Bousquet C et al. (2023), Characterization of Microtubule Lattice Heterog...

XB-ART-60172

Bio Protoc 2023 Jul 20;1314:e4723. doi: 10.21769/BioProtoc.4723.

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Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging.

Bousquet C , Heumann JM , Chrétien D , Guyomar C .

Abstract
Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the αβ-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the αβ-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (α-α, β-β), except at one unique region called the seam, made of heterotypic ones (α-β, β-α). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every αβ-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts. Key features This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments. Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent αβ-tubulin heterodimers. The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft. Graphical overview.

PubMed ID: 37497446
PMC ID: PMC10366996
Article link: Bio Protoc

Species referenced: Xenopus
GO keywords: microtubule

Article Images: [+] show captions

References [+] :

Debs, Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy. 2020, Pubmed

	Figure 1. Tomogram visualization. (A) 3dmod Information Window. Selection of the Model mode (red circle). (B) 3dmod ZaP Window.
	Figure 2. Microtubule selection. (A) Tomogram visualized in the Slicer. (B) Selection of the microtubule (arrow). (C) Microtubule in (B) visualized in the Slicer averaged over 50 slices. (D) Microtubule oriented in cross-section after rotation of 90° around the X-axis and -57.4° around the Z-axis.
	Figure 3. Creation of a protofilament model. (A) Configuration of the Object type menu. (B) Creation of the first point. (C) Information window showing a first contour and a first point for Object 1.
	Figure 4. Modeling the protofilament path. (A) Microtubule cross-section at View axis position 106. (B) Addition of Point 2 on the same protofilament as in Figure 3B. (C) 3dmod Information Window showing a second point in Contour 1, Object 1.
	Figure 5. Microtubule cross-sections at positions listed in Table 1
	Figure 6. Microtubule center modeling. (A) Selection of the Centering button. (B) Selection of Point 1 in Object 1 Contour 1. (C) Selection of Object 2. (D) Selection of Object 1. (E) Copy Contours of Object 1 to Object 2. (F) Increase of the sphere radius of Point 1 in Object 1. (G) Colocalization of Points 1 of Objects 1 (green circle) and 2 (cyan circle). (H) Centering of Point 1 of Object 1 with the right mouse button.
	Figure 7. Addition of points spaced every ~8 nm on the model. The points of Object 1 (green circles) follow the microtubule center path, and those of Object 2 (cyan circles) follow the protofilament path.
	Figure 8. Configuration of the Setup tab of Etomo. (A) Front page of Etomo. (B) Starting PEET menu. (C) Setup tab.
	Figure 9. Configuration of the Run tab of Etomo. (A) Iteration runs. (B) Rotations used to adjust the subvolumes with the reference.
	Figure 10. Configuration of the More Options tab of Etomo
	Figure 11. Subtomogram average of the full-length microtubule. (A) 3dmod Information Window. (B) 3dmod ZaP window displaying the intermediate 40 particles subtomogram average. (C) Isosurface of (B). (D) Isosurface control panel.
	Figure 12. Inspection of the subtomogram average of the full-length microtubule. (A) MV Controls menu. (B) Selection of the 81 particle subtomogram average (MT_AvgVol_81.mrc) in the ZaP window. (C) Visualization of the 81 particle subtomogram average showing a B-type lattice organization. (D) Rotation of the map by 180° around the Y-axis. (E) Visualization of the 81 particle subtomogram average showing two seams of the A-lattice type and the middle protofilament with thinner densities with respect to the adjacent protofilaments.
	Figure 13. Preparation of the first segment. (A) Starting PEET menu. (B) Selection of the MT.epe file of the full-length microtubule. (C) PEET interface of segment 1.
	Figure 14. Configuration of the ‘Run’ tab of segment1
	Figure 15. Segmented subtomogram averaging. (A) Segment 1. (B) Segment 2. (C) Segment 3. (D) Segment 4. Views turned by 180° around the Y-axis are displayed in (A–D). The kinesin motor domain densities in the three front protofilaments of segments (1–3) are shifted longitudinally by ~49.2 Å indicating that the underlying αβ-tubulin molecules share heterotypic interactions of the A-type. Conversely, they are shifted longitudinally by ~9.2 Å in segment 4 indicating that the underlying αβ-tubulin molecules share homotypic interactions of the B-type.