Progress 03/01/15 to 03/31/19
Outputs
Target Audience: Scientists, students, general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student Alyssa Parish, who is working on this proposal was trained in molecular biology and genetics. She was also was mentored in experimental design and project planning while working on her first proposal. How have the results been disseminated to communities of interest?Publications, lectures to undergraduate students, and presentations at the Washington State University. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. We characterized phenotype of a knockout mutant in the microtubule associated protein MACET4. MACET4 localizes to the phragmplast microtubules, but the function of this protein remins unknown. We identified T-DNA knockout line and found that phragmoplast was shorter. We could complemet macet4 knockout mutant with GFP. We also measured impact of MACET4 on microtubule dynamics in cells transiently transfected with proMACET4:MACET4-GFP. These experiments demonstrated that MACET4 reduces microtubule polymerization and depolymerization rates. Furthermore, MACET4 caused a greater frequency of depolymerizing microtubules, and promoted transition from polymerization to pause, from pause to catastrophe, and from polymerization to catastrophe. At the same time, transitions from catastrophe to polymerization and from catastrophe to pause were greater in the cells expressing MACET4-GFP; although there was no overall increase in polymerization events seen with MACET4. Therefore, at physiologically relevant concentrations in differentiated cells, MACET4 shifts the bulk of microtubule dynamics towards depolymerization. 2. We analyzed funtions of a TPX2-like microtubule-binding protein MAP20. We found that MAP20 is expressed during late stages of metaxylem and phloem differentiation in Brachypodium distachyon and localizes to the borders of the pit construction sites. MAP20 lacks Aurora- and importin-binding domains characteristic of canonical TPX2 family proteins, but has retained a conserved C-terminal domain pfam06886 of poorly characterized function. We find that pfam06886 is both necessary and sufficient for the interactions between MAP20 and microtubules. In vitro, MAP20 promotes microtubule nucleation and inhibits microtubule depolymerization. In vivo, MAP20 suppresses microtubule depolymerization in a pfam06886-dependent manner. Knockdown of MAP20 causes increase of pit size, thinner pit membranes, perturbed vasculature development, lower reproductive potential, and higher drought susceptibility. Our findings reveal an essential role of pfam06886 in binding and stabilizing microtubules. Furthermore, we show that MAP20 functions in drought adaptation by regulating pit membrane architecture.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Smertenko A (2019) Phosphoinositides break microtubule dynamics symmetry in the phragmoplast. Trends in Cell Biology, in press.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Scientists, students, farmers, general public Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PD and graduate student Sharol Schmidt attended Gordon Research Conference on Plant and Microbial cytoskeleton, Andover, NH, August 12-17, 2018. During the meeting, they learned about current developments in the filed and about new technologies, got feedback on our research from peers. They also established new professional contacts. PD presented a talk on novel cytokinetic protein MACET4 that was identified in the course of this research program. In addition, PD also attended XXVI Plant and Animal Genome and American Society for Cell Biology Annual Meeting conference where they learned about new technologies in cell biology and plant research, and about funding opportunities for plant sciences. How have the results been disseminated to communities of interest?Pubished research papers. Presented posters and talks at the national conferences. Puslished a popular article. Recorded and published a podcast. What do you plan to do during the next reporting period to accomplish the goals?In the next period the focus will be on two projects: 1. Regulation of microtubule dynamics by phragmoplast protein microtubule-binding protein MACET. In particular we will generate mutants to all members in the gene family using CRISPR/Cas technology and analyze mutant phenotypes. 2. Determining the role of the phragmoplast associated receptor kinase PARK1 in regulation of microtubule dynamics during phragmoplast expansion. To this end we will generate double mutants between PARK1 and its close homologue PARK2 and analysis of thecytokinesis in the double mutant. We also plan to identify PARK1 interactors.
Impacts
What was accomplished under these goals?
