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Microtubules depolymerization caused by the CK1 inhibitor IC261 may be not mediated by CK1 blockage.

Stöter M, Krüger M, Banting G, Henne-Bruns D, Knippschild U - PLoS ONE (2014)

Bottom Line: IC261 treatment of interphase cells affects the morphology of the TGN and Golgi apparatus as well as the localization of CK1δ, which co-localizes with COPI positive membranes.In summary this study provides additional and valuable information about various IC261-induced effects that could be caused by microtubule depolymerization rather than by inhibition of CK1.Data from studies that have used IC261 as an inhibitor of CK1 should be interpreted in light of these observations.

View Article: PubMed Central - PubMed

Affiliation: Department of General and Visceral Surgery, Ulm University Hospital, Ulm, Germany.

ABSTRACT
The ubiquitously expressed serine/threonine specific casein kinase 1 (CK1) family plays important roles in the regulation of various physiological processes. Small-molecule inhibitors, such as the CK1δ/ε selectively inhibitor IC261, have been used to antagonize CK1 phosphorylation events in cells in many studies. Here we present data to show that, similarly to the microtubule destabilizing agent nocodazole, IC261 depolymerizes microtubules in interphase cells. IC261 treatment of interphase cells affects the morphology of the TGN and Golgi apparatus as well as the localization of CK1δ, which co-localizes with COPI positive membranes. IC261-induced depolymerization of microtubules is rapid, reversible and can be antagonized by pre-treatment of cells with taxol. At lower concentrations of IC261, mitotic spindle microtubule dynamics are affected; this leads to cell cycle arrest and, depending on the cellular background, to apoptosis in a dose-dependent manner. In addition, FACS analysis revealed that IC261 could induce apoptosis independent of cell cycle arrest. In summary this study provides additional and valuable information about various IC261-induced effects that could be caused by microtubule depolymerization rather than by inhibition of CK1. Data from studies that have used IC261 as an inhibitor of CK1 should be interpreted in light of these observations.

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Microtubule destabilizing effect of IC261 in mitotic cells.CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cells were treated with DMSO (0.125%), IC261 (1 µM, 3.2 µM, 50 µM), 10 µM taxol or 0.4 µM nocodazole. Here exemplary cells are shown for indicated time points (see video sequence, movies S3 and S4). Treatment with low concentrations of IC261 induced a depolymerization of spindle microtubules within a few minutes (row 2–3) in a concentration dependent manner and interestingly by nocodazole treatment a similar phenotype could be observed (row 6). Cells entering mitosis during IC261 treatment had spindle poles and microtubule nucleating centers, but could not form a spindle (row 4, arrows indicate spindle poles). Treatment with 50 µM IC261 induced the complete depolymerization of microtubules within a few minutes (3–5 min, row 5). When cells were treated with 10 µM taxol during time period “−10 min” to “0 min” prior to treatment with taxol+IC261 at time point “0 min” the MT depolymerizing effect of IC261 could be blocked (row 7). When cells were first treated for 10 min with IC261 resulting in a complete dissolution of the spindle apparatus, and subsequently treated with taxol+IC261 tubulin could re-polymerize at the spindle poles (arrows) and in other MT nucleation centers within the cell (row 8).
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pone-0100090-g004: Microtubule destabilizing effect of IC261 in mitotic cells.CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cells were treated with DMSO (0.125%), IC261 (1 µM, 3.2 µM, 50 µM), 10 µM taxol or 0.4 µM nocodazole. Here exemplary cells are shown for indicated time points (see video sequence, movies S3 and S4). Treatment with low concentrations of IC261 induced a depolymerization of spindle microtubules within a few minutes (row 2–3) in a concentration dependent manner and interestingly by nocodazole treatment a similar phenotype could be observed (row 6). Cells entering mitosis during IC261 treatment had spindle poles and microtubule nucleating centers, but could not form a spindle (row 4, arrows indicate spindle poles). Treatment with 50 µM IC261 induced the complete depolymerization of microtubules within a few minutes (3–5 min, row 5). When cells were treated with 10 µM taxol during time period “−10 min” to “0 min” prior to treatment with taxol+IC261 at time point “0 min” the MT depolymerizing effect of IC261 could be blocked (row 7). When cells were first treated for 10 min with IC261 resulting in a complete dissolution of the spindle apparatus, and subsequently treated with taxol+IC261 tubulin could re-polymerize at the spindle poles (arrows) and in other MT nucleation centers within the cell (row 8).

