Journal: bioRxiv

Article Title: Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

doi: 10.1101/2021.01.20.427433

Figure Lengend Snippet: ( A ) Protein schematic of XRCC4 highlighting key structural and functional features. S: SUMOylation; P: phosphorylation; Ub: ubiquitylation; NLS: nuclear localisation signal. ( B ) Schematic of DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). ( C ) SPR sensorgrams showing preferential binding of XRCC4 to polySUMO2 chains (top) over SUMO monomers (mSUMO1, mSUMO2) and dimers (diSUMO2; bottom), using XRCC4 as ligand and SUMO topologies as analytes. Association and dissociation phases are separated by vertical dashed lines, as indicated. ( D ) Equilibrium analysis of SPR response unit maxima of polySUMO2 binding to immobilised wildtype (WT) and mutant versions of full-length XRCC4, showing binding of XRCC4 to polySUMO2 and polySUMO3 chains with similar affinity. ( E ) SPR sensorgram showing no or minor detectable binding of XRCC4 to ubiquitin monomers (mUb) and tetra-ubiquitin chains linked via ubiquitin’s internal lysines K63, K48, K33, K11, K6 or its N-terminal methionine (M1), using XRCC4 as ligand and ubiquitin topologies as analytes. Association and dissociation phases are separated by vertical dashed lines, as indicated. ( F ) XRCC4 interacts with 4xSUMO2 (6xHis-4xSUMO2-Strep) in solution in vitro. ( G ) XRCC4 preferentially associates with SUMO2 chains over SUMO2 monomers in solution as shown by GST-mSUMO2 and GST-4xSUMO2 pulldowns co-precipitating GFP-XRCC4 ectopically expressed in HEK293T XRCC4 −/− cells in the absence or presence of DNA damage induced by ionizing radiation (IR; 15 Gy, ~15 min). Inputs were 4% of the total. ( H ) GST-4xSUMO2 pulldowns co-precipitate endogenous XRCC4 from HEK293T nuclear extracts in the absence or presence of IR-induced DNA damage (15 Gy, ~15 min). K D : dissociation constant; RUs: response units; S2: SUMO2; SPR: surface plasmon resonance.

Article Snippet: Pulldowns with cellular extracts were carried out as described for GFP-immunoprecipitations using 1-2 μg of GST-fused proteins bound to glutathione magnetic beads (Promega).

Techniques: Functional Assay, Non-Homologous End Joining, Binding Assay, Mutagenesis, In Vitro, SPR Assay

Journal: bioRxiv

Article Title: Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

doi: 10.1101/2021.01.20.427433

Figure Lengend Snippet: ( A ) XRCC4 1-180 residues implicated in SUMO2 monomer (mSUMO2) binding, as indicated by intensity losses in the 1 H- 15 N BEST-TROSY spectra of XRCC4 1-180 after addition of increasing concentrations of mSUMO2. Error bars represent 1 standard deviation from the plotted value, as calculated from the noise levels in the TROSY spectra using the standard error propagation formula. Grey bars indicate residues assigned in XRCC4 1-180 with too low intensity for reliable measurement (arbitrarily set to 1); lack of bars represents residues that could not be assigned. Red and blue bars indicate assigned residues in XRCC4 1-180 with reliable intensity measurements and levels of intensity ratio loss of more or less than 30%, respectively. ( B ) Representative panel of 1 H- 15 N BEST-TROSY spectra of XRCC4 1-180 without (red) and with (blue) 1 molar equivalent of mSUMO2 added. ( C ) mSUMO2 residues implicated in full-length XRCC4 (XRCC FL ) binding, as indicated by intensity losses in the 1 H- 15 N HSQC spectra of mSUMO2 after addition of 0.33 molar equivalents of XRCC4 FL . Colour gradient for mSUMO2 structure (PDB 2N1W) ranges from red (most affected by binding) to blue (unaffected by binding). For details see Methods section. Unassigned prolines shown in black, other unassigned/overlapping residues in grey. ( D ) Electrostatic surface potential of SUMO2 created using APBS electrostatics plugin for Pymol . Dashed line highlights key residues implicated in binding to XRCC4 FL according to (B) and Supplementary Figure S8A. Electrostatic potential values are multiples of kT/e, where kb is the Boltzmann's constant, T the temperature the calculation was run at (300 K) and e the charge of an electron, leading to a conversion factor of 25.85 mV. ( E ) SUMO2 (top), LIG4 (middle), and IFFO1 (bottom) binding to overlapping or spatially proximal regions on XRCC4 coiled-coil (PDB 1IK9 for top and middle and PDB 6ABO for bottom). SUMO2-binding region depicted on XRCC4 entails 163-EKCVSA-168 comprising residues with >30% intensity loss as well as their unassigned neighbouring residues according to (A). LIG4- and IFFO1-binding regions encompass XRCC4 residues 173-195 and 162-196, respectively , . ( F ) Precipitation of LIG4 by GFP-Trap pulldowns of GFP-XRCC4, ectopically expressed in HEK293T cells, from whole cell extracts after addition of increasing amounts of 4xSUMO2. Band intensities are averaged from two independent biological replicates and normalised to pulled-down XRCC4 levels. ( G ) GFP-XRCC4 pulled down with GST-4xSUMO2 from HEK293T whole cell extracts contains negligible amounts of LIG4. Inputs were 4% of the total. ( H ) GFP-Trap pulldowns of GFP-XRCC4 expressed in HEK293T cells contain a substantial fraction of the total LIG4 pool available in cells. Inputs were 4% of the total. S2: SUMO2.

