Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Outreach activities were provided to Middle and High School students: RiverOaks Middle School students (12) visited the lab for hands-on lab experiences for one morning in Fall 2012. One High School student from the North Carolina School of Science and Mathematics spent 10 weeks in the lab conducting research during the Summer of 2013. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has resulted in the training of 3 graduate students, 2 undergraduate students and 1 High School student: Graduate student, Sang Won Han, continued to characterize itt5 mutants and the genetic screen. She has been trained in in molecular biology, genetics and microscopy. Graduate student Irina Boico was trained in molecular biology and microscopy as part of her work on a new project characterizing the trafficking of phosphate transporters in plants. Graduate student Jiameng Zheng was trained in protein biochemistry, microscopy, molecular biology and Genetics. She is the first author of the publication describing a role of phosphoinositides on vacuole fusion currently under review. Undergraduate student Ian Manning graduated in Summer 2013. He gained training in laboratory skills including PCR, molecular cloning, and phenotypic characterization of mutants. He is currently interviewing for jobs at local biotech companies. Undergraduate student Sam Lee is currently assisting with plant growth and tissue culture. He is also carrying out a small project and gaining research experience with plant genetics. High School student Alexandria Thompson conducted research for 10 weeks in the Summer and gained first-hand experience with laboratory research. How have the results been disseminated to communities of interest? Our results were presented at Plant Biology 2013 (international meeting for the American Society of Plant Biologists) and ENPER 2013 (European Plant Endomembrane meeting). Some stduents presented in a local meeting in North Carolina (NCBC Plant Molecular Biology retreat). What do you plan to do during the next reporting period to accomplish the goals? We will continue to characterize mechanism of vacuole membrane fusion and the role of phosphoinositides in this process. We aim to identify proteins that interact with these regulatory lipids and regulate the activity of the vacuolar SNARE complex. In addition, we will continue our efforts to clone the genes responsible for two novel mutations, itt5 and itt6. Candidate genes for these loci have been identified by Next Generation Sequencing and rough mapping. Our next goal is to identify the molecular mechanisms that result in mutant phenotypes and identify the specific functions of the ITT5 and ITT6 proteins in the trafficking of tonoplast proteins.
Impacts
What was accomplished under these goals?
Characterization of our impaired traffic to tonoplast 3 (itt3) mutant indicated that the SNARE protein VTI11 is critical for homotypic vacuole fusion in plants. This indicates that a vacuolar SNARE complex containing VTI11 is important for the biogenesis of lytic and storage vacuoles and for overall plant development. Our work also demonstrated that inhibition of Phosphatidylinositol 3-Kinase by treatment with Wortmannin completely restored a vacuole morphology phenotype initt3mutants. We concluded from this analysis that phosphoinositides are negative regulators of vacuole fusion. A manuscript describing this work is currently under review.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Rojas-Pierce M. 2013. Trafficking of Tonoplast Proteins to the Vacuole. Plant Science. 211: 132-136.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Zheng J, Han S.W., Rodriguez-Wesh, M.F., Rojas-Pierce M. 2013. Phosphatidylinositol 3-Phospate is a Negative Regulator of Vacuole Fusion in Plants. Poster and Invited talk, Plant Biology 2013, American Society of Plant Biologists, Providence, RI.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Many essential roles of the plant tonoplast are due to the activity of integral membrane proteins; yet virtually nothing is known about their trafficking pathway(s). The long-term goal of our research is to identify and characterize proteins that regulate the trafficking of tonoplast proteins. We previously carried out a screen for small chemicals and mutations that affected the trafficking of a tonoplast protein marker. This screen identified unique probes that affected only a subset of tonoplast proteins and make a compelling argument for a Golgi-independent trafficking pathway for these proteins. In addition, viable mutants with novel phenotypes of abnormal tonoplast marker localizations have been identified and partially characterized. 