The research focused on determining the role of receptor kinase PARK1 that associates with the cell plate during cytokinesis. In one direction we focused on the characterization of the knockout mutant. We found that knockout causes smaller meristem. This phenotype can be rescued by growing roots on medium supplemented with GA3. The meristem size in park1 is insensitive to GA synthesis inhibitor paclobutrazol. It means that PARK1 is part of GA signaling for controlling root apical meristem size. We generated dominant-negative mutants of PARK1 under control of estrogen-inducible promoter. We found that induction of the mutant causes cytokinetic defects including disorientation of the cell division planes and formation of the cell wall stubs. These outcomes demonstrate importance of PARK1 in phragmoplast expansion. The second project was focused on the analysis of microtubule-nucleating protein MACET1 in cell division. We produced fluorescently labeled protein and tested its binding to microtubule in vitro. We also analyzed impact of MACET1 on microtubule dynamics in cells.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Smertenko A.*, Hewitt S.L., Jacques C.N., Kacprzyk R., Liu Y., Marcec M.J., Moyo L., Ogden A., Oung H.M., Schmidt S. and Serrano-Romero E.A. (2018) Phragmoplast microtubule dynamics: a game of zones. J. Cell Science 29, 131(2). pii: jcs203331. doi: 10.1242/jcs.203331.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Smertenko A., Sanguinet K. and Jååranta E. (2018) When navigating drought, peroxisomes may save the day. Wheat Life 61 (8;August/September), 57-59.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Gordon Research Conference on Plant and Microbial cytoskeleton, Andover, NH, August 12-17, 2018
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Scientific community Graduate and Undergraduate Students High School Students Educators General public Changes/Problems:N/A What opportunities for training and professional development has the project provided?The project provided opportunity to attend scientific meetings of American Society of Cell Biology Annual in San-Francisco (December 2-7) and in Philadelphia (December 2-6, 2017); and the Midwest Plant Cell Dynamics Meeting, Madison, May 30-June 2, 2017. Attending the meetings help in networking with other scientists working in our field, learn about new technologies, and get information about funding opportunities and insights into the application process. Specifically, during American Society of Cell Biology Annual in San-Francisco PI Smertenko attended a round table about NSF-CAREER program. The knowledge and advice form this event helped him in writing a proposal for the summer 2017 deadline. The proposal was funded. How have the results been disseminated to communities of interest?Outcomes of this project were disseminated through the following mechanisms: 1. Publication of original data and review articles (see list of products). 2. Presentations at conferences. PI Smertenko presented poster at the American Society of Cell Biology Annual Meeting, San-Francisco, December 2-7, 2016 and a talk at the Midwest Plant Cell Dynamics Meeting, Madison, May 30-June 2, 2017. PI Smertenko and graduate student Sharol Schmidt presented posters at the American Society of Cell Biology Annual Meeting, Philadelphia, December 2-6, 2017. 3. Seminars at other universities. PI Smertenko presented results of the project at the University of Washington in Seattle in September 2017 and at the University of California Riverside in December 2016. 4. Local presentations. Graduate student Sharol Schmidt presented a poster at the Washington State University Showcase. This event is open to general public. What do you plan to do during the next reporting period to accomplish the goals?N/A. This project has been terminated.
Impacts
What was accomplished under these goals?