Article Snippet: Pulldowns with cellular extracts were carried out as described for GFP-immunoprecipitations using 1-2 μg of GST-fused proteins bound to glutathione magnetic beads (Promega).

Techniques: Binding Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: DAAM1 activation is required for collagen-induced cell haptotaxis. A–D, type IV collagen triggered haptotaxis of MDA-MB-231 and MDA-MB-453 breast cancer cells. MDA-MB-231, MDA-MB-453, and MCF10A cells were examined for cell haptotaxis for 8 h in 8.0-μm porous Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Migratory cells on the lower side of the membrane were counted per field of the microscope. Bar, 50 μm. Objective lens, magnification, ×20; numerical aperture, 0.75. E–G, DAAM1 activation was significantly elevated by type IV collagen coated on the lower sides of Boyden chamber membranes. MDA-MB-231 or MDA-MB-453 cells were seeded on Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). MDA-MB-231 or MDA-MB-453 cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. H, efficiency of gene knockdown was analyzed by immunoblotting for DAAM1. MDA-MB-231 and MDA-MB-453 cells were transfected with scrambled or DAAM1 shRNAs (#1 and #2). Total protein extracts from MDA-MB-231 and MDA-MB-453 cells transfected with scrambled or DAAM1 shRNAs were analyzed by immunoblotting for DAAM1. I and J, knockdown of DAAM1 significantly inhibited cell haptotaxis induced by collagen coated on the lower sides of Boyden chamber membranes. MDA-MB-231 and MDA-MB-453 cells transfected with DAAM1 shRNA or scrambled shRNAs were allowed to migrate for 8 h, and the migration rate was determined by Boyden chamber assays. K and P, the expression of empty vector and HA-tagged N-DAAM1 (N terminus of DAAM1) was verified using total protein from cells and immunoblotted using anti-HA antibody. The expression level of DAAM1 was tested using immunoblotting and proved the efficiency of N-DAAM1 expression. L and M, the expression of N-DAAM1 significantly inhibited cell haptotaxis induced by collagen coated on the lower sides of Boyden chamber membranes. MDA-MB-231 and MDA-MB-453 cells transfected with N-DAAM1 were allowed to migrate for 8 h, and the number of migratory cells were counted per field of the microscope. N and P, the expression of empty vector and HA-tagged C-DAAM1 (C terminus of DAAM1) was verified using total protein from cells and immunoblotted using anti-HA antibody. The expression level of DAAM1 was tested using immunoblotting and proved the efficiency of C-DAAM1 expression. O, MDA-MB-231 cells transfected with C-DAAM1 were seeded on Boyden chamber membranes and allowed to migrate for 8 h, and the number of migratory cells were counted per field of the microscope. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, Microscopy, Western Blot, Transfection, shRNA, Migration, Expressing, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: DAAM1 is associated with integrin β3 and acts as a downstream target of the integrin receptor. A, integrin inhibitors blocked type IV collagen–induced haptotaxis of breast cancer cells. MDA-MB-231 and/or MDA-MB-453 cells, treated with cilengitide trifluoroacetate (an integrin inhibitor for αvβ3 receptor and αvβ5 receptor, 10 nmol/liter) or cyclo(-RGDfK) (an αvβ3 integrin inhibitor, 20 nmol/liter), were examined for cell haptotaxis for 8 h in 8.0-μm porous Boyden chamber membranes coated with type IV collagen on the lower sides. CT, cilengitide trifluoroacetate. Cy, cyclo(-RGDfK). B and C, collagen-induced DAAM1 activation was blocked by αvβ3 integrin inhibitor treatment. MDA-MB-231 or MDA-MB-453 cells were seeded on Boyden chamber membranes coated with collagen type IV on both sides (Both) or lower sides (Lower) and treated with cyclo(-RGDfK) (20 nmol/liter). MDA-MB-231 or MDA-MB-453 cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. Cy, cyclo(-RGDfK). D and E, the lysates of MDA-MB-231 or MDA-MB-453 cells were subjected to immunoprecipitation (IP) with antibody to DAAM1, followed by immunoblotting with antibody to integrin β3 and DAAM1. F, HA-tagged DAAM1 constructs were expressed in HEK-293T cells. Cell lysates were harvested and cleared, and integrin was immunoprecipitated with anti-HA antibodies. Immune precipitates were analyzed by immunoblotting for anti-HA or anti-integrin β3. Whole-cell lysates were used as a control. G, purified GST or GST-fused C terminus (amino acids 716–762) of integrin β3 was incubated with purified HA-tagged full-length DAAM1. The amounts of HA-DAAM1 co-purified with GST or GST-integrin (pulldown) were analyzed by immunoblotting. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Construct, Purification, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: RHOA is a downstream target of DAAM1, and its activation is required for collagen-induced cell haptotaxis. A–C, RHOA, RAC1, and CDC42 activations were elevated by type IV collagen treatment. MDA-MB-231 cells were seeded on Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA, RAC1, and CDC42 activation assays. D, collagen-induced RHOA activation was inhibited by DAAM1 silence. Stable DAAM1 knockdown MDA-MB-231 cells (DAAM1 shRNA #1 and #2) and control cells were seeded on Boyden chamber membranes coated with collagen type IV on the lower sides. Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA, RAC1, and CDC42 activation assays. E, collagen-induced RHOA activation was inhibited by cyclo(-RGDfK) treatment. MDA-MB-231 cells were seeded on Boyden chamber membranes coated with collagen type IV on the lower sides. Cells treated with cyclo(-RGDfK) (20 nmol/liter) were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for RHOA activation assays. Cy, cyclo(-RGDfK). F and G, CCG-1423 (RHOA-specific inhibitor) or RHOA-N19 (dominant-negative RHOA) inhibited collagen-induced haptotaxis of MDA-MB-231 cells. MDA-MB-231 cells treated with CCG-1423 (1 μmol/liter) or RHOA-N19–overexpressing stable cells were seeded in Boyden chamber membranes coated with type IV collagen on both sides (Both) or lower sides (Lower). Cells were allowed to migrate toward collagen for 8 h. H, CCG-1423 or RHOA-N19 inhibited collagen-induced haptotaxis of MDA-MB-453 cells. MDA-MB-453 cells treated with CCG-1423 (1 μmol/liter) or transfected with RHOA-N19 were seeded in Boyden chamber membranes coated with type IV collagen on both sides (Both) or lower sides (Lower). Cells were allowed to migrate toward collagen for 8 h. I and J, DAAM1 activation was not altered by RHOA-N19 overexpression. RHOA-N19–overexpressing stable cells were seeded on Boyden chamber membranes coated with type IV collagen on both sides (Both) or on lower sides (Lower). Cells were allowed to migrate toward collagen for 4 h. Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. K, RHOA-V14 rescued the haptotaxis of N-DAAM1–expressed MDA-MB-231 cells. MDA-MB-231 cells transfected with N-DAAM1 and/or RHOA-V14 were seeded in Boyden chamber membranes coated with type IV collagen on lower sides. Cells were allowed to migrate toward collagen for 8 h. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, shRNA, Dominant Negative Mutation, Transfection, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