1. This project included the development of a dual-label marker line where a tonoplast marker protein is labeled with GFP (green) and an endoplasmic reticulum (ER) marker is labeled with mCherry (red fluorescence). We reasoned that mis-localization of the tonoplast marker would result in co-localization of the green and red fluorescent proteins at the ER. We used this line in a mutagenesis experiment to identify genetic lesions that result in mis-localization of the tonoplast marker. Close to 600,000 seeds were screened using a root-growth assay and ~8,000 plants with short roots were screened by confocal microscopy. We have selected eight mutants with consistent and heritable phenotypes under the microscope, which will be characterized. One mutant displays a novel fragmented vacuole phenotype, which can be rescued by treatment with Wortmannin. Another mutant accumulates the tonoplast protein in an unknown pre-vacuolar compartment and indicates that the trafficking pathway requires a pre-vacuolar compartment before delivery to the vacuole. A third mutant shows enhanced accumulation of vacuolar structures called "bulbs" and is being used to identify a putative function for these structures. 2. Using the same strategy we have completed a chemical genomic screen for inhibitors that induce the mis-localization of the tonoplast marker at the ER. We identified and characterized one small molecule that is specific to a subset of tonoplast proteins (α-TIP and δ-TIP but not γ-TIP), indicating that we can uncouple multiple pathways of membrane protein trafficking with our chemical probes. We have further shown that α-TIP and δ-TIP but not γ-TIP traffic via a Golgi-independent pathway. Given that Golgi-independent trafficking of proteins seems to be a plant specific process, our inhibitors provide a unique opportunity to study this pathway. PARTICIPANTS: Individuals Marcela Pierce, PI. Dr Pierce designs the experiments, mentors students, writes grant proposals and publications and runs some of the experiments for this project. Training or professional development: Efrain Rivera-Serrano, M.Sc., Graduated May 2012. He participated in the chemical screen and the characterization of one chemical inhibitor, and was been trained in confocal microscopy and molecular biology. Graduate student, Sang Won Han, was trained during the characterization of itt5 mutants and the genetic screen. She performs experiments in molecular biology, genetics and microscopy. Graduate student Irina Boico was trained in molecular biology and microscopy as part of her work on a new project characterizing the trafficking of phosphate transporters in plants. Graduate student JIameng Zheng was trained in microscopy, molecular biology and Genetics. She is involved in the genetic screen. Undergraduate student Ian Manning, has continued to work in this project. He has been trained in laboratory skills including PCR, molecular cloning, and phenotypic characterization of mutants. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The analysis of small chemical probes indicated that vacuolar membrane proteins are trafficked either via a Golgi-dependent or Golgi-independent pathway. Proteins within the TIP family use one of the two pathways exclusively, and we have identified a compound C834 that inhibits the trafficking of the Golgi-independent pathway. Mutant phenotypes indicate the involvement of a pre-vacuolar compartment in the trafficking of tonoplast proteins, and we have identified a new interaction between lipids and vacuolar fusion machinery. The new chemicals and mutants will be used to dissect the pathway for tonoplast protein trafficking in plants.
Publications
- Rivera-Serrano E, Rodriguez-Welsh MF, Hicks G, Raikhel N and Rojas-Pierce M. 2012. A Small Molecule Inhibitor Partitions Two Distinct Pathways for Trafficking of Tonoplast Intrinsic Proteins in Arabidopsis. PloS ONE. 7(9): e44735.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Many essential roles of the plant tonoplast are due to the activity of integral membrane proteins; yet virtually nothing is known about their trafficking pathway(s). The long-term goal of our research is to identify and characterize proteins that regulate the trafficking of tonoplast proteins. We designed a novel screen for small chemicals and mutations that affected the trafficking of a tonoplast protein marker. This screen identified unique probes that affected only a subset of tonoplast proteins and make a compelling argument for a Golgi-independent trafficking pathway for these proteins. In addition, viable mutants with novel phenotypes of abnormal tonoplast marker localizations have been identified. 1. This project included the development of a dual-label marker line where a tonoplast marker protein is labeled with GFP (green) and an endoplasmic reticulum (ER) marker is labeled with mCherry (red fluorescence). We reasoned that mis-localization of the tonoplast marker would result in co-localization of the green and red fluorescent proteins at the ER. We used this line in a mutagenesis experiment to identify genetic lesions that result in mis-localization of the tonoplast marker. Close to 600,000 seeds were screened using a root-growth assay and ~8,000 plants with short roots were screened by confocal microscopy. We have selected eight mutants with consistent and heritable phenotypes under the microscope, which will be characterized. One mutant displays a novel fragmented vacuole phenotype, which can be rescued by treatment with Wortmannin. Another mutant accumulates the tonoplast protein in an unknown pre-vacuolar compartment and indicates that the trafficking pathway requires a pre-vacuolar compartment before delivery to the vacuole. 