title: Microtubule disruption with BAPTA and dimethyl BAPTA by a calcium chelation-independent mechanism in 3T3-L1 adipocytes.
abstract: While the physiological role for calcium in the insulin action on glucose transport has been disputed, it was reassessed in a recent study by using a calcum chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). Although BAPTA has been widely used to study the role for calcium in a variety of cell functions, it has also been suggested to have properties unrelated to the calcium chelating activity. Here, we investigated the effects of BAPTA and dimethyl BAPTA on the cytoskeletons in 3T3-L1 adipocytes. Both calcium chelators were successfully loaded in 3T3-L1 adipocytes and inhibited endothelin-1-induced cytosolic calcium elevation. Confocal fluorescence microscopy revealed that BAPTA and dimethyl BAPTA caused profound depolymerization of the microtubules without affecting the cortical actin filaments in 3T3-L1 adipocytes. Biochemical quantification also showed that BAPTA and dimethyl BAPTA significantly decreased the amount of polymerized tubulin but had little effect on filamentous actin. Consistent with these results, GLUT4-positive perinuclear compartments were dispersed throughout the cytoplasm in BAPTA- or dimethyl BAPTA-loaded adipocytes. Intriguingly, these calcium chelators did not disrupt the microtubules in undifferentiated preadipocytes. The microtubule-depolymerizing property of BAPTA and dimethyl BAPTA is unrelated to calcium chelation, since the microtubules were resistant to depletion of cytosolic calcium by using a calcium ionophore A23187. Insulin-stimulated glucose transport was not affected by cytosolic calcium depletion with A23187, but significantly inhibited with BAPTA and dimethyl BAPTA to the extent similar to that with nocodazole. BAPTA and its derivatives should be used with caution in studies of cytoskeleton-related cell functions.
authors.affiliation: Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Furuta
authors.forename: Marie
authors.initials: M
authors.lastname: Tanaka
authors.forename: Waka
authors.initials: W
authors.lastname: Omata
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagasawa
authors.forename: Itaru
authors.initials: I
authors.lastname: Kojima
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shibata
mesh_terms: D041721:3T3-L1 Cells; D000199:Actins / Q000187:drug effects / Q000378:metabolism; D017667:Adipocytes / Q000187:drug effects; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects*; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism
chemical_list: D000199:Actins; D002614:Chelating Agents; D051275:Glucose Transporter Type 4; C495693:Slc2a4 protein, mouse; D014404:Tubulin; D000001:Calcimycin; D004533:Egtazic Acid; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D005947:Glucose; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium