Visualization of PtdIns3P dynamics in living plant cells
Visualization of PtdIns3P dynamics in living plant cells
To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.
Androstadienes/pharmacology Animals Arabidopsis/cytology/genetics/*metabolism Cell Differentiation Cell Nucleus/metabolism/ultrastructure Cytokinesis/physiology Cytoplasmic Vesicles/chemistry/metabolism/ultrastructure Cytosol/metabolism/ultrastructure Endosomal Sorting Complexes Required for Transport Fabaceae/cytology/genetics/*metabolism Luminescent Proteins/analysis Mice Microscopy, Confocal Phosphatidylinositol Phosphates/*analysis/genetics/metabolism Phosphoproteins/chemistry/genetics Plants, Genetically Modified/cytology/drug effects/metabolism Protein Kinase Inhibitors/pharmacology Protein Structure, Tertiary Protoplasts/metabolism/ultrastructure Recombinant Fusion Proteins/analysis/chemistry Tobacco/cytology/genetics/*metabolism Wortmannin
687-700
Vermeer, J. E.
30259145-19ec-4062-94a3-d94629b3a2e8
van Leeuwen, W.
f95d6544-d707-4f7c-9128-64d4faab2e43
Tobena-Santamaria, R.
c91a517e-79ed-48f0-9ac9-bd0d879bcab4
Laxalt, A. M.
8ced3e06-232d-43a8-a239-7ecb70f30e1b
Jones, D. R.
87aa0b62-ca75-4e58-8533-022a7b5cc354
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Gadella T. W., Jr.
c86c53f2-8ef8-401b-95fb-a041f1300c5f
Munnik, T.
3f33c9a2-167b-400a-a9ee-7cd7ca53089a
19 July 2006
Vermeer, J. E.
30259145-19ec-4062-94a3-d94629b3a2e8
van Leeuwen, W.
f95d6544-d707-4f7c-9128-64d4faab2e43
Tobena-Santamaria, R.
c91a517e-79ed-48f0-9ac9-bd0d879bcab4
Laxalt, A. M.
8ced3e06-232d-43a8-a239-7ecb70f30e1b
Jones, D. R.
87aa0b62-ca75-4e58-8533-022a7b5cc354
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Gadella T. W., Jr.
c86c53f2-8ef8-401b-95fb-a041f1300c5f
Munnik, T.
3f33c9a2-167b-400a-a9ee-7cd7ca53089a
Vermeer, J. E., van Leeuwen, W., Tobena-Santamaria, R., Laxalt, A. M., Jones, D. R., Divecha, N., Gadella T. W., Jr. and Munnik, T.
(2006)
Visualization of PtdIns3P dynamics in living plant cells.
The Plant Journal, 47 (5), .
(doi:10.1111/j.1365-313X.2006.02830.x).
Abstract
To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.
This record has no associated files available for download.
More information
Accepted/In Press date: 10 May 2006
Published date: 19 July 2006
Additional Information:
Vermeer, Joop E M van Leeuwen, Wessel Tobena-Santamaria, Rafa Laxalt, Ana M Jones, David R Divecha, Nullin Gadella, Theodorus W J Jr Munnik, Teun eng Research Support, Non-U.S. Gov't England 2006/07/22 Plant J. 2006 Sep;47(5):687-700. doi: 10.1111/j.1365-313X.2006.02830.x. Epub 2006 Jul 19.
Keywords:
Androstadienes/pharmacology Animals Arabidopsis/cytology/genetics/*metabolism Cell Differentiation Cell Nucleus/metabolism/ultrastructure Cytokinesis/physiology Cytoplasmic Vesicles/chemistry/metabolism/ultrastructure Cytosol/metabolism/ultrastructure Endosomal Sorting Complexes Required for Transport Fabaceae/cytology/genetics/*metabolism Luminescent Proteins/analysis Mice Microscopy, Confocal Phosphatidylinositol Phosphates/*analysis/genetics/metabolism Phosphoproteins/chemistry/genetics Plants, Genetically Modified/cytology/drug effects/metabolism Protein Kinase Inhibitors/pharmacology Protein Structure, Tertiary Protoplasts/metabolism/ultrastructure Recombinant Fusion Proteins/analysis/chemistry Tobacco/cytology/genetics/*metabolism Wortmannin
Identifiers
Local EPrints ID: 479378
URI: http://eprints.soton.ac.uk/id/eprint/479378
ISSN: 0960-7412
PURE UUID: a6a78ec9-dcb5-40ea-81bb-a469d00423d9
Catalogue record
Date deposited: 20 Jul 2023 17:39
Last modified: 17 Mar 2024 03:00
Export record
Altmetrics
Contributors
Author:
J. E. Vermeer
Author:
W. van Leeuwen
Author:
R. Tobena-Santamaria
Author:
A. M. Laxalt
Author:
D. R. Jones
Author:
Jr. Gadella T. W.
Author:
T. Munnik
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics