint teamwork effort with Bni1 and Bnr1, respectively, by interacting with the C-terminal intrinsically disordered region (IDR) of formin, in which two NPFs work together to promote formin-mediated actin nucleation. Although the C-terminal IDRs of Bni1 ...

Research paper thumbnail of Intrinsically Disordered Region of Actin Binding Protein Regulates Dynamic Actin Assembly

Research paper thumbnail of Quantitative Analysis of Clathrin-Mediated Endocytosis in Yeast by Live Cell Fluorescence Microscopy

Methods in molecular biology, 2018

The budding yeast Saccharomyces cerevisiae has provided a useful model for studying clathrin-medi... more The budding yeast Saccharomyces cerevisiae has provided a useful model for studying clathrin-mediated endocytosis due to ease of genetic manipulation and crosssectional imaging of individual endocytic sites. This protocol describes a method for using live cell fluorescence microscopy to analyze clathrin-mediated endocytosis and the contributions of actin to the process.

Research paper thumbnail of Polarisome assembly mediates actin remodeling during polarized yeast and fungal growth

Journal of Cell Science

Dynamic assembly and remodeling of actin is critical for many cellular processes during developme... more Dynamic assembly and remodeling of actin is critical for many cellular processes during development and stress adaptation. In filamentous fungi and budding yeast, actin cables align in a polarized manner along the mother-to-daughter cell axis, and are essential for the establishment and maintenance of polarity; moreover, they rapidly remodel in response to environmental cues to achieve an optimal system response. A formin at the tip region within a macromolecular complex, called the polarisome, is responsible for driving actin cable polymerization during polarity establishment. This polarisome undergoes dynamic assembly through spatial and temporally regulated interactions between its components. Understanding this process is important to comprehend the tuneable activities of the formin-centered nucleation core, which are regulated through divergent molecular interactions and assembly modes within the polarisome. In this Review, we focus on how intrinsically disordered regions (IDRs...

Research paper thumbnail of Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Proceedings of the National Academy of Sciences

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family.... more Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutate...

Research paper thumbnail of Salicylic acid regulates PIN2 auxin transporter hyperclustering and root gravitropic growth via Remorin ‐dependent lipid nanodomain organisation in Arabidopsis thaliana

Research paper thumbnail of Review: F-Actin Remodelling during Plant Signal Transduction via Biomolecular Assembly

Plant Science

During signal transduction, multivalent interactions establish dynamic molecular connectivities t... more During signal transduction, multivalent interactions establish dynamic molecular connectivities that propagate molecular cascades throughout the entire signaling pathway. Such multivalent interactions include the initial activation, cascade signal transduction, and the amplification and assembly of structural machinery. For example, plants rapidly remodel the actin cytoskeleton during signal transduction by perceiving a wide range of mechanical and chemical cues from developmental and defense pathways. Actin treadmilling is stepwise-regulated by interactions between actin and actin-binding proteins (ABPs). Emerging evidence shows that intrinsically disordered regions (IDRs) enable flexible and promiscuous interactions that serve as the functional hub for generating cellular interactomes underlying various signaling events. Though IDRs are present in a majority of ABPs, few of the functional roles of IDR in the interaction and functions of ABPs have been defined. The distinct features of IDRs create diverse and dynamic molecular interactions that introduce a new paradigm to our understanding of the structure-function relationships for actin assembly. In this review, we will create a snapshot of recent advances in IDR-mediated plant actin remodeling and discuss future research directions in studying the complexity of actin assembly via multifaceted biomolecular assembly during signal transduction.

Research paper thumbnail of The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related ... more Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely under-studied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of oligomerization states and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis, with a particular increase of the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol remo...

Research paper thumbnail of Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro

BIO-PROTOCOL

Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capp... more Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin purification and polymerization assays in this protocol, the readers could quickly get the affordable and straightforward assays to study actin dynamics.

