Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics
Mutations within the <i>Leucine-Rich Repeat Kinase 2 (LRRK2)</i> gene are the most common genetic cause of autosomal and sporadic Parkinson’s disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and...
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| Format: | Article |
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MDPI AG
2020-05-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/11/2561 |
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| author | Bethany J. Sanstrum Brandee M. S. S. Goo Diana Z. Y. Holden Donovan D. Delgado Thien P. N. Nguyen Kiana D. Lee Nicholas G. James |
| author_facet | Bethany J. Sanstrum Brandee M. S. S. Goo Diana Z. Y. Holden Donovan D. Delgado Thien P. N. Nguyen Kiana D. Lee Nicholas G. James |
| author_sort | Bethany J. Sanstrum |
| collection | DOAJ |
| description | Mutations within the <i>Leucine-Rich Repeat Kinase 2 (LRRK2)</i> gene are the most common genetic cause of autosomal and sporadic Parkinson’s disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation—all of which could lead to neurodegeneration associated with PD. |
| first_indexed | 2024-03-10T19:28:53Z |
| format | Article |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T19:28:53Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-a14a1f3dac674d79b6c483523946058e2023-11-20T02:21:57ZengMDPI AGMolecules1420-30492020-05-012511256110.3390/molecules25112561Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane DynamicsBethany J. Sanstrum0Brandee M. S. S. Goo1Diana Z. Y. Holden2Donovan D. Delgado3Thien P. N. Nguyen4Kiana D. Lee5Nicholas G. James6Department of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USADepartment of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USADepartment of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USADepartment of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USADepartment of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USADepartment of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USADepartment of Cell and Molecular Biology, University of Hawaii at Manoa, 651 Ilalo Street, Honolulu, HI 96813, USAMutations within the <i>Leucine-Rich Repeat Kinase 2 (LRRK2)</i> gene are the most common genetic cause of autosomal and sporadic Parkinson’s disease (PD). LRRK2 is a large multidomain kinase that has reported interactions with several membrane proteins, including Rab and Endophilin, and has recently been proposed to function as a regulator of vesicular trafficking. It is unclear whether or how the spatiotemporal organization of the protein is altered due to LRRK2 activity. Therefore, we utilized fluctuation-based microscopy along with FLIM/FRET to examine the cellular properties and membrane recruitment of WT LRRK2-GFP (WT) and the PD mutant G2019S LRRK2-GFP (G2019S). We show that both variants can be separated into two distinct populations within the cytosol; a freely diffusing population associated with monomer/dimer species and a slower, likely vesicle-bound population. G2019S shows a significantly higher propensity to self-associate in both the cytosol and membrane regions when compared to WT. G2019S expression also resulted in increased hetero-interactions with Endophilin A1 (EndoA1), reduced cellular vesicles, and altered clathrin puncta dynamics associated with the plasma membrane. This finding was associated with a reduction in transferrin endocytosis in cells expressing G2019S, which indicates disruption of endocytic protein recruitment near the plasma membrane. Overall, this study uncovered multiple dynamic alterations to the LRRK2 protein as a result of the G2019S mutation—all of which could lead to neurodegeneration associated with PD.https://www.mdpi.com/1420-3049/25/11/2561Parkinson’s diseaseLRRK2endocytosisFLIMFRET-Phasorfluorescence fluctuation spectroscopy |
| spellingShingle | Bethany J. Sanstrum Brandee M. S. S. Goo Diana Z. Y. Holden Donovan D. Delgado Thien P. N. Nguyen Kiana D. Lee Nicholas G. James Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics Molecules Parkinson’s disease LRRK2 endocytosis FLIM FRET-Phasor fluorescence fluctuation spectroscopy |
| title | Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics |
| title_full | Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics |
| title_fullStr | Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics |
| title_full_unstemmed | Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics |
| title_short | Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics |
| title_sort | fluctuation imaging of lrrk2 reveals that the g2019s mutation alters spatial and membrane dynamics |
| topic | Parkinson’s disease LRRK2 endocytosis FLIM FRET-Phasor fluorescence fluctuation spectroscopy |
| url | https://www.mdpi.com/1420-3049/25/11/2561 |
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