2. Using the same strategy we have completed a chemical genomic screen for inhibitors that induce the mis-localization of the tonoplast marker at the ER. We screened a collection of 360 pollen growth inhibitors and identified 6 good candidates. Our new inhibitors are specific to tonoplast proteins and one is specific for a subset of these (α-TIP and δ-TIP but not γ-TIP), indicating that we can uncouple multiple pathways of membrane protein trafficking with our chemical probes. We have further shown that α-TIP and δ-TIP but not γ-TIP traffic via a Golgi-independent pathway. Given that Golgi-independent trafficking of proteins seems to be a plant specific process, our inhibitors provide a unique opportunity to study this pathway. PARTICIPANTS: Marcela Pierce, PI. Dr Pierce designs the experiments, mentors students, writes grant proposals and publications and runs some of the experiments for this project. Maria Fernanda Rodriguez Welsh, Lab technitian. Mrs Rodriguez is involved in the day-to day management of the lab, participates in the microscopy-based screens and characterization of chemicals and mutants using genetic approaches. Training or professional development: Graduate student Efrain Rivera-Serrano spearheaded the chemical screen and the characterization of one chemical inhibitor, and has been trained in confocal microscopy and molecular biology. Graduate student, Sang Won Han, was trained during the characterization of grv-hs1 mutants and the genetic screen. She performs experiments in molecular biology, genetics and microscopy. Graduate student Irina Boico was trained in molecular biology and microscopy as part of her work on a new project characterizing the trafficking of phosphate transporters in plants. Graduate student JIameng Zheng was trained in microscopy, molecular biology and Genetics. She is involved in the genetic screen. Undergraduate students Ian Manning and Elizabeth Flores, were involved in this project and were trained in laboratory skills including PCR, and phenotypic characterization of mutants. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The analysis of small chemical probes indicated that vacuolar membrane proteins are trafficked either via a Golgi-dependent or Golgi-independent pathway. Proteins within the TIP family use one of the two pathways exclusively. Mutant phenotypes indicate the involvement of a pre-vacuolar compartment in the trafficking of tonoplast proteins. The new chemicals and mutants will be used to dissect the pathway for tonoplast protein trafficking in plants.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Many essential roles of the plant tonoplast are due to the activity of integral membrane proteins; yet virtually nothing is known about their trafficking pathway(s). The long-term goal of our research is to identify and characterize proteins that regulate the trafficking of tonoplast proteins. We designed a novel screen for small chemicals and mutations that affected the trafficking of a tonoplast protein marker. This screen identified unique probes that affected only a subset of tonoplast proteins and make a compelling argument for a Golgi-independent trafficking pathway for these proteins. In addition, viable mutants with novel phenotypes of abnormal tonoplast marker localizations have been identified. 1. This project included the development of a dual-label marker line where a tonoplast marker protein is labeled with GFP (green) and an endoplasmic reticulum (ER) marker is labeled with mCherry (red fluorescence). We reasoned that mis-localization of the tonoplast marker would result in co-localization of the green and red fluorescent proteins at the ER. We used this line in a mutagenesis experiment to identify genetic lesions that result in mis-localization of the tonoplast marker. Close to 600,000 seeds were screened using a root-growth assay and 6,000 plants with short roots were screened by confocal microscopy. We have selected six mutants with consistent and heritable phenotypes under the microscope, which will be characterized. 2. Using the same strategy we have completed a chemical genomic screen for inhibitors that induce the mis-localization of the tonoplast marker at the ER. We screened a collection of 360 pollen growth inhibitors and identified 6 good candidates. Our new inhibitors are specific to tonoplast proteins and at least two are specific for a subset of these, indicating that we can indeed uncouple multiple pathways of membrane protein trafficking with our chemical probes. PARTICIPANTS: Individuals Marcela Pierce, PI. Dr Pierce designs the experiments, mentors students, writes grant proposals and publications and runs some of the experiments for this project. Maria Fernanda Rodriguez Welsh, Lab technitian. Mrs Rodriguez is involved in the day-to day management of the lab, participates in the microscopy-based screens and characterization of chemicals and mutants using genetic approaches. Training or professional development: Graduate student Efrain Rivera-Serrano spearheaded the chemical screen and the characterization of one chemical inhibitor, and has been trained in confocal microscopy and molecular biology. Graduate student, Sang Won Han, was trained during the characterization of grv-hs1 mutants and the genetic screen. She performs experiments in molecular biology, genetics and microscopy. Undergraduate students, Elizabeth Burguess, Irina Boico and Jennifer Uddom were involved in this project and were trained in laboratory skills including PCR, and phenotypic characterization of mutants. Middle School teacher Mrs Samantha Wilson spent one week in the lab gaining training in cell biology. