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1.
FIGURE 9

FIGURE 9. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1 requires WASp for localization to podosome cores, but WASp localizes independently of HS1. HS1−/−, WASp−/y, or DKO cells were untransduced or transduced with Venus-HS1 (HS1−/− and DKO), GFP-WASp (WASp−/y and DKO), or Venus-HS1W465Y (HS1−/−). Cells were prepared as in Fig. 3A but stained with anti-GFP to visualize the transduced proteins. A, Cells were scored for the presence of HS1 in podosome cores as described in Materials and Methods (**p < 0.01). B, Cells were scored for the presence of WASp in podosome cores as described in Materials and Methods. C, Colocalization of HS1 or WASp with phalloidin staining in podosomes cores was visualized by confocal microscopy. Insets, Enlargements of the indicated regions. Scale bars, 10 μm.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
2.
FIGURE 1

FIGURE 1. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1 is the only cortactin family member expressed in murine BMDCs. BMDCs and T cells were cultured from WT or HS1−/− mice, and whole-cell lysates were analyzed by immunoblotting with anti-HS1 or anti-cortactin Abs. Lysates from the nonhematopoietic cell lines 3T3 and 293T were loaded as positive controls for mouse and human cortactin, respectively. Lysate from Jurkat T cells was loaded as a positive control for human HS1. Recombinant human cortactin and HS1 were loaded as positive controls for Ab specificity. GAPDH was used to verify equal loading.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
3.
FIGURE 6

FIGURE 6. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1−/− DCs exhibit increased lamellipodial dynamics. WT and HS1−/− BMDCs were transduced with GFP-Lifeact to facilitate identification of lamellipodial boundaries, and lamellipodial dynamics were monitored by video microscopy as detailed in Materials and Methods. On the basis of the kymographic analysis, the distance (A) and velocity (B) of lamellipodial protrusions and retractions was determined. Box and whiskers plots represent data from 30 lamellipodial protrusions for each cell type with horizontal lines representing median values. *p < 0.05, **p < 0.01, ***p < 0.005.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
4.
FIGURE 4

FIGURE 4. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1 is required for efficient localization and organization of podosome arrays. WT and HS1−/− BMDCs were cultured on coverslips overnight, fixed, and stained with phalloidin (red) and anti-vinculin (green) to visualize podosome cores and rings, respectively. DAPI (blue) was used to stain nuclei. Images were captured by confocal microscopy. A and B, Cells were scored for position and organization of the podosome array as described in Materials and Methods. C, Examples of podosome array localization as quantified in A. D, Examples of tight and loose packing of podosome arrays as quantified in B. C and D, Original magnification ×300.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
5.
FIGURE 5

FIGURE 5. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

Suppression of HS1 perturbs the organization of podosome arrays in RAW macrophages. RAW/LR5 cells were untransduced or transduced with control or shHS1 retrovirus. A, Whole-cell lysates were immunoblotted with anti-HS1 Ab and with anti-GAPDH to verify equal loading. B and C, Cells were cultured on coverslips overnight, fixed, and stained with phalloidin (red) and anti-vinculin (green) to visualize podosome cores and rings, respectively. DAPI (blue) was used to stain the nucleus. Images were captured by confocal microscopy, and the organization of the podosome arrays was determined as described in Materials and Methods. Original magnification ×1000. *p < 0.05.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
6.
FIGURE 8

FIGURE 8. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1 and WASp cooperate to form organized podosome arrays. A, BMDCs were cultured from WT, HS1−/−, WASp−/y, or HS1−/− WASp−/y (DKO) mice, and whole-cell lysates were analyzed by immunoblotting with anti-HS1 or anti-WASp Abs. GAPDH was used to verify equal loading. B and C, WT, HS1−/−, WASp−/y, or DKO cells were untransduced or transduced with Venus-HS1 (HS1−/− and DKO, gray), GFP-WASp (WASp−/y and DKO, hatched), or Venus-HS1W465Y (HS1−/−, white) and prepared as in Fig. 3A. Cells were scored for the presence of podosomes (B) and array organization in cells containing podosomes (C) as described in Materials and Methods. *p < 0.05, **p < 0.01 compared with WT cells.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
7.
FIGURE 7