Mentions: Whereas DMSO (0.1%) and taxol (10 µM) treatment did not show any effects (figure 2 row 1, 4), IC261 (50 µM) and nocodazole induced the fragmentation of TGN38 structures into vesicles within a few minutes (figure 2 row 2–3), most likely due to the previously described microtubule destabilizing effect of IC261 [48]. When cells were treated in parallel with 10 µM taxol the effect of IC261 could be blocked (figure 2 row 5). We then focused on the characterization of IC261 induced alterations of MT structures in interphase and mitotic cells using CV-1 cells stably expressing EYFP-tubulin (CV-1 766CL5); this was done by time-resolved fluorescence microscopy (figure 3, movie S2). In interphase cells the microtubule network was destabilized by treatment of IC261 (50 µM) and dissolved completely within a few minutes (figure 3 row 2). However, combined treatment of cells with IC261 (50 µM) and taxol (10 µM) nearly completely compensated for the MT destabilizing effect of IC261 (figure 3 row 3). To characterize the effects of IC261 on mitotic spindle microtubules at prophase or pro-metaphase, CV-1 766CL5 cells were observed for 30 min before treatment with DMSO, IC261, nocodazole or taxol (time point “0 min”) and then imaged for at least another 60 min (figure 4, movies S3 and S4). Whereas most DMSO (0.125%) treated cells completed mitosis and cytokinesis within approximately 60 min, IC261 treatment affected mitosis through destabilizing mitotic microtubules in a dose-dependent manner. Even at low IC261 concentrations (1 µM) the spindle structure was apparently altered. The spindle apparatus became smaller, the poles were not clearly detectable and spindle dynamics were impaired (figure 4 row 2) finally leading to an arrest of cells in mitosis. At an IC261 concentration of 3.2 µM the spindle apparatus was considerably dissolved after 3–5 min (figure 4 row 3). Cells entering mitosis during IC261 treatment were unable to build up a spindle apparatus (figure 4 row 4), although spindle poles and several MT nucleating centers with short microtubules were recognizable (figure 4 row 4 arrows). While treatment of cells with higher concentrations of IC261 (50 µM) induced the complete dissolution of the spindle apparatus within a few minutes (figure 4 row 5), prior administration of 10 µM taxol (10 min before IC261 addition) blocked the MT destabilizing effect of IC261 even at high doses of 50 µM. Treatment of cells with taxol alone prevented the formation of a functional spindle apparatus and led to an aggregation of polymerized tubulin in the cell center, which could not be antagonized by IC261 (figure 4 row 7). However, if the spindle was completely dissolved by prior treatment of cells with IC261 (50 µM), subsequent treatment with 10 µM taxol could induce the polymerization of tubulin at the centrosomes and later also at several smaller MT nucleation centers (figure 4 row 8). To compare and quantify the dose-dependent MT depolymerizing effects of IC261 and nocodazole, the relative fluorescence intensity of EYFP-tubulin was measured in a defined region of interest (ROI) around the spindle apparatus and outside this region within the cytoplasm. 3.2 µM IC261 resulted in an almost complete dissolution of the mitotic spindle within a few minutes (figure 4 row 3, which could be quantified as a decrease of relative intensity within the ROI spindle and an increase of relative intensity within the ROI cytoplasm due to depolymerized EYFP-tubulin (figure S4 B, D). Similar results were obtained from measurements of cells treated with MT destabilizing agent nocodazole at 0.4 µM (figure 4 row 6; figure S4 C, E). It should be noted that during the observation time no cell treated with IC261 or nocodazole was able to enter anaphase.