1. We identified a novel phragmoplast protein Initial GeLC MS/MS analysis of the purified phragmoplasts identified ca. 3,000 proteins. Next we identified embryophyte-specific proteins amongst this set using NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A protein was considered embryophyte-specific if the BLAST e-value for a closest homologue protein from non-embryophyte species was greater than 0.01. Altogether we identified 32 such proteins of unknown function in the phragmoplast proteome. Each of the 32 proteins was expressed in tobacco BY-2 cells as an in-frame N-terminal translational GFP fusion to allow for examination of their intracellular localization during cytokinesis using live-cell imaging. Only one protein, MACET1, exhibited a unique localization pattern: it was not associated with microtubules during metaphase, but it was associated with the distal zone of the phragmoplast. Expression of the Arabidopsis MACET1-GFP in Arabidopsis under control of its native promoter also resulted in distal zone localization. Next, we examined ability of MACET to bind microtubules by transiently expressing MACET1-GFP in leaf pavement cells of tobacco plants that were stably transformed with a-Tubulin 5-mCherry. MACET1-GFP co-localized with microtubules. These data demonstrate that we have identified a novel phragmoplast microtubule protein. 2. Analysis of MACET1 activity in vitro. A protein can associate with microtubules in vivo through direct binding or through interaction with a microtubule-binding protein. To discriminate between these options, we expressed recombinant MACET1 in E. coli and used it in microtubule co-sedimentation assays. We found that MACET1 both co-sediments with microtubules and increases amount of microtubules in the pellet. The latter finding suggests that MACET1 can promote microtubule polymerization. We measured the impact of MACET1 on microtubule elongation. Adding MACET1 to ATTO488-tubulin resulted in the formation of asters. The appearance of asters suggests that MACET1 nucleates microtubules. To test this possibility, we mixed green (ATTO488-labeled) microtubules polymerized in the presence of a non-hydrolysable GTP-analogue GMPPNP ("seeds") with a 5 mM mixture containing both Rhodamine-labeled and unlabeled tubulin (ratio 1:4) and GTP. Under these conditions microtubules in control reactions will only polymerize on the seeds. However, when 200 nM MACET1 was added to the tubulin mixture, 32% of all microtubules were nucleated de novo, showing that MACET1 nucleates microtubules in vitro. In agreement with this conclusion, MACET1 localizes to sites of microtubule-nucleation in vivo. This data implies a role of MACET1 in generation of new microtubules during phragmoplast expansion. To determine how MACET1 regulates microtubule dynamics, we performed "on glass" microtubule polymerization assays. We monitored microtubule dynamics using TIRF microscopy. Kymograph analysis demonstrated that MACET1 had no significant impact on the polymerization rate, but reduced the frequency of transition from depolymerization to polymerization events. Thus, MACET1 can both nucleate microtubules and to suppress rescue. 3. Role of MACET1 in phragmoplast expansion. To determine the role of MACET1 in cytokinesis, we identified a tDNA knockout allele for the Arabidopsis MACET1. Roots of the macet1 mutant grew slower and this phenotype was rescued by transforming macet1 with the genomic region of MACET1 fused to GFP. The short root phenotype points our potential role of MACET1 in meristem activity or/and cell elongation. The latter role is unlikely because cell length in the root differentiation zone was not affected in macet1. To test the role of MACET1 in cell division, we first examined promoter activity using a GUS (β-glucuronidase) reporter assay. GUS was detected in meristem tissues and young leaves. Consistent with this finding, MACET1-GFP driven by MACET1 native promoter was not detected in the interphase cells within root meristems. The data suggest that the shorter root phenotype was induced by a function of MACET1 during mitosis. We found that the rate of cell plate expansion in macet1 was slower relative to the rescued lines. Furthermore, immunostaining of macet1 roots with anti-tubulin, showed that macet1 phragmoplasts were significantly shorter. The percentage of slanted cell plates was significantly higher in macet1. Thus, we identified a novel phragmoplast protein, which plays an essential role in cell plate expansion and orientation, and phragmoplast structure.