Flagellar attachment is a visibly striking morphological feature of African trypanosomes but little is known about the requirements for attachment at a molecular level. This study characterizes a previously undescribed membrane protein, FLA3, which plays an essential role in flagellar attachment in Trypanosoma brucei. FLA3 is heavily N-glycosylated, locates to the flagellar attachment zone and appears to be a bloodstream stage specific protein. Ablation of the FLA3 mRNA rapidly led to flagellar detachment and a concomitant failure of cytokinesis in the long slender bloodstream form but had no effect on the procyclic form. Flagellar detachment was obvious shortly after induction of the dsRNA and the newly synthesized flagellum was often completely detached after it emerged from the flagellar pocket. Within 12 h most cells possessed detached flagella alongside the existing attached flagellum. These results suggest that proteins involved in attachment are not shared between the new and old attachment zones. In other respects the detached flagella appear normal, they beat rapidly although directional motion was lost, and they possess an apparently normal axoneme and paraflagellar rod structure. The flagellar attachment zone appeared to be disrupted when FLA3 was depleted. Thus, while flagellar attachment is a constitutive feature of the life cycle of trypanosomes, attachment requires stage specific elements at the protein level.

Bone cells are connected to one another in a network, via their dendritic cellular processes. Previously, we hypothesised that these processes could be ruptured by microcracks. We proposed this as a mechanism by which osteoctyes could detect the presence of microcracks. In order for this mechanism to be effective, the number of ruptured processes would have to increase with microcrack length and also with the applied cyclic stress applied. This paper presents for the first time experimental data which shows that this is indeed the case. We examined samples of bovine, ovine and murine bone ex vivo and observed processes passing across crack faces: some were still intact whilst others had ruptured. The number of intact processes per unit crack length decreased significantly with increasing crack length, and also decreased in samples which had been tested in vitro at higher stress levels. A theoretical model which we had developed previously was able to predict the overall magnitude and general trends in the experimental data. This work has provided further support for our "scissors" model which proposes that microcracks can be detected because they disturb the osteocyte network, specifically by rupturing cellular processes where they pass across the crack faces. AbstractBone cells are connected to one another in a network, via their dendritic cellular processes. Previously, we hypothesised that these processes could be ruptured by microcracks. We proposed this as a mechanism by which osteoctyes could detect the presence of microcracks. In order for this mechanism to be effective, the number of ruptured processes would have to increase with microcrack length and also with the applied cyclic stress applied. This paper presents for the first time experimental data which shows that this is indeed the case. We examined samples of bovine, ovine and murine bone ex vivo and observed processes passing across crack faces: some were still intact whilst others had ruptured. The number of intact processes per unit crack length decreased significantly with increasing crack length, and also decreased in samples which had been tested in vitro at higher stress levels. A theoretical model which we had developed previously was able to predict the overall magnitude and general trends in the experimental data. This work has provided further support for our "scissors" model which proposes that microcracks can be detected because they disturb the osteocyte network, specifically by rupturing cellular processes where they pass across the crack faces.

The physical effects of fatigue failure caused by cyclic strain are important and for most materials well understood. However, nothing is known about this mode of failure in living cells. We developed a novel method that allowed us to apply controlled levels of cyclic displacement to networks of osteocytes in bone. We showed that under cyclic loading, fatigue failure takes place in the dendritic processes of osteocytes at cyclic strain levels as low as one tenth of the strain needed for instantaneous rupture. The number of cycles to failure was inversely correlated with the strain level. Further experiments demonstrated that these failures were not artefacts of our methods of sample preparation and testing, and that fatigue failure of cell processes also occurs in vivo. This work is significant as it is the first time it has been possible to conduct fatigue testing on cellular material of any kind. Many types of cells experience repetitive loading which may cause failure or damage requiring repair. It is clinically important to determine how cyclic strain affects cells and how they respond in order to gain a deeper understanding of the physiological processes stimulated in this manner. The more we understand about the natural repair process in bone the more targeted the intervention methods may become if disruption of the repair process occurred. Our results will help to understand how the osteocyte cell network is disrupted in the vicinity of matrix damage, a crucial step in bone remodelling.

Natural materials such as bone and insect cuticle are capable of self‐repair, a facility that greatly increases their durability and safe working stress. Some engineering materials have also been designed to be self‐healing, although currently they cannot match the performance of natural materials as regards the efficiency and longevity of the healing process. In this paper, we review the state of the art regarding these two types of materials. We discuss the role of fracture mechanics in the development of theoretical models of self‐healing; we identify certain crucial parameters that make natural materials successful and discuss how these lessons can be applied to improve the performance of self‐healing materials for engineering applications.

We have proposed a new model for microcrack detection by osteocytes in bone. According to this model, cell signalling is initiated by the cutting of cellular processes which span the crack. We show that shear displacements of the crack faces are needed to rupture these processes, in an action similar to that of a pair of scissors. Current work involves a combination of cell biology experiments, theoretical and experimental fracture mechanics and system modelling using control theory approaches. The approach will be useful for understanding effects of extreme loading, aging, disease states and drug treatments on bone damage and repair; the present paper presents recent results from experiments and simulations as part of current, ongoing research.