Microtubules depolymerization caused by the CK1 inhibitor IC261 may be not mediated by CK1 blockage.

Stöter M, Krüger M, Banting G, Henne-Bruns D, Knippschild U - PLoS ONE (2014)

Microtubule destabilizing effect of IC261 in mitotic cells.CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cells were treated with DMSO (0.125%), IC261 (1 µM, 3.2 µM, 50 µM), 10 µM taxol or 0.4 µM nocodazole. Here exemplary cells are shown for indicated time points (see video sequence, movies S3 and S4). Treatment with low concentrations of IC261 induced a depolymerization of spindle microtubules within a few minutes (row 2–3) in a concentration dependent manner and interestingly by nocodazole treatment a similar phenotype could be observed (row 6). Cells entering mitosis during IC261 treatment had spindle poles and microtubule nucleating centers, but could not form a spindle (row 4, arrows indicate spindle poles). Treatment with 50 µM IC261 induced the complete depolymerization of microtubules within a few minutes (3–5 min, row 5). When cells were treated with 10 µM taxol during time period “−10 min” to “0 min” prior to treatment with taxol+IC261 at time point “0 min” the MT depolymerizing effect of IC261 could be blocked (row 7). When cells were first treated for 10 min with IC261 resulting in a complete dissolution of the spindle apparatus, and subsequently treated with taxol+IC261 tubulin could re-polymerize at the spindle poles (arrows) and in other MT nucleation centers within the cell (row 8).
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Related In: Results  -  Collection

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pone-0100090-g004: Microtubule destabilizing effect of IC261 in mitotic cells.CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cells were treated with DMSO (0.125%), IC261 (1 µM, 3.2 µM, 50 µM), 10 µM taxol or 0.4 µM nocodazole. Here exemplary cells are shown for indicated time points (see video sequence, movies S3 and S4). Treatment with low concentrations of IC261 induced a depolymerization of spindle microtubules within a few minutes (row 2–3) in a concentration dependent manner and interestingly by nocodazole treatment a similar phenotype could be observed (row 6). Cells entering mitosis during IC261 treatment had spindle poles and microtubule nucleating centers, but could not form a spindle (row 4, arrows indicate spindle poles). Treatment with 50 µM IC261 induced the complete depolymerization of microtubules within a few minutes (3–5 min, row 5). When cells were treated with 10 µM taxol during time period “−10 min” to “0 min” prior to treatment with taxol+IC261 at time point “0 min” the MT depolymerizing effect of IC261 could be blocked (row 7). When cells were first treated for 10 min with IC261 resulting in a complete dissolution of the spindle apparatus, and subsequently treated with taxol+IC261 tubulin could re-polymerize at the spindle poles (arrows) and in other MT nucleation centers within the cell (row 8).
Mentions: Whereas DMSO (0.1%) and taxol (10 µM) treatment did not show any effects (figure 2 row 1, 4), IC261 (50 µM) and nocodazole induced the fragmentation of TGN38 structures into vesicles within a few minutes (figure 2 row 2–3), most likely due to the previously described microtubule destabilizing effect of IC261 [48]. When cells were treated in parallel with 10 µM taxol the effect of IC261 could be blocked (figure 2 row 5). We then focused on the characterization of IC261 induced alterations of MT structures in interphase and mitotic cells using CV-1 cells stably expressing EYFP-tubulin (CV-1 766CL5); this was done by time-resolved fluorescence microscopy (figure 3, movie S2). In interphase cells the microtubule network was destabilized by treatment of IC261 (50 µM) and dissolved completely within a few minutes (figure 3 row 2). However, combined treatment of cells with IC261 (50 µM) and taxol (10 µM) nearly completely compensated for the MT destabilizing effect of IC261 (figure 3 row 3). To characterize the effects of IC261 on mitotic spindle microtubules at prophase or pro-metaphase, CV-1 766CL5 cells were observed for 30 min before treatment with DMSO, IC261, nocodazole or taxol (time point “0 min”) and then imaged for at least another 60 min (figure 4, movies S3 and S4). Whereas most DMSO (0.125%) treated cells completed mitosis and cytokinesis within approximately 60 min, IC261 treatment affected mitosis through destabilizing mitotic microtubules in a dose-dependent manner. Even at low IC261 concentrations (1 µM) the spindle structure was apparently altered. The spindle apparatus became smaller, the poles were not clearly detectable and spindle dynamics were impaired (figure 4 row 2) finally leading to an arrest of cells in mitosis. At an IC261 concentration of 3.2 µM the spindle apparatus was considerably dissolved after 3–5 min (figure 4 row 3). Cells entering mitosis during IC261 treatment were unable to build up a spindle apparatus (figure 4 row 4), although spindle poles and several MT nucleating centers with short microtubules were recognizable (figure 4 row 4 arrows). While treatment of cells with higher concentrations of IC261 (50 µM) induced the complete dissolution of the spindle apparatus within a few minutes (figure 4 row 5), prior administration of 10 µM taxol (10 min before IC261 addition) blocked the MT destabilizing effect of IC261 even at high doses of 50 µM. Treatment of cells with taxol alone prevented the formation of a functional spindle apparatus and led to an aggregation of polymerized tubulin in the cell center, which could not be antagonized by IC261 (figure 4 row 7). However, if the spindle was completely dissolved by prior treatment of cells with IC261 (50 µM), subsequent treatment with 10 µM taxol could induce the polymerization of tubulin at the centrosomes and later also at several smaller MT nucleation centers (figure 4 row 8). To compare and quantify the dose-dependent MT depolymerizing effects of IC261 and nocodazole, the relative fluorescence intensity of EYFP-tubulin was measured in a defined region of interest (ROI) around the spindle apparatus and outside this region within the cytoplasm. 3.2 µM IC261 resulted in an almost complete dissolution of the mitotic spindle within a few minutes (figure 4 row 3, which could be quantified as a decrease of relative intensity within the ROI spindle and an increase of relative intensity within the ROI cytoplasm due to depolymerized EYFP-tubulin (figure S4 B, D). Similar results were obtained from measurements of cells treated with MT destabilizing agent nocodazole at 0.4 µM (figure 4 row 6; figure S4 C, E). It should be noted that during the observation time no cell treated with IC261 or nocodazole was able to enter anaphase.

