Background:The mechanism by which Rab GTPases and their effectors act in tethering is not well understood. Results: An in vitro assay was developed to study vesicle clustering by the Lgl family member Sro7. Conclusion: Clustering in vitro and in vivo depends on the conformation of the Rab GTPase and Sro7. Significance: This assay provides a new tool to dissect the role of Rab and Lgl family function.

Sro7, a downstream effector of the Sec4 Rab GTPase, acts during Golgi-to-cell surface transport. The binding site for Sec4 occurs within a cleft on Sro7 formed by the junction of two adjacent β-propeller structures. In silico docking and mutational validation experiments point to a precise model for interaction of Sec4-GTP with Sro7.

The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

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Tetracalcium phosphate (TetCP, Ca4(PO4)2O) reacts rapidly with polyacrylic acid (PAA). Complete reaction results in the formation of hydroxyapatite (HAp) and calcium polyacrylate. Consequently, this combination of reactants can react to form a dental cement. However, reaction occurs so rapidly that it would be difficult to achieve a homogeneous mixture of reactants suitable for use in restorations. In order to explore extending the working time, the effects of prehydrating the TetCP to form surface layers of HAp on the TetCP particles was explored. Prehydration was found to be an effective means of allowing workability. Therefore, the effects of the proportions of TetCP and PAA, with and without HAp filler, on cement properties were investigated. The extents of the reactions were investigated by X-ray diffraction analysis; the extents of PAA neutralization were studied by Fourier transform infra-red spectroscopy (FTIR); pore structures were determined by mercury intrusion porosimetry; microstructures were observed by scanning microscopy, and compressive strengths were determined. After curing for 17 days at room temperature PAA neutralization was almost complete; however, residual TetCP could be detected by X-ray diffraction and PAA by FTIR. As expected, the compressive strengths of the cements showed a dependence on the liquid (water+polymer)-to-solid (TetCP+HAp filler) used. The presence of HAp filler caused a significant decrease in compressive strength and increasing the proportion of HAp filler resulted in a decrease in the compressive strength. The characteristics of the load-deflection curves showed a dependence on the presence of HAp filler. In the absence of filler, two slopes were observed in the curves whereas a linear curve, typical of a ceramic, was observed when HAp filler was present. Mercury intrusion porosimetry (MIP) indicated the majority of the porosity was present in pores larger than 0.1 microm. Porosity increased with increasing liquid-to-solids ratio and with an increasing proportion of HAp filler at a constant liquid-to-solids ratio. Microstructural observations indicated the effect of HAp filler on increasing porosity was the result of porosity present in the filler itself. Thus, poorly consolidated HAp filler contributed to increased porosity and reduced compressive strength.