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Te analysis of small chemical probes indicated that vacuolar membrane proteins are trafficked either via a Golgi-dependent or Golgi-independent pathway. Proteins within the TIP family use one of the two pathways exclusively. The new chemicals and mutants will be used to dissect the pathway for tonoplast protein trafficking in plants.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Many essential roles of the plant tonoplast are due to the activity of integral membrane proteins; yet virtually nothing is known about their trafficking pathway(s). Our hypothesis is that the trafficking of integral membrane proteins is strictly regulated and distinct from that of soluble vacuolar proteins. This has been demonstrated in part by our identification of several inhibitors of tonoplast protein trafficking that do not inhibit soluble protein targeting to the vacuole. We are using both traditional and chemical genetic approaches to disrupt tonoplast protein trafficking in Arabidopsis with mutants or bioactive compounds. One such molecule was identified previously by the PI and colleagues as Gravacin, a potent inhibitor of the targeting of multiple tonoplast marker proteins. EMS mutants with altered sensitivity to this inhibitor were previously identified. We have now identified 5 candidate mutations in one if these mutants by map-based cloning and whole genome sequencing (collaboration with Dr Glenn Hicks, UC Riverside). We are currently testing each mutation by complementation and by characterization of other insertion-mutant alleles. This mutant displays multiple phenotypes such as reduced root growth, abnormal patterns of cell division in the root and branched and supernumerary root hairs. These phenotypes are reminiscence of cell polarity defects and indicate a putative role of this locus on vesicle trafficking mechanisms that specify cell polarity in the root. We plan to submit a manuscript describing these phenotypes in 2010. Another screen of ca. 5100 Activation Tagging lines yielded two more mutants with hypersensitivity to Gravacin. We have identified flanking sequences for one of these insertions using TAIL PCR. More experiments will be carried out to determine if this insertion is the cause of the phenotype. 1) First, co-segregation experiments will determine if the T-DNA insertion and the Gravacin-hypersensitive phenotype are linked. 2) If the phenotype and the T-DNA insertion are linked, we will test the effect of the T-DNA insertion on the expression of both At5g25600 and At5g25610 using total RNA extracts from different tissues and developmental stages by RT-PCR and quantitative PCR. It is likely that mutations in some of the components of membrane protein trafficking pathways are not viable or do not result in mutant phenotypes because of gene redundancy. Therefore, a chemical genetic approach is also being utilized. We have screened a collection of 360 pollen inhibitors (as a collaboration with Dr. Glenn Hicks, UC Riverside) and identified 37 hits. We have found that several of them are specific to tonoplast proteins because they do not affect localization of plasma membrane, Golgi and other subcellular markers. At least three appear to be specific for a subset of tonoplast proteins, but do not affect all, indicating that we can indeed uncouple multiple pathways of membrane proteins with our chemical probes. PARTICIPANTS: Marcela Pierce, principal investigator. Designed experiments, trained graduate and undergraduate students and wrote reports. Involved in gene mapping and analysis of mutants. Presented results at the Annual Meeting of the Society of Cell Biologists. Designed and taught a course in Plant Molecular Biology to nine graduate students in the College. Maria Fernanda Rodruiguez-Welsh, Laboratory technician, initiated and completed chemical screens under confocal microscope, participated in gene mapping of one of the mutants and administered the laboratory. Mutagenized seed population for genetic screen. Efrain Rivera-Serrano, graduate student, was involved in chemical screen and in implementing a new gene silencing approach for trafficking machinery. Three undergraduate students were trained in laboratory research: Allison Vestal and Briana Taylor from NCSU and Tracey Leonardo from Columbia U. TARGET AUDIENCES: Two minority students (one undergraduate and one graduate) were trained during the course of this reporting period. Five minority High-school students from the North Carolina School of Science and Mathematics participated in a research activity for two days in the laboratory. The PI also participated in one outreach activity at a local Elementary school in the Spring of 2009. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have identified small chemical probes that can inhibit the targeting of a subset of tonoplast proteins. These probes could be used in the future in bioassays for cell biological applications.
Publications
- No publications reported this period
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