Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis.

International audienceBone sialoprotein (BSP) and osteopontin (OPN) are both highly expressed in bone, but their functional specificities are unknown. OPN knockout ((-/-)) mice do not lose bone in a model of hindlimb disuse (tail suspension), showing the importance of OPN in bone remodeling. We report that BSP(-/-) mice are viable and breed normally, but their weight and size are lower than wild-type (WT) mice. Bone is undermineralized in fetuses and young adults, but not in older (>/=12 mo) BSP(-/-) mice. At 4 mo, BSP(-/-) mice display thinner cortical bones than WT, but greater trabecular bone volume with very low bone formation rate, which indicates reduced resorption, as confirmed by lower osteoclast surfaces. Although the frequency of total colonies and committed osteoblast colonies is the same, fewer mineralized colonies expressing decreased levels of osteoblast markers form in BSP(-/-) versus WT bone marrow stromal cultures. BSP(-/-) hematopoietic progenitors form fewer osteoclasts, but their resorptive activity on dentin is normal. Tail-suspended BSP(-/-) mice lose bone in hindlimbs, as expected. In conclusion, BSP deficiency impairs bone growth and mineralization, concomitant with dramatically reduced bone formation. It does not, however, prevent the bone loss resulting from loss of mechanical stimulation, a phenotype that is clearly different from OPN(-/-) mice

Coordination by Cdc42 of actin, contractility, and adhesion for melanoblast movement in mouse skin

YesThe individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics.Cancer Research UK (to L.M.M. [A17196], R.H.I. [A19257], and S.W.G.T.) and NIH grants P01-GM103723 and P41-EB002025 (to K.M.H.). N.R.P. is supported by a Pancreatic Cancer Research Fund grant (to L.M.M.). Funding to Prof. Rottner by the Deutsche Forschungsgemeinschaft (grant RO2414/3-2)

Accelerating Live Single-Cell Signalling Studies.

The dynamics of signalling networks that couple environmental conditions with cellular behaviour can now be characterised in exquisite detail using live single-cell imaging experiments. Recent improvements in our abilities to introduce fluorescent sensors into cells, coupled with advances in pipelines for quantifying and extracting single-cell data, mean that high-throughput systematic analyses of signalling dynamics are becoming possible. In this review, we consider current technologies that are driving progress in the scale and range of such studies. Moreover, we discuss novel approaches that are allowing us to explore how pathways respond to changes in inputs and even predict the fate of a cell based upon its signalling history and state

A study of the IgM interaction with complement using mouse IgM/IgG2b domain-switched hybrids

grantor: University of TorontoThe IgM and IgG classes of immunoglobulins share an important biological function that is distinct from that of other classes. When bound to antigen, both can initiate the classical complement pathway by interacting with C1q, a subunit of the first component of complement (C1). To study the C1q binding properties of IgM in a form comparable to that of IgG, a 'monomeric' form of mouse IgM was prepared and its function was compared to that of a mouse IgG with the same antigen binding site. Monomeric IgM was isolated from a mutant (IgM P544G). The monomeric form of this mutant was not able to bind C1q or initiate complement-mediated lysis (CML) on haptenated SRBC. Under the same conditions, the positive control (IgG2b) bound C1q and initiated CML in a dose-dependent manner. Polymeric IgM isolated from this mutant was able to bind C1q and initiate CML as well as wildtype IgM. To understand monomeric IgM inactivity, mouse IgM/IgG2b hybrid mutants were created and analyzed for their C1q binding properties. Cã2, the intrinsically active C1q binding domain of IgG2b, did not bind C1q or initiate CML when placed in the monomeric IgM background ([mu][mu]ã[mu]). This suggests that the activity of an intrinsically active C1q binding domain is hidden by one or both of the neighbouring C[mu]2 and C[mu]4 domains. C[mu]3, the putative C1q binding domain of IgM, remained inactive when it was placed in the non-inhibitory IgG2b background (ãã[mu]ã), demonstrating that a pre-formed C1q binding site is not found in the C[mu]3 domain. Collectively, the results suggest that if the C[mu]3 contains the entire C1q binding site for IgM, the site is unformed and hidden. To determine if one or both of the C[mu]2 and CU[mu]4 domains are responsible for the inactivity of the [mu][mu]ã[mu] hybrid monomer two hybrids ([mu]ãã[mu] and [mu][mu]ãã) were created in which these domains were individually replaced by their corresponding IgG2b domains (ã-hinge and Cã3). The results show that C[mu]4 interferes with the interaction between C1 and the Cã2 domain whereas C[mu]2 interferes with the subsequent activation of C1. C[mu]2 and C[mu]4 may therefore have similar inhibitory effects on the C[mu]3 domain in the IgM monomer. The transplantation of C[mu]3 together with C[mu]4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although both the monomer and the polymer forms were not able to initiate CML; conversely, all IgM polymers with a transplanted Cã2 domain were active in both C1q binding and CML and demonstrated apparent Kd values for C1q (1-2 * 10-9 M) that were similar to that of wild-type IgM. The findings reported in this thesis are discussed in relation to the activity of the IgM polymer and are consistent with the view that the 'star' to 'staple' conformational change resulting when IgM binds to antigen is necessary for the expression of the C1q binding site. (Abstract shortened by UMI.)Ph.D

Partitioning of the source of leaf calcium of American beech and sugar maple using leaf Ca/Sr ratios: A predominantly surficial but variable depth of Ca uptake

Background and Aims Reduced availability of calcium (Ca) has been linked to maple forest decline. We therefore aimed at assessing the contribution of the different soil horizons to leaf Ca of competing beech (Fagus grandifolia Ehrh.) and sugar maple (Acer saccharum Marsh.) to better understand the dynamics of Ca uptake. Methods Leaf Ca was partitioned using the Ca/Sr ratio approach in two mature forests of southern Quebec. A mass balance was also used at one site to validate the results obtained with the Ca/Sr approach. Results The L and F horizons contributed most of the leaf Ca of beech and maple with likely small contributions from the upper B and/or H/Ahe horizons. Leaf Ca/Sr ratios of beech were however more variable than those of maple. Using a mass balance, the organic horizons and upper mineral soil horizons were found to provide ca. 80 and 20%of tree Ca uptake, respectively. Conclusion Beech and maple Ca uptake depth apportionment is on average similar but beech is likely more plastic in sourcing soil Ca. The low contribution of the mineral soil to leaf Ca at our sites can be linked to less favorable conditions for Ca uptake likely associated with low Ca/Al ratios