Limits...
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.

Show MeSH

Related in: MedlinePlus

Microtubule depolymerization by IC261 treatment is reversible.(A) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S5). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). (B) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4061085&req=5

pone-0100090-g005: Microtubule depolymerization by IC261 treatment is reversible.(A) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S5). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). (B) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.

Mentions: Nocodazole mediated effects are known to be readily reversible [66], [67], raising the question whether this is also the case for IC261. CV-1 766CL5 cells were treated for 10 min with 3.2 µM IC261 and investigated by time-resolved fluorescence microscopy (figure 5, movie S5). Almost immediately after administration of IC261 the spindle apparatus dissolved, but removing IC261 led to the restoration of the spindle apparatus within 20 min (time point “30 min”, figure 5 A). 140 min after removal of IC261 cells completed their cell division by cytokinesis indicating that IC261 induced MT depolymerization and mitotic arrest for a short period of time is reversible.


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 depolymerization by IC261 treatment is reversible.(A) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S5). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). (B) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061085&req=5

pone-0100090-g005: Microtubule depolymerization by IC261 treatment is reversible.(A) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S5). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). (B) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.
Mentions: Nocodazole mediated effects are known to be readily reversible [66], [67], raising the question whether this is also the case for IC261. CV-1 766CL5 cells were treated for 10 min with 3.2 µM IC261 and investigated by time-resolved fluorescence microscopy (figure 5, movie S5). Almost immediately after administration of IC261 the spindle apparatus dissolved, but removing IC261 led to the restoration of the spindle apparatus within 20 min (time point “30 min”, figure 5 A). 140 min after removal of IC261 cells completed their cell division by cytokinesis indicating that IC261 induced MT depolymerization and mitotic arrest for a short period of time is reversible.

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