FIGURE 7. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

The SH3 domain of HS1 binds to the WIP/WASp heterodimer. A, WASp and WIP interact with the HS1 SH3 domain. Cells were transfected with empty vector or FLAG-tagged WIP (F.WIP) or WASP (F.WASP), and lysates were incubated with GST-tagged HS1 SH3 domain (SH3) or GST alone. Bound WIP and WASP were detected via anti-FLAG immunoblot. Bottom panel, Coomassie stain. B, WASp and WIP coimmunoprecipitate with HS1. Cells were cotransfected with myc-WIP and HA-WASp, together with FLAG-tagged full length HS1 (F.HS1), the C-terminal half of HS1 (F.HS1ΔN), or the corresponding SH3 domain mutants (F.HS1 W→Y or F.HS1ΔN W→Y). Lysates were immunoprecipitated with anti-FLAG and blotted with the indicated Abs. C, HS1 does not interact with a WASp mutant that cannot bind WIP. Cells were transfected with FLAG-tagged WASp or with a mutant that fails to bind WIP (Δ40-154). Lysates were incubated with GST-tagged HS1 SH3 domain and blotted with anti-FLAG. Bottom panel, Coomassie stain. D, HS1 binds to WIP mutants that cannot bind WASp. Cells were transfected with FLAG-tagged WIP or with mutants that fail to bind WASp (P436A or Δ460). Lysates were incubated with GST-tagged HS1 SH3 domain and blotted with anti-FLAG or immunoprecipitated with anti-FLAG and blotted with the indicated Abs. Arrow, FLAG-WIPΔ460; HC, IgH. E, WIP interacts directly with the HS1 SH3 domain. Cells were transfected with empty vector or FLAG-tagged WIP, and lysates were probed by gel overlay with recombinant GST alone, WT GST-HS1 SH3 domain, or the SH3 domain mutant.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
8.
FIGURE 2

FIGURE 2. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1 colocalizes with F-actin in structures associated with cell migration. A and B, BMDCs were cultured overnight on coverslips, fixed, and stained with anti-HS1 (mAb 3A3, green) and phalloidin to visualize F-actin structures (red). Colocalization of HS1 with F-actin in podosomes (A) and lamellipodial edges (B) is shown. C, To verify Ab specificity, BMDCs from WT or HS1−/− mice were labeled with the indicated Abs (green), together with phalloidin (red). Note that the anti-cortactin Ab GK-18 labels podosomes in WT but not HS1−/− cells, whereas the more specific Ab 4F11 fails to label actin-rich structures, even in WT BMDCs. Both anti-cortactin Abs give a fine punctate pattern in WT and HS1−/− DCs, and 4F11 labels the nuclear envelope. Given the reactivity of these two Abs by Western blot and the fact that these structures do not colocalize with F-actin, this is likely to be nonspecfic labeling. Scale bars, 10 μm. Insets, Higher magnification views of the boxed areas.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
9.
FIGURE 3

FIGURE 3. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

Podosome biogenesis and turnover in HS1−/− BMDCs. A, WT and HS1−/− BMDCs were cultured on coverslips overnight, fixed, and stained with phalloidin (red) and anti-vinculin (green) to visualize podosome cores and rings, respectively. DAPI (blue) was used to stain nuclei. Images were captured by confocal microscopy. Arrows mark the leading edge of the cells. Scale bars, 10 μm. B, Cells were prepared and imaged as in A. Podosome-containing cells were chosen at random, and the number of podosomes per cell was counted as described in Materials and Methods. Each dot represents a single cell. C, WT and HS1−/− BMDCs cultured overnight on coverslips were treated for 30 min at 37°C with cytochalasin D, after which time the drug was washed out, and cells were allowed to recover. At the indicated times, cells were fixed and labeled with phalloidin and anti-vinculin, and the percentage of cells containing podosome arrays was determined. (n ≥ 200 cells/time point). D, Podosome lifetimes were calculated as described in Materials and Methods. Data represent >4000 podosomes from 15 cells of each type with medians indicated with a line. **p < 0.01, ****p < 0.0001.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.
10.
FIGURE 10

FIGURE 10. From: Hematopoietic Lineage Cell-Specific Protein 1 Functions in Concert with the Wiskott-Aldrich Syndrome Protein To Promote Podosome Array Organization and Chemotaxis in Dendritic Cells.

HS1−/− BMDCs show altered migration in a chemokine gradient. A and B, WT and HS1−/− BMDCs were treated with 100 ng/ml LPS (mature) or left untreated (immature). Migration toward media alone or media containing CXCL12 (A) or CCL19 (B) was assessed by Transwell assay. The percentage of cells that migrated was determined by dividing the number of cells in the bottom well by the number of input cells. Data are means 6 SD of replicates from one representative experiment (out of four). C-E, WT, HS1−/−, WASp−/y, or DKO BMDCs were injected into a fibronectin-coated microfluidic chamber with a gradient of CCL19 (0–20 nM). Cells were allowed to settle and loosely adhered cells were cleared from the chamber. Phase contrast images were collected every minute for 1 h, migration was analyzed, and trajectory plots of 100 cells were analyzed with positive movement along the y-axis corresponding to movement up the gradient (C). The average velocity of motile cells in C was determined (D). The chemotactic index, defined as the distance migrated toward the chemokine source divided by the absolute distance traveled for each motile cell in C, was calculated (E). Data represent averages of multiple cells ± SEM. ***p < 0.00001.

Deborah A. Klos Dehring, et al. J Immunol. 2011 April 15;186(8):4805-4818.

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