The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

Background: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment

High-Resolution X-Ray Structure of the Trimeric Scar/WAVE-Complex Precursor Brk1

The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex

Crystallographic reconstruction study of the effects of finish rolling temperature on the variant selection during bainite transformation in C-Mn high-strength steels

The effect of finish rolling temperature (FRT) on the austenite- () to-bainite () phase transformation is quantitatively investigated in high-strength C-Mn steels. In particular, the present study aims to clarify the respective contributions of the conditioning during the hot rolling and the variant selection (VS) during the phase transformation to the inherited texture. To this end, an alternative crystallographic reconstruction procedure, which can be directly applied to experimental electron backscatter diffraction (EBSD) mappings, is developed by combining the best features of the existing models: the orientation relationship (OR) refinement, the local pixel-by-pixel analysis and the nuclei identification and spreading strategy. The applicability of this method is demonstrated on both quenching and partitioning (Q&P) and as-quenched lath-martensite steels. The results obtained on the C-Mn steels confirm that the sample finish rolled at the lowest temperature (829{\deg}C) exhibits the sharpest transformation texture. It is shown that this sharp texture is exclusively due to a strong VS from parent brass {110}, S {213} and Goss {110} grains, whereas the VS from the copper {112} grains is insensitive to the FRT. In addition, a statistical VS analysis proves that the habit planes of the selected variants do not systematically correspond to the predicted active slip planes using the Taylor model. In contrast, a correlation between the Bain group to which the selected variants belong and the FRT is clearly revealed, regardless of the parent orientation. These results are discussed in terms of polygranular accommodation mechanisms, especially in view of the observed development in the hot-rolled samples of high-angle grain boundaries with misorientation axes between and