Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin.

In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor

Involvement of Girdin in the Determination of Cell Polarity during Cell Migration

Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement

TRIM27/MRTF-B-Dependent Integrin β1 Expression Defines Leading Cells in Cancer Cell Collectives

SummaryFor collective invasion, cancer cells form cohesive groups comprised of leading cells (LCs) at the forefront and following cells (FCs) at the rear. However, the molecular mechanisms that define LCs and FCs remain elusive. Here, we demonstrated that LCs, but not FCs, upregulated the expression of integrin β1 after the loss of intercellular adhesion. The LC-specific expression of integrin β1 was posttranscriptionally regulated by the TRIM27/MRTF-B complex in response to the loss of intercellular adhesion, thereby regulating the stability and translation of integrin β1 mRNA via microRNA-124 in LCs. Accordingly, depletion of TRIM27 and MRTF-B abrogated the upregulation of integrin β1 in LCs and blocked the invasion of cancer cell groups in vitro and in vivo. Therefore, our findings revealed that the specific function of LCs was defined by intrinsic mechanisms related to the presence of the cell’s free surface, providing insights into the regulation of intratumor heterogeneity

Identifying the Par-3 domain responsible for the interaction with Girdin.

<p>A. Fragments of the Par-3 4N domain used in the study are shown. The 4N domain of Par-3 was further divided into three subdomains (4N/1, 4N/2, and 4N/3). The corresponding amino acid numbers for the fragments are indicated in parenthesis. B. HEK293T cells were transfected with the indicated combination of each subdomain of myc-Par-3 4N, GST, and GST-CT. Lysates were precipitated with glutathione-Sepharose beads and eluted proteins were analysed with the indicated antibodies. Par-3 4N and 4N/2 domains that bound to GST-CT are indicated by asterisks. C. Direct interaction between the 4N/2 region of Par-3 and Girdin. Purified recombinant MBP (maltose binding protein)-fusion proteins containing the 4N, 4N/2, and 4N/1 regions of Par-3 (300 pmol) were incubated with 50 pmol of recombinant GST of GST-CT conjugated with glutathione beads for 1 hr at 4°C, washed three times, eluted with 10 mM reduced glutathione, separated on SDS-polyacrylamide gels, and subjected to Western blot analyses using the indicated antibodies. Asterisks, bound MBP-fusion proteins. White asterisks, MBP-fusion protein input. D. Total cell lysates from HEK293T cells that expressed Girdin-V5 and either myc-GST, myc-Par-3, or myc-Par-3 Δ4N/2 were immunoprecipitated with anti-myc antibody, followed by Western blot analyses using the indicated antibodies. myc-Par-3 (wild type and Δ4N/2) and Girdin-V5 are indicated by asterisks and stars, respectively.</p

Defects in cell polarity of neuroblasts migrating through the RMS in Girdin^−/− mice.

<p>A. Upper panel. Schematic illustration of the pathway taken by neuroblasts born in the SVZ that migrate to the OB through the RMS. Lower panels. Nissl-stained brain sections of wild-type (left) and Girdin^−/− (right) P6 mice show significant deficits in the chain migration of neuroblasts through the RMS toward the OB in Girdin^−/− mice. SVZ, subventricular zone; HP, hippocampus; RMS, rostral migratory stream; OB, olfactory bulb. Scale bar, 500 µm. B. Lysates prepared from the brains of wild-type and and Girdin^−/− P2 mice were subjected to Western blot analysis using anti-Girdin antibody. Mr, molecular mass. C, D. Defects in cell polarity of SVZ neuroblasts in Girdin^−/− mice. Sagittal sections through the RMS of wild-type (left panels) and Girdin^−/− (right panels) P15 mice were stained with anti-Dcx (a marker of immature neuroblasts) (red) and anti-γ-tubulin (green) antibodies. MTOC were frequently found in discrete positions in Girdin^−/− mice (yellow arrowheads) in contrast to wild-type mice (white arrowheads), in which most of the MTOC were found in front of the nuclei. Scale bars, 100 µm (upper panels); 20 µm (middle panels). Shown in D is a schematic illustration of the scoring principle: cells with the MTOC located within one third of the cytoplasm relative to the tangential direction were counted. An arrow indicates the direction of the migratory stream. The graph shows the percentage of polarized neuroblasts in wild-type and Girdin^−/− mice. A minimum of 40–50 neuroblasts of randomly picked sections from each animal and at least 200 neuroblasts were analyzed for each group. An asterisk indicates statistical significance (Student's t test; P<0.05).</p

Girdin depletion from MCF10A cells: effects on the formation of polarized acinar structures.

<p>A. Analysis of the efficiency of retrovirus-delivered shRNAs in silencing endogenous Girdin and Par-3 in MCF10A cells. MCF10A cells were infected with retroviruses expressing control, Girdin, and Par-3 shRNAs. Cell lysates were collected seven days post-infection and subjected to Western blot analysis using the indicated antibodies. B. Morphology of acinar or sheet structures formed by control, Girdin, and Par-3 shRNA-expressing MCF10A cells plated on Matrigel for 15 days. The acini (arrowheads, far left panel) or sheet structures (middle panel and far left panels) were labeled for actin (green), GM130 (red), and nuclei (blue) and representative confocal images are shown. The regions within white boxes are shown at a higher magnification in lower panels. C. Numbers of acini formed by control, Girdin, and Par-3 shRNA-expressing MCF10A cells in Matrigel were determined on day 15. The experiment was repeated three times. Data are expressed as the mean ± S.E.M., and comparison between control shRNA and Girdin/Par-3 shRNA was done by Student's t-test. Asterisks indicate significant differences (P<0.05). D. Localization of Par-3, aPKC, and E-cadherin in Girdin-depleted MCF10A cells. Control and Girdin shRNA-expressing MCF10A cells plated on Matrigel for 15 days were fixed and stained with the indicated antibodies (green) and DAPI (blue).</p

Mapping of interacting domains of Girdin and Par-3.

