Abstract
SummaryMicrotubule nucleation by the γ-tubulin complex occurs primarily at centrosomes, but more diverse types of microtubule organizing centers (MTOCs) also exist, especially in differentiated cells [1–4]. Mechanisms generating MTOC diversity are poorly understood. Fission yeast Schizosaccharomyces pombe has multiple types of cytoplasmic MTOCs, and these vary through the cell cycle [5, 6]. Cytoplasmic microtubule nucleation in fission yeast depends on a complex of proteins Mto1 and Mto2 (Mto1/2), which localizes to MTOCs and interacts with the γ-tubulin complex [7–12]. Localization of Mto1 to prospective MTOC sites has been proposed as a key step in γ-tubulin complex recruitment and MTOC formation [9, 13], but how Mto1 localizes to such sites has not been investigated. Here we identify a short conserved C-terminal sequence in Mto1, termed MASC, important for targeting Mto1 to multiple distinct MTOCs. Different subregions of MASC target Mto1 to different MTOCs, and multimerization of MASC is important for efficient targeting. Mto1 targeting to the cell equator during division depends on direct interaction with unconventional type II myosin Myp2. Targeting to the spindle pole body during mitosis depends on Sid4 and Cdc11, components of the septation initiation network (SIN), but not on other SIN components.
Highlights
All three fusion proteins localized to SPBs and to equatorial MTOCs (eMTOCs) sites when expressed in mto1D cells (Figure S2E), demonstrating that sequence-independent multimerization promotes MASC-dependent Mto1 localization, most likely by increasing avidity of Mto1 binding to microtubule organizing centers (MTOCs) sites
Mto1 eMTOC Localization Depends on Interaction with Unconventional Myosin Myp2 We found that Mto1 localization to eMTOC sites was abolished by disruption of the actin cytoskeleton with latrunculin B (Figure 3A) and that Mto1 colocalized with markers of the contractile actin ring (CAR) during cytokinesis (Figure 3B; Figure S3A), suggesting that Mto1 localization to eMTOC sites depends on association with a CAR component
GFP-tagged Alp4 was observed at eMTOC sites in w66% of wild-type cells with a CAR, it was completely absent from eMTOC sites in myp2D cells; myp2D mutants failed to nucleate postanaphase arrays (PAAs) MTs (Figure S3C)
Summary
A triple-point mutant (R1056A, E1059A, E1061A), termed Mto1-427, localized to iSPBs and mSPBs, but not to eMTOC sites (Figures 1F and 1G; additional data not shown), and mto1-427 cells nucleated astral MTs in mitosis, but not PAA MTs (Figure 1H). These results demonstrate that different mechanisms and subregions of MASC regulate Mto1 localization to different subcellular sites—iSPBs, mSPBs, and eMTOCs–and that targeting to SPBs and eMTOC sites can occur independently of each other (Figure 1I). Large fragments such as GFP-Mto1(919-1115) showed robust localization to iSPBs, mSPBs, and eMTOC
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