Protein 4.1R regulates interphase microtubule organization at the centrosome

Abstract

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 γ-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 γ-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.

Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase

“…We tested our hypothesis by analyzing microtubule regrowth which was similar to that in controls. Interestingly, ectopic overexpression of a variant 4.1R isoform also does not impair microtubule nucleation/regrowth (52). We did find that 4.1R-depleted cells had a loss of focused microtubule arrays, thus confirming that 4.1R is involved in microtubule anchoring at centrosomes.…”

Fodrin in Centrosomes: Implication of a Role of Fodrin in the Transport of Gamma-Tubulin Complex in Brain

“…They participate in the dynamic interrelationships between the centrosome and the cytoarchitecture [24]. Spectrin and dynein is known to involve with early form of vesicles and aid in their transport to respective sites [25].…”

Protein 4.1R regulates cell migration and IQGAP1 recruitment to the leading edge

“…Although the expression pattern of 4.1R in mature mammalian red cells is relatively simple, multiple isoforms of 4.1R with variable N-terminal extensions and internal sequences are expressed in nonerythroid cells, mainly as a result of extensive alternative splicing of the 4.1R-encoding pre-mRNA (Conboy, 1999;Tang et al, 1990). The localization of protein 4.1R in nonerythroid cells is not restricted to the subjacent area beneath the plasma membrane, as 4.1R has also been identified at the nucleoskeleton (De Carcer et al, 1995), the centrosome Perez-Ferreiro et al, 2004), the endoplasmic reticulum (Luque et al, 1999) and microtubules (Perez-Ferreiro et al, 2001), thereby showing that 4.1R functions at multiple sites in the cell. The interaction of protein 4.1R with zonula occludens (ZO)-2 (Mattagajasingh et al, 2000) and with -catenin and E-cadherin (Yang et al, 2009) suggested that 4.1R might act as a linker between tight and adherens junctions and the actin cytoskeleton.…”