No abstract

In this study we used Triton X-100 extracted sea urchin spermatozoa to investigate the mechanical behavior of the basic 9+2 axoneme. The dynein motors were disabled by vanadate so that the flagellum is rendered a passive structure. We find that when a proximal portion of the flagellum is bent with a glass microprobe, the remainder of the flagellum distal to the probe exhibits a bend in the opposite direction (a counterbend). The counterbend can be understood from the prevailing sliding doublet model of axoneme mechanics, but does require the existence of elastic linkages between the outer doublets. Analysis of the shapes of counterbends provides a consensus value of 0.03-0.08/microm(2) for the ratio of the interdoublet shear resistance (E(S)) to the bending resistance (E(B)) and we find that the ratio E(S)/E(B) is relatively conserved for both passive flagella and transiently quiescent live flagella. This ratio expresses a fundamental mechanical property of the eukaryotic axoneme. It defines the contributions to total bending resistance derived from bending the microtubules and from stretching the interdoublet linkages, respectively. Using this ratio, and computer simulations of earlier experiments that measured the total stiffness of the flagellum, we obtain estimates of approximately 1 x 10(8) pN nm(2)/rad for E(B) and 6 pN/rad for E(S), assuming that both elasticities are linear. Our results indicate that the behavior of the flagellum is close to that predicted by a linear model for shear elasticity.

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 muM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 +/- 0.29 nN/microm in 0.1 mM ATP and increasing to 2.9 +/- 1.2 nN/microm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed.

Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6–13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.