RESUMEN
Fatigue is a common mode of mechanical failure which occurs when a material is subjected to repeated cycles at a strain level less than that needed for monotonic fracture. Fatigue has been observed and measured in many different materials but, until recently, not in cells. We devised a novel experiment which allowed us to create both monotonic failure and fatigue in the cellular processes of osteocytes within samples of bone (Dooley et al., European Cells and Materials 2014). In the present paper, we describe the results of further experiments and a computer simulation, which has allowed us to estimate the strain history of each sample tested and thus present, for the first time, strain/life data for cells. Failure occurred during the first cycle at strains of 0.1-0.2; at lower strains failure occurred after a number of cycles which depended inversely on the applied strain range. Scatter in the strain/life data was reduced when we allowed for the effects of mean stress using the Smith-Watson-Topper parameter. We confirmed that aspects of our experimental method (the types of microcrack used and the testing of fresh versus frozen samples) did not affect the results. Such information is useful because many cell types, including the cellular processes of osteocytes, experience cyclic strain in vivo.
Asunto(s)
Huesos/patología , Osteocitos/patología , Tibia/patología , Animales , Bovinos , Simulación por Computador , Fracturas Óseas , Congelación , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Estrés Mecánico , Soporte de PesoRESUMEN
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.
Asunto(s)
Flagelos/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/fisiología , Animales , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Transporte de Proteínas , Interferencia de ARN , RatasRESUMEN
Bone cells are connected to one another in a network, via their dendritic cellular processes. Previously, we hypothesized 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. 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 that 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.