Assembly of Yeast Spindle Pole Body and its Components Revealed by Electron Cryo-Tomography

Abstract

Spindle pole body (SPB) is the microtubule organizing center (MTOC) in yeast. It plays essential roles during many cellular processes, ranging from mitosis to karyogamy. Here, we used electron cryo-tomography (cryo-ET) and image processing to study SPB and its component purified from the budding yeast. The 3D images and models of SPB at various cell cycle stages were reconstructed by cryo-ET and were analyzed. The results reveal SPB as a cylindrical shaped structure composed of multiple layers. The central layers are arranged with a degree of crystalline order. By using subtomogram averaging methods, we studied the purified "sheet" from over-expressed Spc42p. Our analysis of the SPBs and its components provides new insights into the assembly of this organelle and its cellular function as an MTOC.

Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication

“…We analyzed images for each Spc42 variant construct as single particles (see Materials and Methods ) to obtain four class averages per Spc42 variant (Supplemental Figure S2C), the best of which was chosen by eye as a reference image for subsequent alignment and averaging (Figure 2C). All constructs that formed an array of hexagonal symmetry exhibited unit cell dimensions in real space of a = b = ∼145 Å (1/126 Å −1 reciprocal dimensions), identical to the dimensions of lattices formed in vivo by Spc42 (Bullitt et al , 1997; Li and Fernandez, 2018) (Table 2 and Figure 2D).…”

“…Cryo-EM of this superplaque revealed a hexagonal lattice with 1/126 Å −1 reciprocal dimensions (for Fourier transform of real space), comparable to the hexagonal pattern seen in the central layer of the SPB by cryo-electron tomography (Bullitt et al , 1997). These findings were recently corroborated by cryo-electron tomography studies describing 1/132 Å −1 reciprocal dimensions (Li and Fernandez, 2018).…”

“…To elucidate which parts of Spc42 were essential for assembly of higher-order arrays, we adopted methods used for 2D crystallization, in which a protein of interest is localized to a lipid monolayer formed at the air–water interface by use of an affinity tag (Kubalek et al , 1994; Kelly et al , 2010). A series of Spc42 constructs, varied by the inclusion or exclusion of symmetry elements (DCC, TCC, ACC) and undetermined regions (UR1–UR4), were prepared (Figure 2A and Supplemental Table S1), assembled onto lipid monolayers, and examined by negative-stain transmission EM to determine whether array formation occurred and whether arrays were similar to those observed previously in vivo (Bullitt et al , 1997; Li and Fernandez, 2018). Several Spc42 variant constructs formed hexagonal arrays, as evidenced by images and fast Fourier transforms (FFTs) (Table 2, Supplemental Figure S2, A and B, and Figure 2B).…”

“…The SPB appears as three layers when viewed by electron microscopy (EM): the inner and outer plaques nucleate respective nuclear and cytoplasmic microtubules, and the central plaque anchors the SPB to the nuclear envelope via two hooklike appendages (Byers and Goetsch, 1974, 1975). Two additional intermediate layers, IL1 and IL2, are observed between the central and outer plaques when viewed by cryo-EM and electron or cryo-electron tomography (Bullitt et al , 1997; O’Toole et al , 1999; Li and Fernandez, 2018). The SPB core, formed by the central plaque, IL2 and IL1, contains the yeast pericentrin-related protein Spc110, calmodulin (Cmd1), and three yeast-specific coiled-coil–containing proteins, Spc42, Spc29, and Cnm67 (Figure 1A).…”

“…It is essential for SPB formation, and during SPB duplication, Spc42 is the first core protein to localize to the new SPB (Donaldson and Kilmartin, 1996; Burns et al , 2015). Spc42 overproduction in yeast results in the lateral expansion of a two-dimensional (2D) crystal into a “superplaque” structure (Donaldson and Kilmartin, 1996; Bullitt et al , 1997; Castillo et al , 2002; Jaspersen et al , 2004; Li and Fernandez, 2018). Cryo-EM of this superplaque revealed a hexagonal lattice with 1/126 Å −1 reciprocal dimensions (for Fourier transform of real space), comparable to the hexagonal pattern seen in the central layer of the SPB by cryo-electron tomography (Bullitt et al , 1997).…”