In-cell cryo-electron tomography reveals differential effects of type I and type II kinase inhibitors on LRRK2 filament formation and microtubule association
- Department of Chemistry and Biochemistry, University of California San Diego, United States
- Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, United States
- Howard Hughes Medical Institute, United States
- Department of Molecular Biology, University of California San Diego, United States
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, United States
Figures
Figure 1 with 1 supplement
LRRK2IT expression in 293T cells responds differently to treatment with type I and type II kinase inhibitors.
(A) LRRK2 contains multiple functional domains, including armadillo (ARM), ankyrin repeats (ANK), leucine-rich repeats (LRR), Ras of complex (ROC), COR, kinase, and WD40 domain. (B) Schematic of the LRRK2 protein construct used in this paper, which includes a hyperactive I2020T mutation and an N-terminal GFP-tag. (C) Type I inhibitors (red) capture the LRRK2 kinase in active, closed conformation, while type II inhibitors (blue) trap the kinase of LRRK2 in an inactive, open-kinase conformation. (D–E) Cryo-fluorescence microscopy (cryo-FM) image of a vitreous cell on an electron microscopy grid expressing LRRK2IT treated with MLi-2. The GFP-LRRK2IT signal (green) is present as filaments as well as puncta. (F–G) Cryo-FM image of a cell expressing LRRK2IT treated with GZD-824. The GFP-LRRK2IT signal (green) shows a punctate distribution of the protein throughout the cytosol of the cell. (H–I) Cryo-TEM overview of the lamella of a cell expressing LRRK2IT treated with MLi-2 (H) and GZD-824 (I). Microtubule bundles are highlighted in green, with some displaying dense LRRK2 decoration. Green: microtubule bundles; red: mitochondria; pink: vesicles; blue: plasma membrane; purple: endoplasmic reticulum. Scale bars: D, F, 20 μm; E, G, 5 μm; H, I, 500 nm.
Figure 1—figure supplement 1
LRRK2IT expression in 293T cells responds differently to treatment with type I and type II kinase inhibitors.
(A) LRRK2IT microtubule-associated filaments (green) in 293T fixed cells treated with MLi-2. (B–D) Cryo-fluorescence micrographs of GFP-LRRK2IT-expressing cells treated with MLi-2 show a distribution of LRRK2IT as filaments, puncta, and diffuse in the cytosol. (E–H) Cryo-fluorescence microscope images of GFP-LRRK2IT-expressing cells treated with GZD-824 displaying diffuse and punctate distribution of LRRK2IT. (I–J) Cryo-TEM lamella overview of LRRK2IT-expressing cell treated with either MLi-2 (I) or GZD-824 (J). GFP-LRRK2IT signal (green) is overlaid from cryo-FM imaging to guide localization of LRRK2IT. (K) Length of LRRK2IT filaments evaluated from the cryo-TEM overview in (I) cells treated with MLi-2. Each data point represents a single filament. (22 data points; vertical bar represents mean with standard deviation 2.17±1.4 μm). MVB: multivesicular body V: vesicles, ER: endoplasmic reticulum, Mito: mitochondria, PM: plasma membrane. Scale bars: A, 40 μm; B–H, 20 μm; I–J, 500 nm.
Figure 2 with 3 supplements
Treatment with the type I kinase inhibitor MLi-2 promotes the formation of higher-order LRRK2IT-decorated microtubule bundles, whereas the type II kinase inhibitor GZD-824 reduces LRRK2IT filamentation and microtubule bundling in cells.
(A, C) Representative tomogram slices through the cytosol of a cell treated with MLi-2, showing a bundle of microtubules decorated with LRRK2IT. (B, D) Segmentation of the tomogram in A, C, highlighting the bundles of microtubules decorated with a well-ordered LRRK2IT lattice. (E) Tomogram slice of a cell treated with GZD-824, showing sparse LRRK2IT decoration on microtubules. (F) Segmentation of the tomogram in E. LRRK2IT decoration is present on three microtubules while there are nine undecorated microtubules present in the proximity. (G) Representative image of a cell treated with GZD-824. Single microtubule is decorated with LRRK2IT. This distribution pattern contrasts sharply with the extensive microtubule-associated filaments observed following type I inhibitor treatment. (H) Segmentation of a tomogram in G of a cell treated with GZD-824. Six undecorated microtubules are present near a single LRRK2-decorated microtubule. Lime green: LRRK2IT; green: microtubule; red: mitochondria; light pink: vesicles; blue: multivesicular bodies; pink: ribosomes. Scale bars: 100 nm.
Figure 2—figure supplement 1
LRRK2IT filament formation around microtubules is extensive when treated with type I, but not type II inhibitor.
