Fission Yeast Mto1 Regulates Diversity of Cytoplasmic Microtubule Organizing Centers

Summary 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 [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.

(E) Mto1 is co-immunoprecipitated with Myp2-YFP. Anti-GFP immunoprecipitations from indicated strains, probed with antibodies to Mto1 and GFP. "Cell extract" lanes (left) are loaded with equal amounts of cell extract. To compensate for different endogenous expression levels of YFP-and GFP-tagged proteins, "anti-GFP IP" lanes (right) are loaded with different amounts of cell-extract equivalents. "1X" indicates 100-fold loading relative to cell extracts; "0.1X" indicates 10-fold loading relative to cell extracts, etc. Ain1 (alphaactinin) was independently found to interact with Mto1 in a yeast two-hybrid screen (not shown). Asterisks indicate full-length Myp2-YFP and major degradation products. Molecular weights are shown in kDa. All bars, 10 µm. Semi-quantitative assays using ortho-nitrophenyl beta-galactoside (ONPG) as substrate. Mto1 fragments that interact with Cdc11 correspond to those that localize to mSPBs (Fig. 1). Truncation of the Cdc11 fragment from either end abrogates the interaction. (I) Position of leucine-rich repeats in Cdc11 C-terminus. While the Cdc11 C-terminus has been noted to contain leucine-rich repeats [31], our analysis suggests that these repeats are more extensive and continuous than previously recognized. Relevant amino acids are colorcoded by similarity. Leucine-rich repeats typically fold into a single curved solenoid structure [37,38], which may explain why truncation from either end of the Cdc11 fragment abrogates interaction with Mto1. All bars, 10 µm.

Yeast Strains
Standard fission yeast classical and molecular-genetic techniques were used throughout [39,40]. Table S1 contains a list of strains used. Deletion, truncation and tagging of genes at Nand C-termini were performed using PCR-based targeting methods [41]. Fluorescent tags included GFP as well as dsRed, mCherry (mCh), tandem dimer Tomato (tdT), and tandem dimer 2(12) (td2(12))[34, 42,43]. Additional strains were purchased from Bioneer or derived from strains kindly provided by P. Fantes (University of Edinburgh, UK) T. Pollard (Yale University, USA), K. Gould (Vanderbilt University, USA), and M. Balasubramanian and S. Oliferenko (Temasek Life Science Laboratory, Singapore). Strains were confirmed by PCR and Western blotting as appropriate.
To construct plasmids expressing GFP fused to Mto1-C-terminal fragments under control of the nmt81 promoter [44], specified regions of Mto1 were amplified by PCR and subcloned 3' to the GFP-coding sequence of plasmid pKS71, an enhanced-GFP version of pWGA [45]. mto1∆ strains were used for plasmid transformation. To construct plasmids containing coiledcoil sequences between GFP and Mto1 sequences, synthetic DNA fragments (GeneArt) encoding coiled-coils were inserted at a SacII site in the linker sequence between GFP and Mto1 coding sequence in the above plasmids. Nucleotide sequences for the coiled-coil sequences are (SacII sites underlined): To construct strains with GFP-VTD-Mto1-C-terminal fragments integrated at the mto1 locus (in place of the endogenous mto1+ gene), recombinant genes encoding various GFP-coiledcoil-Mto1-C-terminal fragments were amplified by PCR, using the multicopy plasmids described above as templates. Reverse PCR primers contained 80 nucleotides homology to a region 3' to mto1+. PCR products were used to transform an nmt81:GFP-mto1(1-800):ura4+ strain, followed by 5-FOA selection. Homologous recombination within the GFP coding sequences and within mto1+ 3' sequence replaced the mto1(1-800):ura4+ sequence with coiled-coil-Mto1-C-terminal fragments (confirmed by sequencing).
Phenotypes of cdc11∆ and sid4∆ were analyzed primarily by loss of rescuing plasmids from haploid deletion strains, although in a few instances spore germination of heterozygous deletion strains was used. Heterozygous deletion strains cdc11∆ / cdc11+ and sid4∆ / sid4+ were created by replacing one copy of the respective ORFs with a ura4+ cassette in diploid backgrounds. To generate rescuing plasmids, cdc11+ and sid4+ genes were amplified by genomic PCR and cloned into the S. pombe shuttle vector plasmid pAL-KS (selection for leucine prototrophy; [46,47]). The plasmid pKS560 contains cdc11+ coding sequence and 1091 bp of 5' sequence; pKS1031 contains sid4+ coding sequence and 5' and 3' flanking sequences of 1065 bp and 165 bp, respectively. The heterozygous deletion strains were transformed with the appropriate plasmids and sporulated to generate haploid deletion strains containing the rescuing plasmid. For plasmid loss, haploid deletion strains containing the rescuing plasmid were cultured in medium supplemented with leucine, to allow growth of auxotrophs. For spore germination experiments (Suppl. Fig. 4C, 4D) heterozygous diploid cells (without rescuing plasmids) were sporulated on SPA plates. Asci were scraped from plates and treated with 0.2 % helicase. Spores were inoculated in YE5S and incubated at 32°C for 17.5 hrs before imaging.

