This publication raises questions about the specificity of IC261 and the interpretation of the reported effects. The situation is complicated by the fact that several studies have suggested that CK1d/e could be directly involved in microtubule dynamics. CK1d co-localizes with spindle microtubules and phosphorylates a- and b-tubulin in vitro. Furthermore, direct interactions between CK1d and microtubule associated proteins, such as MAP1A, MAP4 and end binding SCH772984 moa protein 1 have been reported. In the present study, re-investigation of the subcellular localization of CK1d using high resolution confocal microscopy revealed that CK1d is located in the perinuclear region close to the TGN and Golgi apparatus, but does not co-localize with these compartments. Instead, CK1d partly co-localizes with COPI positive membranes and b-COP. Further studies of the IC261mediated effects on microtubules showed that high concentrations of IC261 disrupt interphase microtubules, finally leading to a dispersed phenotype of perinuclear membranes compartments. This effect of IC261 can be blocked by pretreatment of cells with taxol. Low concentrations of IC261 disrupt spindle microtubules leading to mitotic arrest, post-mitotic arrest or DAPT apoptosis. The effect of IC261 on microtubules is reversible. These results are in line with the recent finding that IC261 can act as a microtubule depolymerizing agent. Therefore, the effects on cells induced by IC261 should be interpreted carefully as such effects may be due to either inhibition of CK1 or the depolymerization of microtubules, or a combination of the two. The evolutionary conserved serine/threonine-specific kinase family CK1 is involved in a broad range of intracellular processes and can be regulated by intracellular compartmentalization. We here provide evidence that CK1d is localized at perinuclear membrane compartments and co-localizes with b-COP, a subunit of the coatomer protein complex coating COPI vesicles. Treatment of cells with the CK1-inhibitor IC261 induces changes in CK1d localization as well as changes of other membrane compartments such as the TGN and Golgi apparatus, most likely due to depolymerization of microtubules. The aim of the present study was to unravel the various effects of IC261 described in recent years on CK1d, on microtubule dynamics, and on membrane transport processes. Since it has been reported that CK1d is localized on several intracellular membrane compartments, e.g. TGN or GA, we investigated the subcellular localization of CK1d by fluorescence microscopy at high resolution and found that CK1d neither co-localizes with the TGN nor GA structures, but is in close proximity to both compartments. This finding was confirmed by using multiple antibodies for CK1d and for typical TGN and GA markers in two rat cell lines. Whereas the GA and TGN compartments looked like the well-known stack of cisternae, CK1d-positive structures appeared more vesicular and in close proximity to the TGN and GA. Furthermore CK1d seemed to be closer to the GA markers than the TGN marker. Interestingly, CK1d showed partial co-localization with bCOP positive vesicles. b-COP is a subunit of the coatomer complex coating COPI vesicles, which are responsible for retrograde GA-to-ER or intra-GA membrane transport processes. The hypothesis that CK1d could be 
involved in GA-ER transport is supported by CK1d co-localizes with another coatomer protein b’-COP, and by the report of CK1d regulating membrane binding of ARF GAP1 – a protein stimulating GTPase activity of ARF1, which is required for the uncoating of COPI vesicles. However, in the latter report IC261 was used at high concentration for experiments in cells. The authors argue that in vitro experiments use a lower ATP concentration, whereas intracellular ATP concentrations in vivo are higher.