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Smertenko A., Assaad F., Baluska F., Bezanilla M., Buschmann H., Drakakaki G., Hauser M.-T., Janson M., Mineyuki Y., Moore I, M�ller S., Murata T., Otegui M.S., Panteris E., Rasmussen C., Schmit A.-C., `amaj J., Samuels L., Staehelin A., Van Damme D., Wasteneys G., and }�rsk� V. (2017) Plant cytokinesis: terminology for structures and processes. Trends Cell Biology 27, 885-894.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Smertenko A. (2017) Can Peroxisomes Inform Cellular Response to Drought? Trends Plant Science 22, 1005-1007.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Lehman, T.A., Smertenko, A., and Sanguinet K.A. (2017) Auxin, microtubules, and vesicle trafficking: conspirators behind the cell wall. J Experimental Botany 68, 3321-3329.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2018
Citation:
Smertenko A., Hewitt S.L., Jacques C.N., Kacprzyk R., Liu Y., Marcec M.J., Moyo L., Ogden A., Oung H.M., Schmidt S., and Serrano-Romero E.A. (2018) Phragmoplast microtubule dynamics: a game of zones. J. Cell Science In press.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Oles V., Panchenko A. and Smertenko, A. (2017) Modeling hormonal control of cambium proliferation. PLoS One 12: e0171927, doi:10.1371/journal.pone.0171927.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Fahy D., Sanad M. N., Duscha K., Lyons M., Liu F., Bozhkov P., Kunz H. H., Hu J., Neuhaus H. E., Steel P. G., Smertenko, A. (2017) Impact of salt stress, cell death, and autophagy on peroxisomes: quantitative and morphological analyses using small fluorescent probe N-BODIPY. Scientific Reports 7, 39069, doi:10.1038/srep39069.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Moroz, N., Fritch, K.R., Marcec, M.J., Tripathi, D., Smertenko, A. and Tanaka, K. (2017) Extracellular alkalinization as a defense response in potato cells. Frontiers in Plant Science 8:32. doi: 10.3389/fpls.2017.00032
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:We targeted the following groups: professional scientists, employees of industry, graduate students, undergraduate students, and high school students. We conducted the following activities: 1. Outreach activity in collaboration with Native American Programs at Washington State University. We host a group of high school students from local Native American communities. This program aims at encouraging careers in STEM subjects including agriculture. The event includes a workshop, lectures, mini-projects, and introduction to the life on campus. 2. Lecturing to undergraduate students. Project Director delivers host lectures to undergraduate students on campus. 3. Research experience for the undergraduate students. We host several students through the year to get hands on laboratory experience. Students participation is encouraged through opportunities to present data on the local and national meetings, and co-authorship of publications. 4. Workshops, classroom instructions and mentoring of graduate students. Project Directors teaches graduate class, and advises graduate student in the lab. 5. Publishing papers in peer-review journals and book chapter. 6. Presenting outcomes of our research at research conferences. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Project director, graduate student, and undergraduate student working on the project have attended Plant Cell Dynamics meeting. During the meeting students presented their findings as oral talks and as posters. Project director has also attended a Gordon Research Conference on Plant, Fungal, and Microbial Cytoskeleton and American Society of Cell Biology meeting. This provided an opportunity to learn about novel developments in the field and discuss results of the project with peers. How have the results been disseminated to communities of interest?Results of the research performed in the course of this project have been disseminated through publications and presentation at the conferences What do you plan to do during the next reporting period to accomplish the goals?In the following year we will aim at maintaining high productivity of our research and specifically focus on the following areas: 1. Continue molecular and functional characterization of MACET and PARK. 2. Publish two papers in peer-reviewed journals. 3. Take part in the outreach activities with students and local communities. 4. Attend conferences.
Impacts
What was accomplished under these goals?