Imaging and Physiology Experiments
For live-cell microscopy, EMM2 minimal medium with sodium glutamate as nitrogen source was used. Single time-point and time-lapse imaging of GFP-and RFP-fusion proteins was essentially as described previously [48,49]. Cells were mounted on medium-agarose pads and sealed with VALAP before time-lapse imaging. Images were collected on a Nikon TE300 inverted microscope with automated filter and z-axis control, running MetaMorph software (Universal Imaging, Downingtown, PA). Time-lapse images were taken with intervals of 15-30 sec for single-channel and 20-30 sec for two-channel movies. Image sequences were further deconvolved using Softworx (Applied Precision, Issaquah, WA). Temperature-sensitive strains were observed using a heated objective at 36°C (Bioptechs).
For quantitation of Mto1-GFP and truncations at interphase SPBs (Fig. S1E), Sad1-dsRed was used as SPB marker. In some experiments (as indicated), to discriminate Mto1-GFP signal at the SPB from Mto1-GFP signal on microtubules near the SPB, microtubules were disrupted by addition of methyl benzimidazol-2-yl carbamate (MBC; 50 µg/ml final concentration), 30 min prior to imaging. 90-113 cells were scored for each strain. For quantitation of Mto1-GFP and truncations at mitotic SPBs (Fig. S1F), mCherry-tubulin (Atb2) was used both to confirm mitotic state and indicate spindle poles. In experiments with other Mto1-variants (Fig. 2, Fig. S2), additional quantitation of SPB localization was done using either Sad1-dsRed or Cut12-tdT as SPB marker (shown in table below): To score Mto1-GFP and Mto1-427-GFP presence at eMTOC sites as a function of spindle length ( Fig. 1G and additional data not shown), image projections were used to measure the distance between SPBs (marked with Cut12-tdT) in mitotic cells, using MetaMorph software. 72 mto1-GFP and 61 mto1-427-GFP cells were scored. Based on the observed difference in Mto1-GFP localization in wild-type cells with short vs. long spindles (Fig. 1G), we used an SPB-SPB distance of > 8 µm as a criterion for additional quantitation of eMTOC localization of several Mto1-variants (shown in table below).
To measure Mto1-GFP signals at mSPBs in cdc11∆ and sid4∆ strains (Fig. 4A, 4B), the Mto1-GFP signal was measured within a 0.65 µm 2 circular region at the end of short spindles (< 6.5 µm in length, judging by mCherry-tubulin fluorescence). The signal from an identically-sized neighboring region was subtracted to calculate the net SPB-associated signal. 84 mSPBs in 8 cdc11∆ cells with multiple spindles, 22 mSPBs in 11 cdc11∆ cells with one spindle, 124 mSPBs in 14 sid4∆ cells with multiple spindles, and 20 mSPBs in 10 sid4∆ cells with one spindle were scored. Box in Fig. 4B shows median and interquartile range, and whiskers show interdecile range. Orange line shows upper bound (ninety-fifth percentile) from comparable measurements of non-SPB background areas, indicating that the "noise" in these measurements is relatively small.
Assays of microtubule re-growth after cold-shock (Fig. S1J) were as described previously [7]. Exponentially growing cells were chilled in ice water bath for 30 min and transferred to a pre-warmed flask and incubated at 32°C for the specified time before collection by filtration. Cells were fixed in methanol at -70°C and processed for anti-tubulin immunofluorescence, exactly as described previously [7,51].

