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. 2014 Apr 14;24(8):896–903. doi: 10.1016/j.cub.2014.03.006

Figure 1.

Figure 1

Mto1[bonsai], a “Minimal” Mto1 Truncation Mutant, Promotes Spatially Random Cytoplasmic Microtubule Nucleation

(A) Schematic of full-length Mto1 and Mto1 truncation mutants. Dashed lines indicate N- and C-terminal truncation sites. Red boxes indicate predicted coiled coils. Gray boxes indicate regions involved in binding to γ-TuC (centrosomin motif 1; CM1), binding to Mto2, and localization to the equatorial microtubule-organizing center and spindle pole body (eMTOC and SPB). The region involved in binding to microtubules (MTs) is approximately defined [10].

(B) Cartoon summarizing the different means by which Mto1/2 complexes can become enriched on the nuclear envelope.

(C) Localization of mildly overexpressed Mto1-GFP and Mto1[NΔ130]-GFP in control cells (+DMSO) and after MT depolymerization by methyl benzimidazol-2-yl-carbamate (MBC). Arrows indicate Mto1[NΔ130]-GFP at spindle pole bodies, but not more generally on the nuclear envelope (NE).

(D) Anti-tubulin immunofluorescence showing time course and spatial distribution of MT regrowth after cold-induced MT depolymerization in mto1-GFP and mto1[NΔ130]-GFP cells. Arrows indicate regrowth from NE.

(E) Time-lapse images (15 s interval) of GFP-tubulin in an mto1[bonsai]-GFP cell. Arrows indicate de novo MT nucleation events free in the cytoplasm. Circles indicate position of nucleus. Relative to GFP-tubulin, Mto1[bonsai]-GFP is too faint to be seen. See Movie S1 for corresponding movie.

(F) Frequency of de novo MT nucleation (±SEM) free in the cytoplasm versus in the NE region in the cell genotypes indicated (see also Movie S1). The apparent low total MT nucleation frequency in mto1-GFP and mto1[NΔ130]-GFP cells is artifactual; in these cells, a significant proportion of nucleation occurs on preexisting MTs but cannot be quantified and thus is excluded from analysis (see Supplemental Experimental Procedures).

(G) Localization of Mto1[bonsai]-GFP in wild-type (alp16+) and in alp16Δ cells, together with γ-TuC protein Alp4 (GCP2 homolog) fused to tandem-dimer Tomato (Alp4-tdT). Green arrows indicate Mto1[bonsai] puncta without Alp4. Yellow arrows indicate Mto1[bonsai] puncta with Alp4. Red arrows indicate spindle pole bodies (nucleoplasmic face), which contain Alp4 but not Mto1[bonsai]. Scale bars, 5 μm.