Handong Su, Yang Liu,  Chunhui Wang, Yalin Liu, Chao Feng, Yishuang Sun, Jing Yuan,  James A. Birchler, and Fangpu Han.

PNAS May 18, 2021 118 (20) e2022357118; GENETICS

Significance

The spindle assembly checkpoint (SAC) signaling controls the attachments of kinetochores to spindle microtubules rigorously. However, how SAC proteins connect to the kinetochores is largely unknown in plants. Here, we identified the constitutive component of the central kinetochore protein Knl1 in maize and revealed a conserved function of Knl1 in cell division. A helical conformation with hydrophobic amino acids in the middle of a Knl1 protein was involved in the recruitment of an SAC protein, which is different from the Mps1-mediated phosphorylation of MELT modules that are described in yeast and mammalian cells. This region, detected in maize Knl1, is conserved in monocots but displays high divergence in eudicots, suggesting distinct kinetochore architectures with SAC signaling in plant lineages.

Abstract

The Knl1-Mis12-Ndc80 (KMN) network is an essential component of the kinetochore–microtubule attachment interface, which is required for genomic stability in eukaryotes. However, little is known about plant Knl1 proteins because of their complex evolutionary history. Here, we cloned the Knl1 homolog from maize (Zea mays) and confirmed it as a constitutive central kinetochore component. Functional assays demonstrated their conserved role in chromosomal congression and segregation during nuclear division, thus causing defective cell division during kernel development when Knl1 transcript was depleted. A 145 aa region in the middle of maize Knl1, that did not involve the MELT repeats, was associated with the interaction of spindle assembly checkpoint (SAC) components Bub1/Mad3 family proteins 1 and 2 (Bmf1/2) but not with the Bmf3 protein. They may form a helical conformation with a hydrophobic interface with the TPR domain of Bmf1/2, which is similar to that of vertebrates. However, this region detected in monocots shows extensive divergence in eudicots, suggesting that distinct modes of the SAC to kinetochore connection are present within plant lineages. These findings elucidate the conserved role of the KMN network in cell division and a striking dynamic of evolutionary patterns in the SAC signaling and kinetochore network.

See https://www.pnas.org/content/118/20/e2022357118

Figure 1:

Identification and annotation of the Knl1 homolog in maize. (A) Schematic diagram of Knl1 and SAC components in humans (H. sapiens), Arabidopsis (A. thaliana), and maize (Z. mays). Different-colored rectangles on human Knl1 represent the following: SILK and RVSF motif for PP1 binding domain; MELT repeats and KI1 motif for Bub3–Bub1 complex binding; KI2 motif for Bub3–BubR1 complex binding; coiled-coil domain for Zwint1 binding; and RWD domain for Mis12 complex binding. Ovals on Bub1 and BubR1 represent the TPR domains. Rectangles on Bub1 and BubR1 represent the Bub3-binding domain. Only RVSF, coiled-coil, and RWD domains are present in Arabidopsis and maize Knl1. The dotted lines and virtual boxes indicate there are no KI motifs in plant Knl1. (B) Model for evolution of the Bub/Mad family protein in humans, Arabidopsis, and maize. Some functional domains were shown with different-colored rectangles in the presumed ancestral Bub1/Mad3 protein. TPR domain is for Knl1 binding, GLEBS motif is for Bub3 binding, and a kinase domain is for histone H2A phosphorylation. A pseudokinase domain with a gray rectangle was shown in human BubR1. (C–E) Colocalization of KMN network components on maize leptotene and pachytene chromosomes: ZmMis12 (red) and ZmKnl1 (green) (C); ZmNdc80 (red) and ZmKnl1 (green) (D); and ZmMis12 (red) and ZmNdc80 (green) (E). (F) Colocalization signals of ZmKnl1 (green) and ZmBmf1 (red) in maize pachytene chromosomes. The Insets indicate a higher-magnification view of colocalization signals of different proteins. Chromosomes stained with DAPI are shown in blue. (Scale bar, 10 μm.)