doi: 10.1074/jbc.RA117.000327

Figure Lengend Snippet: DAAM1 activation is essential for collagen-induced invadopodia extension. A and B, type IV collagen triggered invadopodia extension of MDA-MB-231 and MDA-MB-453 cells. MDA-MB-231 or MDA-MB-453 cells were examined for invadopodia extension for 6 h in 3.0-μm porous Boyden chamber membranes coated with vehicle (Uncoated) or type IV collagen on upper sides (Upper), both sides (Both), or lower sides (Lower). Invadopodia on the lower sides of the membrane were stained with cortactin antibodies and counted per field of the microscope. Bar, 10 μm. Objective lens, magnification, ×40; numerical aperture, 0.95. C, DAAM1 silence significantly inhibited collagen-induced invadopodia extension. Stable DAAM1 knockdown MDA-MB-231 cells (DAAM1 shRNA #1 and #2) or control cells were allowed to extend invadopodia for 6 h. The number of extended invadopodia was determined by 3.0-μm porous Boyden chamber assays and counted per field of the microscope. D, cyclo(-RGDfK) suppressed collagen-induced invadopodia extension. MDA-MB-231 cells were treated with 20 nmol/liter cyclo(-RGDfK) or vehicle and allowed to extend invadopodia for 6 h. The number of extended invadopodia was determined by 3.0-μm porous Boyden chamber assays and counted per field of the microscope. E, DAAM1 silence or cyclo(-RGDfK) significantly inhibited collagen-induced invadopodia extension. MDA-MB-453 cells transfected with DAAM1 shRNA or treated with cyclo(-RGDfK) were allowed to extend invadopodia for 6 h. The number of extended invadopodia was determined by 3.0-μm porous Boyden chamber assays and counted per field of the microscope. Bo, collagen coated on both sides. Lo, collagen coated on the lower sides. F–H, abundant active DAAM1 were located in invadopodia. MDA-MB-231 or MDA-MB-453 cells were seeded on 3.0-μm porous Boyden chamber membranes coated with collagen type IV on the lower sides. The invadopodia of MDA-MB-231 or MDA-MB-453 cells were allowed to extend toward collagen for 4 h. Cell bodies and invadopodia were separated as described under “Experimental procedures.” Cellular lysates were assayed for the active DAAM1 by a pulldown assay using a GST-RHOA as a bait. Error bars, S.D.

Article Snippet: Equal amounts of total cellular protein were incubated with GTPγS-incubated GST-RHOA beads captured on MagneGST GSH particles (Promega, Madison, WI) at 4 °C with constant rotation for 90 min.

Techniques: Activation Assay, Staining, Microscopy, shRNA, Transfection