Objective 1. We have identified two novel proteins that specifically localize to the phragmoplast. The first protein is a novel microtubule binding protein MACERATOR (MACET). Live cell imaging demonstrated that MACET- GFP localizes to the phragmoplast starting from the end of anaphase and persists on the phragmoplast microtubules until the end of the cytokinesis. The second protein is PARK (Phragmoplast-Associated Receptor Kinase). PARK is recruited to the phragmoplast midzone at the end of the anaphase and persists in the cell plate after depolymerization of microtubules until the onset of G1. Objective 2. Genetics and in vitro analysis have been used to analyze the functions of the identified proteins. We produced recombinant MACET and demonstrated that it binds microtubules via two evolutionarily conserved N-terminal and C-terminal domains. We have shown that MACET promotes microtubule nucleation and restricts microtubule elongation by inhibiting rescue events. The knock-out Arabidopsis mutants of MACET exhibit defect in the phragmoplast expansion and form bifurcated cell plates. Combined data demonstrate that MACET plays a key role in cell plate synthesis by controlling phragmoplast expansion. We have generated dominant-negative mutants of PARK in both Arabidopsis and tobacco tissue culture cells (strain BY-2). Arabidopsis transformants were embryonic-lethal. Tobacco transgenic cell lines expressing PARK dominant negative mutants showed formation of ectopic phragmoplasts, abnormal phragmoplast expansion, and cell wall stubs. Our data suggest that PARK controls microtubule turnover and fidelity of microtubule nucleation in the region of forming cell plate.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Smertenko A., Moschou P., Zhang L., Fahy D. and Bozhkov, P. (2016) Characterization of cytokinetic mutants using small fluorescent probes. Methods of Molecular Biology, 1370, 199-208.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Moschou P., Gutierrez-Beltran E., Bozhkov P.V., Smertenko A. (2016) Separase Promotes Microtubule Polymerization by Activating CENP-E-Related Kinesin Kin7. Developmental Cell 37, 350-361.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Moschou, P.N., Savenkov, E.I., Minina, E.A., Fukada, K., Reza, S.H., Gutierrez-Beltran, E., Sanchez-Vera, V., Suarez, M.F., Hussey, P.J., Smertenko, A.P. and Bozhkov, P.V. (2016) EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression. New Phytologist. 212, 232-243.
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Progress 03/01/15 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for one graduate student and one intern undergraduate student. How have the results been disseminated to communities of interest?The results of the work have been presented at the Annual Meeting of the American Society for Cell Biology. What do you plan to do during the next reporting period to accomplish the goals?1. Submit one manuscript on the role of formins in plant cytokinesis. 2. Identify more cytokinetic mutant alleles for 7 proteins localized to the phragmoplast. The alleles will be searched in the collection of available tDNA lines as well as generated by RNAi or amiRNA approaches. 3. Continue with the detailed analysis of intracellular localization of the identified proteins.
Impacts
What was accomplished under these goals?
We have analysed intracellular localization of 46 phragmoplast proteins using N. benthamiana leaf pavement cells transient system and stably transformed tobacco tissue culture cells. Altogether we identified 7 genes which show localization to the phragmoplast microtubules or the midzone. We have identified a strong cytokinetic mutant allele for a novel plant-specific phragmoplast protein MACET. Conducted detailed intracellular localization and dynamics for a novel protein kinase that localizes to the midzone. Analysed the role of formins in the cell plate synthesis. Now this work is in the preparation for the publication.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Smertenko A., Moschou P., Zhang L., Fahy D. and Bozhkov, P. (2015) Characterization of cytokinetic mutants using small fluorescent probes. Methods in Molecular Biology, 1370, 199-208.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Ovecka M., Vaskebova L., Komis G., Luptovciak I., Smertenko A. and Samaj J. (2015) Preparation of plants for developmental and cellular imaging by light-sheet microscopy. Nature Protocols 10, 1234-1247.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Moschou P.N., Savenkov E.I., Minina E.A., Fukada K., Reza E.H., Gutierrez-Beltran1 E., Sanchez-Vera V., Suarez M.F., Hussey P.J., Smertenko A.P., Bozhkov P.V. EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently from its role in anaphase progression. New Phytologist, submitted.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gutierrez-Beltran E., Moschou, P.N., Smertenko, A.P. and Bozhkov, P.V. (2015) Tudor Staphylococcal Nuclease links formation of stress granules and processing bodies with mRNA catabolism in Arabidopsis. Plant Cell 27, 926-943.
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