<p>A. Domain structures of human Girdin and Par-3. Fragments and domains of Girdin (upper panel) and Par-3 (lower panel) used in this study are shown. The corresponding amino acid numbers for the fragments and domains are given in parentheses. B. Girdin-binding sites map to the 2N and the 4N domains of Par-3. Lysates from COS7 cells transfected with Girdin-V5 were incubated with purified recombinant Par-3 fragments fused with GST (300 pmol) for 1 hr at 4°C, followed by precipitation with glutathione-Sepharose beads. Coomassie brilliant blue staining (lower panel) shows the input GST-fusion proteins (asterisks) used in the assay. Girdin-V5 that bound to GST-Par-3 2N and 4N is indicated by stars. C. HEK293T cells were transfected with Girdin-V5 and each domain of Par-3, fused to a myc epitope at the N-terminus. Fourty-eight hrs after transfection, the cell were lysed and immunoprecipitated with anti-myc antibody. Immunoprecipitated Par-3 fragments and bound Girdin are indicated by white asterisks and black stars, respectively. D. Requirement of the Girdin CT domain for interaction with Par-3. Lysates from HEK293T cells transfected with the myc-Par-3 4N domain and each domain of Girdin, fused to GST tag at the N-terminus, were precipitated with glutathione-Sepharose beads. The precipitates and the total cell lysates were analysed by Western blot using the indicated antibodies. An asterisk indicates myc-Par-3 4N that bound to GST-Girdin CT. E. No apparent effects of Girdin phosphorylation on its interaction with Par-3. HEK293T cells were transfected with the indicated combination of myc-Par-3, GST, Girdin CT domain fused with GST (GST-CT), and a dominant active form of Akt (Akt DA). Fourty-eight hrs after transfection, the lysates were precipitated with glutathione-Sepharose beads and eluted proteins were analysed by the indicated antibodies.</p

Involvement of Girdin in the Par protein complex.

<p>A. Interaction among exogenously expressed Girdin and Par-3, Par-6, and PKCλ in HEK293T cells. Total cell lysates from HEK293T cells expressing Girdin-V5 and either myc-GST, myc-Par-3, myc-Par-6, or myc-PKCλ were immunoprecipitated with anti-myc antibody, followed by Western blot analyses with anti-V5 antibody. Asterisks indicated the immunoprecipitated proteins. Coimmunoprecipitated Girdin-V5 was indicated by stars. B. Girdin interacts with Par-3 independently of the kinase activity of aPKC. Total cell lysates from HEK293T cells expressing the indicated combination of plasmids were immunoprecipitated with anti-myc antibody, followed by Western blot analyses with anti-V5 antibody. White asterisks indicated the immunoprecipitated proteins. Stars indicate coimmunoprecipitated Girdin-V5.</p

Involvement of Girdin in the establishment of cell polarity.

<p>A. Girdin depletion inhibited directional cell migration. Control or Girdin-depleted Vero cells (eight dishes for each group) were scratched to induce cell migration and incubated in medium supplemented with 3% FBS for nine hr. The wound's width was measured in each group at zero, seven, and nine hr after the wounding. Asterisks indicate significant difference compared with control cells (Student's t test; P<0.05). B. Schematic illustration of scoring principle of cell polarity in Vero cells. Monolayers of confluent Vero cells on glass-based dishes were scratched to initiate sheet migration into the wound (left), incubated for eight hrs, fixed, and immunostained for GM130 (a marker of the Golgi apparatus). Polarization of leading cells that face the wound was analyzed by the location of the Golgi apparatus (middle). Cells with the Golgi apparatus locating within one third of the cytoplasm relative to the direction of sheet migration were counted (right). C, D. Depletion of Girdin and Par-3 from Vero cells: effects on cell polarization. Monolayers of control- (far left panel), Girdin- (sh1 and sh7, middle panels), and Par-3 shRNA-expressing Vero cells (far right panel) were scratched to induce migration and the polarization of leading cells was evaluated by immunostaining for GM130 (red). Nuclei and actin filaments were visualized by staining with DAPI (blue) and phalloidin (green), respectively. Properly polarized and non-polarized leading cells are indicated by white and yellow arrowheads, respectively. In D, the graph shows the percentage of leading cells with polarized Golgi apparatus in each group (300 cells from three independent experiments). Inset shows the data from Western blot analysis showing stable depletion of endogenous Par-3 by the infection of a retrovirus expressing Par-3 shRNA. Asterisks indicate statistical significance (Student's t test; P<0.05). E, F, G. Overexpression of Par-3 Δ4N and Δ4N/2 mutants in Vero cells: effects on cell polarization. Shown in E is a schematic illustration of Par-3 Δ4N and Δ4N/2 mutants used in the study. Monolayers of control (myc-GST), Par-3 Δ4N-, and Par-3 Δ4N/2-overexpressing Vero cells were scratched to induce sheet migration. The polarization of leading cells, immunostained for GM130 (F), and the percentage of leading cells with polarized Golgi apparatus was quantified in each group (150 cells each) (G). The asterisk indicates statistical significance (Student's t test; P<0.05).</p