Astral Microtubules in cdc11∆ Mutants
The initial characterization of cdc11∆ mutants described astral MTs to frequently detach from SPBs during anaphase [30]. However, our analysis of microtubule dynamics in truncation mutants such as mto1(1-1085)-GFP shows that astral MT nucleation is tightly 13 correlated with Mto1 mSPB localization (Fig. 1E, Fig. S1G, S1H). Therefore, our finding that Cdc11 is required for Mto1 mSPB localization would lead us to expect no astral MTs at all in cdc11∆ mutants. To clarify this apparent contradiction we reinvestigated astral MT nucleation in cdc11∆ mutants, using strains expressing GFP-tubulin at physiological concentrations [52], which may not have been used in earlier experiments [30].
We made two relevant observations: First, we found that cdc11∆ multinucleated cells are able to nucleate what appear to be astral MTs, but only in later stages of spindle elongation, and this corresponds with a return of Mto1-GFP to SPBs in cdc11∆ cells (Fig. S4A, S4C,  S4D). A likely explanation for the reappearance of Mto1-GFP at this stage is that, biochemically, these cells are already in the next interphase (i.e., with significantly reduced cyclin-dependent kinase activity), a stage when Cdc11 is no longer required for Mto1 SPB localization. The continued presence of spindles in these cells may also be due to lower-thannormal spindle disassembly rates, as the absence of a stable or well-organized CAR in SIN mutants [36] precludes the generation of robust PAAs [27], and PAAs may normally contribute to spindle disassembly by titrating away tubulin dimers; similarly slow spindle disassembly is observed in mto1∆ mutants [7]. Consistent with this view, we found that Mto1 localization and MT nucleation in mto1(1-1085)-GFP cdc11∆ double mutants were largely indistinguishable from cdc11∆ single mutants, even though mto1(1-1085)-GFP mutants lack the Mto1 mSPB localization signal (Fig. S4B, S4D).
Second, although we did not observe astral MTs detaching from SPBs in cdc11∆ mutants, we found that both cdc11∆ and mto1(1-1085)-GFP cdc11∆ mutants often nucleate MTs very close to but not at SPBs as spindles were elongating (Fig. S4D). This nucleation may occur from the surface of the nuclear envelope and/or the cell cortex, as it was also observed in mutants that lack any SPB-associated Mto1, such as mto1(1-1051)-GFP (Fig. 1D, Fig. S1G, Movie S1). Depending on temporal resolution during imaging, this nucleation could be misinterpreted as released astral MTs [8,53]. Thus overall, the earlier analysis of cdc11∆ mutants [30] can be reinterpreted in a manner that is consistent with our current results.

Biochemical Methods
To assay Mto1 levels in mto1-truncation strains, pelleted yeast cells were boiled for 5 minutes and then disrupted by bead-beating by using 0.5-mm zirconium beads in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1 mM PMSF. Pulverized cells were boiled in Laemmli buffer without reducing agent for 5 min. Protein concentration of the cleared cell extract was measured by BCA assay and equal amounts loaded for SDS PAGE and Western blotting.