Bottom Line: IC261 treatment of interphase cells affects the morphology of the TGN and Golgi apparatus as well as the localization of CK1δ, which co-localizes with COPI positive membranes.In summary this study provides additional and valuable information about various IC261-induced effects that could be caused by microtubule depolymerization rather than by inhibition of CK1.Data from studies that have used IC261 as an inhibitor of CK1 should be interpreted in light of these observations.

View Article: PubMed Central - PubMed

Affiliation: Department of General and Visceral Surgery, Ulm University Hospital, Ulm, Germany.

ABSTRACT
The ubiquitously expressed serine/threonine specific casein kinase 1 (CK1) family plays important roles in the regulation of various physiological processes. Small-molecule inhibitors, such as the CK1δ/ε selectively inhibitor IC261, have been used to antagonize CK1 phosphorylation events in cells in many studies. Here we present data to show that, similarly to the microtubule destabilizing agent nocodazole, IC261 depolymerizes microtubules in interphase cells. IC261 treatment of interphase cells affects the morphology of the TGN and Golgi apparatus as well as the localization of CK1δ, which co-localizes with COPI positive membranes. IC261-induced depolymerization of microtubules is rapid, reversible and can be antagonized by pre-treatment of cells with taxol. At lower concentrations of IC261, mitotic spindle microtubule dynamics are affected; this leads to cell cycle arrest and, depending on the cellular background, to apoptosis in a dose-dependent manner. In addition, FACS analysis revealed that IC261 could induce apoptosis independent of cell cycle arrest. In summary this study provides additional and valuable information about various IC261-induced effects that could be caused by microtubule depolymerization rather than by inhibition of CK1. Data from studies that have used IC261 as an inhibitor of CK1 should be interpreted in light of these observations.

Show MeSH
Related in: MedlinePlus