The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization

Abstract

The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged β-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.

MLKS2 is an ARM domain and F-actin-associated KASH protein that functions in stomatal complex development and meiotic chromosome segregation

“…Similarly, mutations in AtPSS1 leads to phenotypes shared with mlks2, including production of univalents and aneuploid cells after meiosis [63]. Findings such as these, together with the large body of evidence implicating microtubules in plant meiotic chromosome behavior [50,[64][65][66][67][68] suggest that meiotic chromosome behavior likely requires both actin and tubulin cytoskeletal systems. Indeed, the LINC complex in general, and MLKS2 in particular, may provide a coordinating structure that integrates cytoskeletal systems to affect nuclear architecture.…”

Functional diversification of the kinesin‐14 family in land plants

“…Arabidopsis atk1 mutants exhibit abnormalities in meiosis, with broadened spindles poles and reduced male fertility , as well as mild spindle bipolarity defects during mitosis . A kinesin‐14 member encoded by the maize Divergent spindle‐1 ( Dv1 ) gene is likely a functional homolog of ATK1 as dv1 mutants have broadened spindle poles and altered spindle length and width during meiosis . A related A. thaliana kinesin‐14 family member, ATK5, has a more pronounced role in mitotic spindle morphogenesis .…”

“…A). However, none of these motors are absolutely required for proper chromosome segregation, indicating either functional redundancy among kinesin‐14s or other redundant pathways leading to proper spindle formation in plants .…”

Anaphase asymmetry and dynamic repositioning of the division plane during maize meiosis

“…Plants were genotyped for the CFPtubulin transgene using a CTAB DNA extraction protocol (Clarke, 2009) on leaf tissue and primers that annealed within the CFP (5′-GGAGTAC-AACTACATCAGCCACAACGTC) and tubulin (5′-CCGGACTGACC-GAAGACGAAGTTGT) sequences. The maize line containing dv1 was obtained from the Maize Genetics Cooperation Stock Center (University of Illinois) and genotyped as previously described (Higgins et al, 2016). All chemicals and reagents, unless otherwise stated, were purchased from Sigma Aldrich.…”

“…Immunolocalization was performed as previously described (Higgins et al, 2016). Anthers from immature tassels were fixed for 60 min in 4% paraformaldehyde-PHEMS buffer (60 mM PIPES, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl 2 , 0.35 M Sorbitol, pH 6.8), washed three times in 1× PBS and dissected for meiotic cells.…”