(A–B) Representative tomogram slice through the cytosol highlighting multiple LRRK2IT-decorated microtubules in a cell treated with MLi-2. (C–D) Tomogram snapshots of a cell treated with GZD-824 where the majority of microtubules are not decorated with LRRK2IT. Scale bars: A–D, 100 nm.
Figure 2—figure supplement 2
Type I inhibitor-treated cells have single microtubules decorated with LRRK2IT.
(A) Tomogram slice of a cell treated with MLi-2 showing six single non-bundled microtubules decorated with LRRK2IT. (B) Segmentation of the tomogram shown in A highlighting LRRK2IT filamentation around microtubules. (C, D) Tomogram snapshot of an LRRK2IT lattice in MLi-2-treated cell. Lime green: LRRK2IT; green: microtubule; light pink: vesicles; blue: multivesicular bodies; pink: ribosomes. Scale bars: A–D, 100 nm.
Figure 2—figure supplement 3
A single instance of LRRK2IT-decorated microtubules was captured in cells treated with type II inhibitor.
(A) Tomogram snapshot of a cell treated with GZD-824 showing an LRRK2IT-decorated microtubule bundle. This is the single instance of LRRK2IT bundle captured in cells treated with GZD-824. (B) Segmented representation of the tomogram shown in A. (C) Side view of the LRRK2IT-decorated microtubule bundle shown in B. Lime green: LRRK2IT; green: microtubule; light pink: vesicles; blue: multivesicular bodies. Scale bars: A–C, 100 nm.
Figure 3 with 1 supplement
LRRK2IT lattice that decorates microtubules is highly ordered in cells treated with type I as compared to type II inhibitor.
(A) Segmented representation of an LRRK2IT-associated microtubule bundle in MLi-2-treated cells. Individual LRRK2IT subunits are highly ordered around the microtubules. (B) Top-down view of the segmented representation of an LRRK2IT-associated microtubule bundle in MLi-2-treated cells. The individual microtubules are separated from each other by 65–70 nm. (C) Segmented representation of an LRRK2IT-associated microtubule bundle in MLi-2-treated cells. (D–E) Close-up view of the LRRK2IT decoration on microtubules in MLi-2-treated cells. (F–G) Segmented representation of LRRK2IT-associated microtubules in GZD-824-treated cells. LRRK2IT lattice is less ordered compared to MLi-2-treated lattice in (D–E). (H–I) Subtomogram average of the LRRK2IT associated with microtubules from MLi-2 (H) or GZD-824 (I) treated cells. MLi-2-treated cells exhibit a more ordered lattice of LRRK2IT than the lattice found in GZD-824-treated cells. (J–K) Nearest-neighbor analysis of a central LRRK2IT subunit (highlighted in black) in MLi-2- and GZD-824-treated cells. In MLi-2-treated cells, the central LRRK2IT subunit has 16 nearest neighbors highlighted in orange. While in GZD-824-treated cells, the central LRRK2IT subunit has only eight nearest neighbors shown in pink. (L) Evaluation of the center-to-center distance of undecorated and LRRK2IT-decorated microtubule bundles in MLi-2- and GZD-824-treated cells (MLi-2 n=80, GZD-824 n=7, undecorated n=13; vertical line at median). (M) Quantification of LRRK2IT-decorated microtubule (MT) bundles and isolated microtubules in MLi-2- and GZD-824-treated cells (bundles quantified in MLi-2-treated cells n=12, isolated MTs quantified in MLi-2-treated cells n=16, isolated MTs quantified in GZD-824 n=6, bundle of MTs quantified in GZD-824 bundle n=1, vertical line at median). Scale bars: A–C, 40 nm; D–G, 20 nm; H–K, 10 nm.
Figure 3—figure supplement 1
Nearest-neighbor analysis of a central LRRK2IT subunit.
(A–B) Nearest-neighbor analysis of a central LRRK2IT subunit (highlighted in black) in MLi-2- and GZD-824-treated cells. (C–D) Annotation of immediate neighboring LRRK2IT subunits within each lattice. In MLi-2-treated cells, the central LRRK2IT subunit is surrounded by 16 nearest neighbors highlighted in orange. While in GZD-824-treated cells, the central LRRK2IT subunit has only eight nearest neighbors shown in pink.
Figure 4 with 1 supplement
Subtomogram analysis of LRRK2 MLi-2 reveals structural details about the N-terminal domains of closed-kinase LRRK2IT.
