Holland AJ , Reis RM , Niessen S , Pereira C , Andres DA , Spielmann HP , Cleveland DW , Desai A , Gassmann R .

R37 GM029513 NIGMS NIH HHS , R01 GM029513 NIGMS NIH HHS , GM29513 NIGMS NIH HHS , R01 GM074215 NIGMS NIH HHS , P30 NS047101 NINDS NIH HHS , GM074215 NIGMS NIH HHS , NINDS P30 NS047101 NINDS NIH HHS

	FIGURE 1:. Human Spindly interacts with and is modified by farnesyltransferase. (A) Results of mass spectrometric analysis of immunoprecipitates from mitotic HeLa cells with two affinity-purified antibodies raised against nonoverlapping N- and C-terminal epitopes of Spindly (amino acids 2–254 and 450–605). Sequence coverage and spectrum count for selected proteins are shown. (B) Sequence alignment of Spindly C-termini in eukaryotes showing widespread conservation of a putative farnesylation motif that deviates from the canonical CAAX box (Hougland et al., 2009). The amino acid number of the fifth residue from the C-terminus is indicated. The farnesyl cysteine is present in vertebrates (represented by Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio). In the two invertebrate chordate groups, the motif is present in tunicates (Ciona intestinalis) but is absent in amphioxus (B. floridae). Of the two closest nonchordate invertebrate relatives of amphioxus, echinoderms (Strongylocentrotus purpuratus) have the motif but hemichordates do not (S. kowalevskii). We could not find the motif in insects (D. melanogaster), nematodes (C. elegans), or cnidaria (H. magnipapillata). However, the motif appears in the primitive multicellular animal Trichoplax adhaerens, the only extant member of Placozoa, as well as in choanoflagellates (Monosiga brevicollis, Salpingoeca spinifera), the closest unicellular relatives of multicellular animals. The motif is also present in the chytrid fungus Batrachochytrium dendrobatidis. (C) Schematic of experimental approach for the detection of farnesylated Spindly based on in vivo labeling with the unnatural farnesyl diphosphate analogue AGOH and AG-specific antibodies. (D) Immunoblots detecting endogenous and transgenic Spindly and corresponding AG signals in cell lysate input fractions (I) and fractions obtained after immunoprecipitation with anti-Myc tag antibody (Myc) or beads-only controls (Con).
	FIGURE 2:. Farnesylation of Spindly is required for its localization to microtubule-unattached kinetochores. (A–C) HeLa cells immunostained for the kinetochore proteins Spindly (A), CENP-E (B), and CENP-F (C) after treatment for 48 h with 10 μM farnesyltransferase inhibitor FTI-277 or DMSO. Cells were incubated in 1 μM nocodazole for 4 h to maximize the accumulation of the proteins at kinetochores and costained with anti-centromere antibodies (ACAs). Scale bars, 5 μm. (D) Quantification of protein levels at kinetochores in the conditions shown in A–C using immunofluorescence intensity measurements. Each condition represents a total of 100 kinetochore measurements from 20 different cells. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***p < 0.0001). (E, F) HeLa cells immunostained for the kinetochore proteins Zwilch (E) and Hec1 (F) after treatment for 48 h with 10 μM farnesyltransferase inhibitor FTI-277 or DMSO. Scale bars, 5 μm. (G) Kinetochore level quantification of the conditions in E and F displayed as described for D (ns, not statistically significant). (H) Kinetochore localization of RNAi-resistant, MycGFP-tagged wild-type (WT) and mutant (C602S) Spindly in nocodazole-treated HeLa cells after depletion of endogenous Spindly, visualized by immunofluorescence with an anti-GFP antibody (see Figure 5A for corresponding RNAi immunoblot). Scale bar, 5 μm. (I) Kinetochore level quantification of the condition in H displayed as described for D.
	FIGURE 3:. Farnesyltransferase inhibition or prevention of Spindly farnesylation impairs kinetochore recruitment of dynein and its cofactor dynactin. (A–D) HeLa cells immunostained for dynein IC (DIC) and the dynactin subunit p150Glued in cells treated with FTI-277 or DMSO (A, B) and in cells expressing wild-type (WT) and mutant (C602S) MycGFP::Spindly after RNAi-mediated depletion of endogenous Spindly (C, D). Cells were incubated in 1 μM nocodazole for 4 h to maximize the accumulation of the proteins at kinetochores and costained with ACAs. Scale bars, 5 μm. (E, F) Quantification of dynein and dynactin levels at kinetochores in the conditions shown in A–D, as well as in the Spindly mutant C602S after treatment with FTI-277, using immunofluorescence intensity measurements. Each condition represents a total of 100 kinetochore measurements from 20 different cells. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***p < 0.0001; ns, not significant).
	FIGURE 4:. CENP-E farnesylation is important for chromosome segregation. (A) Experimental approach for the characterization of DLD-1 Flp-In T-Rex cells with integrated RNAi-resistant CENP-E constructs whose expression is inducible with tetracycline. (B) Immunoblot showing successful replacement of endogenous CENP-E with RNAi-resistant MycGFP::CENP-E in DLD-1 cells. C2261S corresponds to the CENP-E farnesylation mutant and T93N to motor-dead “rigor” CENP-E. α-Tubulin was used as a loading control. (C) Immunofluorescence of DLD-1 cells showing loss of kinetochore-localized CENP-E 48 h after transfection with CENP-E siRNA. Cells were costained with ACAs. Scale bar, 5 μm. (D) Quantification of mitotic duration (nuclear envelope breakdown to anaphase onset) from time-lapse fluorescence microscopy experiments conducted with DLD-1 cells expressing histone H2b::mRFP with or without the CENP-E transgenes described in B. Mitotic duration is shown as a scatter plot with the average and SD for the indicated experimental conditions. All conditions were filmed in parallel during the same imaging session using a multiwell chamber. One hundred cells were scored for each condition. The t test was used to determine statistical significance (***p < 0.0001). (E) Quantification of mitotic duration for the indicated conditions from a time-lapse experiment, as described in D (ns, not statistically significant). (F) Selected images from a time-lapse series of DLD-1 cells coexpressing histone H2b::mRFP and CENP-E transgenes showing chromosome congression defects in cells expressing nonfarnesylated CENP-E. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Lowercase letters refer to the conditions labeled with the same letters in D and E. Scale bar, 5 μm.
	FIGURE 5:. Preventing farnesylation of Spindly and inhibiting farnesyltransferase result in a similar mitotic delay. (A) Immunoblot showing expression levels of endogenous and RNAi-resistant, transgene-encoded MycGFP::Spindly. Cells were treated with control or Spindly siRNA for 22 h, followed by induction of transgene expression with 0.2 μg/ml tetracycline for 8 h. α-Tubulin was used as a loading control. (B) Quantitative analysis of mitotic duration (nuclear envelope breakdown to anaphase onset) for time-lapse fluorescence microscopy experiments performed with HeLa cells expressing histone H2b::mRFP with or without Spindly transgenes. RNAi and induction of transgene expression was performed as for the immunoblot in A. Data are shown as scatter plots with average and SD. Two independent experiments were performed for each condition, and the total number (n) of cells scored is indicated. The t test was used to determine statistical significance (***p < 0.0001; *p < 0.05). (C) Selected images from the time-lapse experiments described in B, showing chromosome congression defects in cells expressing the Spindly farnesylation mutant C602S. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Scale bar, 5 μm.

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