SH1 does not interfere with Mklp2/MPP1 independent cellular processes

SH compounds inhibit Mklp2 and MPP1 in vitro and induce a binuclear phenotype in vivo.

Since both proteins are reportedly required for normal progression through cytokinesis (Abaza et al., 2003; Hill et al., 2000), it was plausible that Mklp2 and MPP1 are the relevant targets of SH1 and SH2 in vivo. To collect further evidence that SH1 and SH2 are selective

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in their mode of action, we analyzed cellular structures and processes known to be independent of Mklp2 and MPP1. Due to the similar characteristics of all SH compounds in vitro and in vivo, we focused on the initially identified molecule SH1 for these analyses.

The cytoskeleton of the cell, composed of actin and MTs, is the structural basic of many cellular processes including cytokinesis. Its structure is based on its dynamic behavior, as well as the functionality of a number of associated proteins. Immunofluorescence analyses of both actin and MT cytoskeleton in interphase cells revealed no differences between SH1 or DMSO treated cells, excluding that the cytoskeleton or any of the proteins required for its structure is affected by the compound (Figure 34.A).

As MTs and their associated motor proteins are involved in the correct cellular localization of organelles such as lysosomes or the Golgi apparatus (Hirokawa, 1998), we tested whether SH1 affects Golgi or lysosome structures in cells. While the disassembly of the MT network by nocodazole resulted in fragmentation and displacement of these structures, the localization of the Golgi apparatus and the lysosomes in SH1-treated cells was similar to the one in the DMSO control (Figure 34.B). Thus, these results imply that none of the proteins involved in the assembly and localization of these MT dependent organelles is affected by SH1.

Cell migration is a process that requires the interplay between actin, MTs and their associated proteins, including motor proteins, to guide the movement of a cell in a specific direction (Ridley et al., 2003). To analyze if any of the proteins, involved in this process is affected by the treatment of cells with SH1, we induced a cut in the cell layer of compound treated BSC-1 cells and monitored the time required to close this gap (wound healing assay). (Rodriguez et al., 2005). While cells treated with nocodazole were not able to close the gap within 22 hours after applying the cut, there was no obvious difference in the efficiency in healing when comparing SH1 or DMSO treated cells (Figure 34.C). Thus SH compounds do not interfere with the function of any protein that is required for this process.

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Figure 34. Specificity analysis of SH1.

(A) SH1 does not affect the interphase cytoskeleton. Immunofluorescence analysis of BSC-1 cells treated with 100 µM SH1 or DMSO as solvent control. Tubulin is shown in green (upper panel), actin in red (lower panel) and DNA in blue. (B) SH1 does not affect the localization of the golgi apparatus and lysosomes. BSC-1 cells treated with the indicated concentrations of SH1, nocodazole, or DMSO as solvent control were fixed and analyzed by immunofluorescence. GM-130 as marker protein for the golgi apparatus is shown in green (upper panel) and calnexin as marker for lysosomes in red (lower panel) and DNA is shown in blue. Cells in the lower panel were also stained for α-tubulin. (C) SH1 does not affect wound healing. A cut was introduced in a layer of BSC-1 cells

treated with the indicated compounds (0 h) and the ability to close this gap was monitored at 1 h, 12 h and 20 h.

(D) SH1 treatment neither causes a mitotic arrest nor overrides the SAC. Hela cells were treated with the combinations of the indicated compounds after the release from thymidine and fixed after 14 h. The percentage of interphase cells with one nucleus (mononucleated) and more than one nucleus (bi-/multinucleated) as well as the percentage of mitotic cells was determined. Error bars represent SD.

The SAC senses incorrect attachment of chromosomes to the mitotic spindle apparatus and delays anaphase onset until error free segregation of the genome into the daughter cells can be ensured (Musacchio and Salmon, 2007). Since neither Mklp2 nor MPP1 have a described role during early mitotic progression, SH treatment should not lead to defects in this process. In order to prove this, compound treated HeLa cells were released for 16 h from a thymidine arrest and the mitotic index, describing the ratio of cells in mitosis compared to other stages of the cell cycle, was determined by immunofluorecence. As expected, VS-83 as well as

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nocodazole treatment, known to delay mitotic progression by the activation of the SAC, lead to an increase in the mitotic index (Figure 34.D). In contrast, SH1-treated cells progress normally through mitosis and the mitotic index was similar to that of cells treated with DMSO as solvent control (Figure 34.D). Moreover, there was a clear increase in the number of binucleated interphase cells after of SH treatment confirming that the compounds were active under the experimental conditions (Figure 34.D). Thus we can rule out that SH drugs interfere with the functionality of proteins known to be required for normal mitotic progression.

Defects in chromosome segregation can induce the formation of binucleated cells (King, 2008). Thus, we investigated whether the SH1-induced binuclear phenotype is due to the override of the SAC. In order to test this possibility, the spindle assembly checkpoint was activated by either nocodazole or VS-83 and the maintenance of the checkpoint was analyzed in the presence of SH1. As a positive control we used ZM447439 which inhibits Aurora B kinase, whose activity is required to sustain the SAC in the presence of mal- attached chromosomes (Ditchfield et al., 2003). As shown in Figure 34.D, the mitotic index of nocodazole- or VS-83-treated cells treat did not change significantly in the presence of SH1 or DMSO, indicating that the SAC can be efficiently sustained in the presence of SH1. In contrast, ZM447439 clearly affected the mitotic arrest induced by VS-83 but not the treatment of nocodazole arrested cells, as reported (Ditchfield et al., 2003) (Figure 34.D). In summary, SH1 seems not to affect any of the analyzed Mklp2- or MPP1-independent cellular processes.

3.6 Binding of SH compounds to the target proteins is mediated by a kinesin 6-family