(A) Subtomogram average map of the microtubule-bound LRRK2 from MLi-2-treated cells (threshold value 0.24). (B–C) Molecular model of two central protomers of active kinase LRRK2 RCKW fit into map A. The protomers of LRRK2 fit well within the density corresponding to the active conformation of the LRRK2 kinase. The domains are colored according to Figure 1A. (D) Subtomogram average of LRRK2 displaying densities for the N-terminal domains emanating from the catalytic half of the protein (threshold value 0.14). (E–F) Molecular model of the two central protomers of full-length LRRK2 fit into the map shown in D. The model includes RCKW domains and N-terminal LRR-ANK domains. (G) Molecular model of a single subunit of full-length LRRK2 in active conformation interacting with a microtubule (gray) via ROC domain (green). (H) Comparison between the RCKW domains of LRRK2 in intermediate active kinase conformation (gray) and fully active full-length in-cell model of LRRK2 (colored). The kinase domain in the cellular map is fully closed, demonstrated by a 15 Å shift between the COR domains. (I–J) Comparison of the LRR and ARM domains of LRRK2 in the in-cell model (colored) versus the intermediate kinase-active LRRK2 structure (gray). The COR–ROC domains are aligned in both structures to highlight the positional shifts in the LRR and ARM domains (K–L). Subtomogram average of microtubule-bound LRRK2 from GZD-824-treated cells. (M–N) Molecular model of the active kinase RCKW domains fitted into the map shown in panel K. The molecular model aligns well with the active kinase conformation of LRRK2. The N-terminal domains are not visible in this map. Rotated views are shown. Scale bars: A–F, 5 nm; G–J, 2 nm; K–L, 10 nm; M–N, 5 nm.
Figure 4—figure supplement 1
Subtomogram analysis of LRRK2IT in MLi-2-treated cells centered on WD40-WD40 domain interaction interface.
(A) Subtomogram average map of LRRK2IT centered on the WD40-WD40 domain interface (threshold value: 0.12). (B, C) Molecular model of the closed-kinase LRRK2 fit in the map in A, demonstrating interaction interface between WD40-WD40 domains. Domain architecture color scheme is the same as given in Figure 1A. (D) Subtomogram average map at threshold value 0.24, showing the densities for N-terminal domains of LRRK2IT. (E, F) Molecular model of full-length LRRK2 fit in the map in D, showing the architecture of the N-terminal domains. (G–I) Molecular model of the four LRRK2 protomers fit into subtomogram map shown in D, demonstrating the overall architecture of the full-length LRRK2IT assembly around a microtubule. Scale bars: A–I, 5 nm.
Figure 5 with 2 supplements
Model of full-length LRRK2IT associated with microtubules in its active-like conformation.
(A) Subtomogram average of LRRK2IT lattice decorating a microtubule. (B) Cross-sectional view of the LRRK2IT lattice. Catalytic domains are closest to the microtubule, while the outer layer represents the N-terminal protein-protein interaction domains of LRRK2. (C) Side view of the microtubule demonstrating the extension of the N-terminal layer of LRRK2. (D–F) Atomic model fit of full-length LRRK2IT in the subtomogram average map. LRRK2 domain colors are as in Figure 1A. Scale bars: A–C, 10 nm; D–F, 10 nm.
Figure 5—figure supplement 1
Model of full-length LRRK2IT associated with microtubules in its active-like conformation.
(A–B) Subtomogram average of LRRK2IT lattice decorating a microtubule and a corresponding cross-section. (C–E) Molecular model of the closed-kinase LRRK2 fit in the map in A, demonstrating domain organization of the N-terminal LRR-ANK-ARM domains. (F) Molecular model of a single full-length LRRK2 fitted into the map shown in A, highlighting the architecture of the N-terminal domains. Domains are colored as in Figure 1A, and the microtubule is presented in gray (EMDB 25908). Scale bars: A–F, 10 nm.
Figure 5—figure supplement 2
Data-processing workflow for LRRK2IT in type I inhibitor-treated cells.
(A) Cryo-electron tomography (cryo-ET) data processing workflow for LRRK2IT-decorated microtubules in MLi-2-treated cells. (B) Fourier shell correlation (FSC) curve for the Dynamo-refined MLi-2-treated LRRK2 subtomogram average presented in Figure 5. (C) Final maps and the gold-standard Fourier shell correlation (FSC) curves (0.143 cutoff) show the final resolution of the COR-COR domain interaction interface-centered LRRK2IT refinement. (D) Final maps and the gold-standard FSC curves (0.143 cutoff) show the final resolution of the WD40-WD40 domain interaction interface-centered LRRK2IT refinement.
Additional files
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Supplementary file 1
Supplementary Materials: Materials Used and Data Acquisition Parameters.
Table A. List of key reagents and tools used to perform this study. Table B. Data acquisition table for cryo-electron tomography (cryo-ET) datasets used in this study.
- https://cdn.elifesciences.org/articles/111075/elife-111075-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/111075/elife-111075-mdarchecklist1-v1.docx
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In-cell cryo-electron tomography reveals differential effects of type I and type II kinase inhibitors on LRRK2 filament formation and microtubule association
eLife 15:RP111075.
https://doi.org/10.7554/eLife.111075.3
