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 that 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 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 20μm; E, G 5μm; H, I 500nm.

Treatment with the type I kinase inhibitor MLi-2 promotes 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 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: 100nm.

LRRK2IT lattice that decorates microtubules is highly ordered in cells treated with type I as compared to type II inhibitor.

(A) Segmented representation of a 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 a LRRK2IT associated microtubule bundle in MLi-2 treated cells. The individual microtubules are separated from each other by 65-70nm. (C) Segmented representation of a 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). (continued) (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 8 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 40nm; D-G 20nm; H-K 10nm.

Subtomogram analysis of LRRK2 MLi-2 reveals structural details about a 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 Fig. 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). (continued) (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 15Å 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 5nm; G-J 2nm; K-L 10nm; M-N 5nm.

Model of full-length LRRK2IT associated with microtubules in its active-like conformation.

(A) Subtomogram average of LRRK2IT lattice decorating a microtubule. (B) Cross-section view of the LRRK2IT lattice. Catalytic domains are closest to the microtubule while 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 Fig. 1A. Scale bars: A-C 10nm; D-F 10nm.