Research paper thumbnail of An Effective Recombinant Protein Expression and Purification System in Saccharomyces cerevisiae

Current protocols in molecular biology, 2018

The expression and purification of recombinant proteins using bacterial vectors is a mature and p... more The expression and purification of recombinant proteins using bacterial vectors is a mature and preferred system to obtain folded and stable proteins. However, functional post-translational protein modifications, such as glycosylation or phosphorylation, can only be achieved using eukaryotic expression systems. In addition, insolubility is another challenge when using proteins expressed in Escherichia coli, such as certain intrinsically disordered proteins, which are more prone to aggregation than folded proteins. Eukaryotic protein expression systems, including human cells, baculovirus/insect cells, and yeast, have become indispensable for the production of functional eukaryotic proteins. This article describes a detailed protocol for performing cytosolic protein expression, protein purification, and protein characterization using the budding yeast Saccharomyces cerevisiae. The introduced protein expression and purification system in yeast are advantageous due to the low cost, high...

Research paper thumbnail of Molecular characterization of plant vacuolar sorting receptor (VSR) proteins

... Yansong Miao1 • Minghui Yang1 • Kwun Yee Li1 • Pui Kit Suen1 • Junli ... University of Hong K... more ... Yansong Miao1 • Minghui Yang1 • Kwun Yee Li1 • Pui Kit Suen1 • Junli ... University of Hong Kong, Shatin, NT, Hong Kong, China 2 State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen ...

Research paper thumbnail of Orchestrated actin nucleation by the Candida albicans polarisome complex enables filamentous growth

Journal of Biological Chemistry

Candida albicans is a dimorphic fungus that converts from a yeast form to a hyphae form during in... more Candida albicans is a dimorphic fungus that converts from a yeast form to a hyphae form during infection. This switch requires the formation of actin cable to coordinate polarized cell growth. It's known that nucleation of this cable requires a multiprotein complex localized at the tip called the polarisome, but the mechanisms underpinning this process were unclear. Here, we found that C. albicans Aip5, a homolog of polarisome component ScAip5 in Saccharomyces cerevisiae that nucleates actin polymerization and synergizes with the formin ScBni1, regulates actin assembly and hyphae growth synergistically with other polarisome proteins Bni1, Bud6, and Spa2. The C terminus of Aip5 binds directly to G-actin, Bni1, and the C-terminal of Bud6, which form the core of the nucleation complex to polymerize F-actin. Based on insights from structural biology and molecular dynamic simulations, we propose a possible complex conformation of the actin nucleation core, which provides cooperative ...

Research paper thumbnail of Localization of Green Fluorescent Protein Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY2 Cells1(W)(OA)

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.

Research paper thumbnail of Overexpression of Arabidopsis AGD7 Causes Relocation of Golgi-Localized Proteins to the Endoplasmic Reticulum and Inhibits Protein Trafficking in Plant Cells1(C)(OA)

ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bou... more ADP ribosylation factor (Arf) GTPase-activating proteins (GAPs) promote the hydrolysis of GTP bound to Arfs to GDP, which plays a pivotal role in regulating Arfs by converting the active GTP-bound forms of these proteins into their inactive GDP- bound forms. Here, we investigated the biological role of AGD7, an Arf GAP homolog, in Arabidopsis (Arabidopsis thaliana). We show that AGD7

Research paper thumbnail of Molecular Characterization of Plant Prevacuolar and Endosomal Compartments

Journal of Integrative Plant Biology, 2007

Prevacuolar compartments (PVCs) and endosomal compartments are membrane-bound organelles mediatin... more Prevacuolar compartments (PVCs) and endosomal compartments are membrane-bound organelles mediating protein traffic to vacuoles in the secretory and endocytic pathways of plant cells. Over the years, great progress has been made towards our understanding in these two compartments in plant cells. In this review, we will summarize our contributions toward the identification and characterization of plant prevacuolar and endosomal compartments. Our studies will serve as important steps in future molecular characterization of PVC biogenesis and PVC-mediated protein trafficking in plant cells.

Research paper thumbnail of Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

Plant physiology, 2006

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT s...

Research paper thumbnail of Localization of GFP Fusions with the Seven Arabidopsis Vacuolar Sorting Receptors to Prevacuolar Compartments in Tobacco BY2 Cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis thaliana contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on previously proved hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to GFP (green fluorescent protein) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types including Arabidopsis, rice, pea, and mungbean. Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells and wortmannin-induced vacuolation of PVCs is a general response in plants. www.plant.org on July 10, 2015 -Published by www.plantphysiol.org Downloaded from

Research paper thumbnail of Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

Plant Physiology, 2006

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on... more We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.