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. 2016 Sep 14;6:33259.
doi: 10.1038/srep33259.

Hook2, a Microtubule-Binding Protein, Interacts With Par6α and Controls Centrosome Orientation During Polarized Cell Migration

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Free PMC article

Hook2, a Microtubule-Binding Protein, Interacts With Par6α and Controls Centrosome Orientation During Polarized Cell Migration

Emilie Pallesi-Pocachard et al. Sci Rep. .
Free PMC article

Abstract

Polarity protein complexes function during polarized cell migration and a subset of these proteins localizes to the reoriented centrosome during this process. Despite these observations, the mechanisms behind the recruitment of these polarity complexes such as the aPKC/PAR6α complex to the centrosome are not well understood. Here we identify Hook2 as an interactor for the aPKC/PAR6α complex that functions to localize this complex at the centrosome. We first demonstrate that Hook2 is essential for the polarized Golgi re-orientation towards the migration front. Depletion of Hook2 results in a decrease of PAR6α at the centrosome during cell migration, while overexpression of Hook2 in cells induced the formation of aggresomes with the recruitment of PAR6α, aPKC and PAR3. In addition, we demonstrate that the interaction between the C-terminal domain of Hook2 and the aPKC-binding domain of PAR6α localizes PAR6α to the centrosome during cell migration. Our data suggests that Hook2, a microtubule binding protein, plays an important role in the regulation of PAR6α recruitment to the centrosome to bridge microtubules and the aPKC/PAR complex. This data reveals how some of the polarity protein complexes are recruited to the centrosome and might regulate pericentriolar and microtubule organization and potentially impact on polarized migration.

Figures

Figure 1
Figure 1. Hook2 controls Golgi orientation during polarized cell migration.
(a) MCF7 cells were transiently transfected with siCT and siHook2 (H1 + H2) and motility of the transfected cells was illustrated by phase contrast images just after a wound healing assay (0 hour) and after 20 hours on cell monolayer (Bar 40 μm). (b) The estimation of cell-migration capacity was obtained by measurement of the distance covered 5, 10, 15 and 20 hours after scratching. (n = 3; *p = 0.0206 for 5 hours; *p = 0.0430 for 10 hours; *p = 0.0129 for 15 hours; **p = 0.0055 for 20 hours). (c) Golgi reorientation was analyzed after wounding for each of siCT or siRNA Hook2 (H1, H2, H1 + H2) transfected MCF7 cells in the same conditions as above. Cells were allowed to migrate for 15 hours (Bar 10 μm). Note in Hook2 depleted cells that the Golgi apparatus is compacted compared to control cells. Images shown are representative of siCT and siHook2 (H1 + H2) cells. The arrow shows the direction of cell migration. Schematic indicates analysis method to determine Golgi apparatus offset. (d) The percentage of cells with the Golgi apparatus in the forward facing 120 °C sector was measured in first row of cells adjacent to the scratch. For each experiment, 100 cells were scored. When the Golgi apparatus position could not be determined it was categorized as such (N.D.). Results shown are the mean of 3 independent experiments (n = 306 cells; *p = 0.0158; **p = 0.0085) for siCT and siHook2 (H1 + H2).
Figure 2
Figure 2. Hook2 is essential for PAR6α localization at the centrosome in migrating MCF7 cells.
(a,b) MCF7 cells transiently transfected with siRNA control (siCT) or Hook2 (siHook2) for 3 days were co-stained with antibodies against γ-tubulin (to visualize the centrosome), TGN46 (to visualize the Golgi apparatus and more precisely its compaction due to Hook2 depletion) and Hook2 (in a) or PAR6α (in b) after methanol fixation. The arrowheads point to the centrosome and arrows indicate direction of migration. Bars = 10 μm and insert magnification, x5000. In the b lower panel, the asterisk indicates one cell non-transfected by siHook2 seen by the dispersed TGN46 labelling, note presence of PAR6α at the centrosome of this cell in comparison of its absence at the centrosome of adjacent transfected cell. (c,d) Quantification of immuno-localization of Hook2 (c) or PAR6α (d) at the centrosome in migrating transfected MCF7 cells (siCT and siHook2) in 3 independent experiments (****p < 0.0001; **p = 0.0087; *p = 0.0204).
Figure 3
Figure 3. Hook2 interacts with PAR6α.
(a) HEK cells transiently transfected with HA::PAR6α and/or human Hook2 plasmids were lysed (Lysate) after one day and their products were immunoprecipitated with mouse anti-HA antibodies (IP-HA) or rat anti-Hook2 (IP-Hk2) and processed for immunoblotting with rabbit anti-Hk2 and anti-HA antibodies. Asterisks indicate HA-PAR6α co-immunoprecipitated with overexpressed human Hook2 or endogenous human Hook2. (b) Hook2 Cter domain (amino acids 438 to 719) interacts with PAR6α Nter domain in two-hybrid assays. Hook2 Cter interacts (+) with PAR6α WT, Nter, and ΔPDZ but not (−) with PAR6α Cter and PAR6 PDZ motif.
Figure 4
Figure 4. Hook2 acts as an adaptor for the PAR complex.
(a) Hook2 mediates aPKC binding to PAR6α. siCT or siHook2 RNA transiently transfected MCF7 cell (3 days) lysates were used to perform GST-pull down assays with the GST-PAR6α Nter (wild type or K19A) fusion proteins (called GST-PAR6α Nter and GST-PAR6α Nter K19A) or GST alone. The Ponceau red-stained blots (lower panel) show the relative quantity of GST constructs used in each lane. (b) Exogenous mouse Hook2 recruits the PAR6α/PAR3/aPKC complex. MCF7 cells were fixed by ethanol/acetic acid after transient overexpression of mouse Hook2 (mHk2) for 16 hours. mHk2 is visualized with Hook2 rat antibodies (in green) and PAR6α or aPKC or PAR3 or PCM1 by rabbit polyclonal antibodies (in red). Note that PCM1 does not accumulate at the aggresomes in contrast to PAR6α, PAR3 and aPKC. Bars = 10 μm.
Figure 5
Figure 5. Hook2 is essential for PAR3 localization at the centrosome in MCF7 cells.
(a,b) MCF7 cells transiently transfected with siRNA control (siCT) or Hook2 (siHook2) for 3 days were co-stained with antibodies against γ-tubulin, TGN46 and PAR3 after methanol fixation in migrating (a) or resting (b) MCF7 cells. The arrowheads point to the centrosome (in a,b) and arrows indicate direction of migration (in a). Bars = 10 μm and insert magnification, x5000. (c,d) Quantification of immuno-localization of PAR3 at the centrosome in migrating (c) or resting (d) transfected MCF7 cells (siCT and siHook2) in 3 independent experiments (****p < 0.0001).
Figure 6
Figure 6. Model for PAR6α recruitment at the centrosome.
In the migrating cell, Hook2 and PAR6α are represented by red and dark blue color, respectively. The upper part of magnification of centrosomal region illustrates participation of Hook2 in transport of the PAR6 complex to the centrosome via dynein/dynactin complex and in its stabilization. In contrast, the lower part illustrates Hook2-independent targeting of PCM1-PAR6α as suggested by the work of Kodani et al..

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