AKAP350 enables p150glued /EB1 interaction at the spindle poles
Introduction
P150glued is the main subunit of dynactin, which is a protein complex essential for dynein-mediated retrograde transport of vesicles and organelles along microtubules [1]. By localizing at microtubule plus-ends, p150glued also has a prominent role in the regulation of microtubule dynamic instability [2]. Although p150glued can directly interact with microtubules, its association with microtubule plus-ends is predominantly dependent on its interaction with EB1 [3], a key member of the family of microtubule plus-end tracking proteins [4]. p150glued also interacts with EB1 at the centrosome. This interaction is required for microtubule minus-end anchoring during the assembly and maintenance of the radial microtubule array of interphase cells [5]. EB1 and p150glued also participate in mitotic spindle formation and orientation [6,7] and in the initiation of cytokinesis [8]. Nevertheless, to our knowledge, there is no mechanistic data on how p150glued is recruited to the spindle poles. AKAP350 (AKAP450, AKAP9 or CG-NAP) is an A-kinase-anchoring protein that scaffolds protein complexes at the Golgi apparatus and at the centrosome [9], playing a central role in the regulation of microtubule dynamics [[10], [11], [12]]. AKAP350 interacts with p150glued, which is relevant to AKAP350 localization at the Golgi apparatus [13]. Considering that AKAP350 interacts with EB1 at the spindle poles [14], in the present study, we analysed AKAP350 participation in p150glued localization at the centrosome and in p150glued/EB1 interaction at the spindle poles, and evaluated the relevance of this protein complex in the regulation of astral microtubule dynamics during mitosis.
Section snippets
Cell culture
MDCK cells, obtained from Keith Mostov lab (UCSF, CA), were cultured and tested for contamination [14]. Cells were treated and cell lines were prepared as described in Supplementary Methods.
Immunofluorescence
Cells were grown on glass coverslips, fixed with 4% paraformaldehyde or 100% methanol, permeabilized and blocked [14]. Cell staining was performed as described in Supplementary Methods.
Image acquisition and analysis
Images of p150glued or EB1 and γ-tubulin or α-tubulin co-staining were obtained by confocal laser microscopy (Nikon C1SiR
AKAP350 facilitates p150glued localization at the spindle poles
MDCK cells with decreased AKAP350 expression (AKAP350KD) were generated using a lentiviral-based short hairpin RNA expression system. Western blot analysis of AKAP350 expression in AKAP350KD cells demonstrated a reduction to 5%–20% of the control levels (Fig. 1A). Subsequently, p150glued levels in centrosome-enriched fractions of control and AKAP350KD cells were analysed by western blot. Our results showed that centrosomal p150glued was dramatically reduced in AKAP350KD cells (Fig. 1B). The
Conclusion
The role of p150glued in the regulation of microtubule remodelling and spindle orientation has been widely characterized [2,[5], [6], [7], [8]]. In this regard, p150glued interaction with EB1 is highly relevant for the regulation of p150glued function [3,[5], [6], [7], [8]]. Even though it has been shown that p150glued localizes at the spindle poles [18], the factors governing p150glued localization and interaction with EB1 at these structures remained unknown. In the present study, we describe
Author contributions
Conceived and designed the experiments: EA, MCL. Performed the experiments: EA, AP, TRM, FH, RV. Supervised the experiments: CF, MCL. Analysed the data: EA, TRM, MCL. Wrote the paper: EA, MCL. All authors read and approved the final manuscript.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
We thank A. B. Almada for the language correction of the manuscript.
This work was supported by Grants PUE 0089 from CONICET and PICT2015-2755 from ANPCyT to MCL. The funders had no role in study design, data collection and analysis, decision to publish, or paper preparation.
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