Protein 4.1R regulates interphase microtubule organization at the centrosome.
In human red blood cells, protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, we analysed the effect of ectopic expression of 4.1R isoforms on interphase microtubules in fibroblastic cells. We found that specific 4.1R isoforms disturbed the microtubule architecture but not the actin cytoskeleton. Biochemical sedimentation and/or confocal microscopy analyses showed that the pericentriolar components gamma-tubulin and pericentrin remained at centrosomes, whereas the distributions of proteins p150Glued and the dynein intermediate chain were altered. Remarkably, 4.1R was displaced from the centrosome. In microtubule depolymerizing-repolymerizing assays, 4.1R-transfected cells showed an ability to depolymerize and nucleate microtubules that was similar to that of untransfected cells; however, microtubules became disorganized soon after regrowth. In microtubule-depolymerized transfected cells and during the initial steps of microtubule regrowth, centrosomal 4.1R localized with gamma-tubulin but did not when microtubules became disorganized. To learn more about centrosomal 4.1R function, isolated centrosomes were examined by confocal microscopy, western blot and in vitro microtubule aster-assembly assays. The experiments showed that 4.1R was present in isolated centrosome preparations, that it remained in the center of in-vitro-assembled microtubule asters and that more asters were assembled by the addition of protein 4.1R fused to glutathione-S-transferase. Together, these results indicate that 4.1R plays a key role at the centrosome, contributing to the maintenance of a radial microtubule organization.
Pérez-Ferreiro
Carmen M
CM
Departamento de Biología Molecular, Centro de Biología Molecular 'Severo Ochoa' (UAM/CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain.
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D000199:Actins / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism* / Q000502:physiology*; D015153:Blotting, Western; D019556:COS Cells; D045744:Cell Line, Tumor; D018385:Centrosome / Q000378:metabolism / Q000648:ultrastructure*; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007399:Interphase; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000378:metabolism
D000199:Actins; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D000072159:Dynactin Complex; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014404:Tubulin; C028841:erythrocyte membrane band 4.1 protein; D005982:Glutathione Transferase; D004398:Dyneins