Fission yeast Mto1 regulates diversity of cytoplasmic microtubule organizing centers - PubMed (original) (raw)

Fission yeast Mto1 regulates diversity of cytoplasmic microtubule organizing centers

Itaru Samejima et al. Curr Biol. 2010.

Abstract

Microtubule 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. Mechanisms generating MTOC diversity are poorly understood. Fission yeast Schizosaccharomyces pombe has multiple types of cytoplasmic MTOCs, and these vary through the cell cycle. 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. Localization of Mto1 to prospective MTOC sites has been proposed as a key step in γ-tubulin complex recruitment and MTOC formation, 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.

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Figures

Figure 1

A Modular Sequence Motif in the Mto1 C Terminus Regulates Differential Localization to Multiple MTOCs (A) Position of conserved MASC motif in Mto1 and truncation mutants. (B) Cytoplasmic distribution of Mto1(1-1051)-GFP and Mto1-GFP (green) and SPB marker Cut12-tdTomato (tdT; red). Mitotic cells are marked with asterisks. (C and D) Time-lapse images of GFP-tubulin in interphase and mitotic mto1(1-1051)-GFP and mto1(1-1095)-GFP cells (2 min intervals). GFP signal from Mto1 itself is too faint to be seen here. Note mitotic astral and postanaphase array (PAA) MTs (red arrowheads) in mto1(1-1095)-GFP but random cortical MT nucleation in mto1(1-1051)-GFP cells (blue arrowheads). (E) Localization or absence of indicated Mto1-GFP truncations (green) at interphase SPBs (iSPB), mitotic SPBs (mSPB), and equatorial microtubule organizing centers (eMTOC). SPB marker Sad1-dsRed is in red. (F and G) Triple-point mutant Mto1-427-GFP (green) is present at mSPBs in metaphase and anaphase but absent from eMTOCs (white arrowhead). (G) Wild-type Mto1-GFP appears at eMTOCs as spindles elongate. (H) Time-lapse images of GFP-tubulin in mitotic mto1-427 cells (2 min intervals). PAA MTs are absent. (I) Localization summary of mutant Mto1 proteins. Additional data are shown in Figure S1. Scale bars represent 10 μm.

Figure 2

Multimerization of the Mto1 C Terminus Is Critical for Robust SPB and eMTOC Localization (A) Localization of indicated Mto1 C-terminal fragments fused to GFP. SPB marker Cut12-tdT is shown in red. (B) Mto1 localization to SPBs but not eMTOCs (arrowhead, top) and to SPBs and eMTOCs (bottom) in the indicated “Mto1 GFP-insertion” strains. SPB marker Sad1-dsRed is shown in red. (C) Time-lapse images (2 min intervals) of GFP-tubulin showing astral MT nucleation (left) and astral and PAA MT nucleation (right) in the Mto1 GFP-insertion strains. Red arrowhead indicates PAA MTs. Mto1-GFP itself is too faint to be seen. (D) Localization of small Mto1 C-terminal fragments fused to GFP plus tetrameric coiled coil from human VASP (VTD). Arrowheads in merged images indicate absence of GFP-VTD-Mto1 fragments. (E) Summary of localization of Mto1 GFP-insertion and GFP-fusion protein fragments. Blue boxes denote predicted coiled-coil regions; purple box denotes MASC. CM1 and Mto2-binding regions of Mto1 are required for association with γ-tubulin complex and microtubule nucleation [13]. Additional data are shown in Figure S2. Scale bars represent 10 μm.

Figure 3

Mto1 Localization to eMTOCs Depends on Interaction with Type II Myosin Myp2 (A) Localization of GFP-Mto1(784-1115) to eMTOCs in binucleate cells treated with latrunculin B (LatB) or control (DMSO). (B) Mto1-GFP (green) colocalizes with the contractile actin ring (CAR) component Rlc1-mCherry (mCh, red) in dividing wild-type but not _myp2_Δ cells. SPB marker Cut12-tdT is also in red. (C) Colocalization of Mto1-CFP (green) with Myp2-YFP (red) in dividing cells. (D) Absence of GFP-VTD-Mto1(1028-1095) (green) from eMTOC sites in _myp2_Δ cells. Rlc1-mCh and Cut12-tdT are shown in red. (E) Coimmunoprecipitation of myc-tagged Myp2 with GFP-VTD-Mto1(1028-1095) by anti-GFP antibody. Western blots were probed with anti-myc (top) and anti-GFP (bottom). Lane 1: GFP-VTD-Mto1(1028-1095), Myp2-myc; lane 2: GFP-VTD-Mto1(1049-1095), Myp2-myc; lane 3: GFP-VTD-Mto1(1028-1095), Sid4-myc; lane 4: GFP-VTD-Mto1(1028-1065), Sid4-myc. Asterisks indicate full-length Myp2-myc and a C-terminal degradation product. Additional data are shown in Figure S3. Scale bars represent 10 μm.

Figure 4

Sid4 and Cdc11 Regulate Mto1 mSPB Localization Independently of Their Role in the Septation Initiation Network (A) Absence or near absence of Mto1-GFP from mSPBs in multinucleate mitotic _cdc11_Δ and _sid4_Δ cells (left), with spindles and/or spindle poles shown underneath (mCh-Atb2, Cut12-tdT). Mononucleate cells from the same culture (e.g., cells that have not lost rescuing plasmids) retain Mto1-GFP at SPBs (right). (B) Quantitation of Mto1-GFP mSPB signal in early-to-mid mitotic cells from the experiment in (A), scoring mononucleate one-spindle (1sp) and multinucleate two-or-more-spindle (≥2sp) cells. Error bars show interdecile range. Orange line shows upper bound (95th percentile) from comparable measurements of non-SPB background areas. (C) Mto1-GFP (green) colocalizes with SPB marker Cut12-tdT (red) at iSPBs in _cdc11_Δ and _sid4_Δ. Enlarged images of each SPB are shown underneath. (D) GFP-VTD-Mto1(1049-1095) has strongly reduced mSPB localization in multinucleate mitotic _sid4_Δ cells. Insets show mononucleate mitotic cell from the same culture. (E) Mto1-GFP is present at iSPBs, but not at mSPBs, in sid4-SA1 mutants at 36°C. (F) Mto1-GFP SPB localization (green) in interphase (I) and mitosis (M) in wild-type and cdc11-123 mutants at 25°C and 36°C. Right column shows merge with SPB marker Sad1-dsRed (red). (G) Mto1-GFP (green) is present at mSPBs in cdc7-24, sid1-239, and sid2-250 mutants at 36°C. RFP-Atb2 spindles are shown in red. (H) Mto1-RFP (green) and Sid2-GFP (red) localize to different equatorial structures during septation. (I) Model for generation of diversity of MTOC by multiple localization signals in the Mto1 C terminus and different cognate _trans_-acting factors. Additional data are shown in Figure S4. Scale bars represent 10 μm, except (C) insets, which represent 2 μm.

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Fission yeast Mto1 regulates diversity of cytoplasmic microtubule organizing centers - PubMed (original) (raw)