Osteocytes play a pivotal role in the regulation of skeletal mass. Osteocyte processes are thought to sense the flow of interstitial fluid that is driven through the osteocyte canaliculi by mechanical stimuli placed upon bone, but how this flow elicits a cellular response is virtually unknown. Modern theoretical models assume that osteocyte canaliculi contain ultrastructural features that amplify the fluid flow-derived mechanical signal. Unfortunately the calcified bone matrix has considerably hampered studies on the osteocyte process within its canaliculus. Using one of the few ultra high voltage electron microscopes (UHVEM) available worldwide, we applied UHVEM tomography at 2 MeV to reconstruct unique three-dimensional images of osteocyte canaliculi in 1 μm sections of human bone. A realistic three-dimensional image-based model of a single canaliculus was constructed, and the fluid dynamics of a Newtonian fluid flow within the canaliculus was analyzed. We created virtual 2.2 nm thick sections through a canaliculus and found that traditional TEM techniques create a false impression that osteocyte processes are directly attached to the canalicular wall. The canalicular wall had a highly irregular surface and contained protruding axisymmetric structures similar in size and shape to collagen fibrils. We also found that the microscopic surface roughness of the canalicular wall strongly influenced the fluid flow profiles, whereby highly inhomogeneous flow patterns emerged. These inhomogeneous flow patterns may induce deformation of cytoskeletal elements in the osteocyte process, thereby amplifying mechanical signals. Based on these observations, new and realistic models can be developed that will significantly enhance our understanding of the process of mechanotransduction in bone.

The self-incompatibility system of the plant species Brassica is controlled by the S-locus, which contains S-RECEPTOR KINASE (SRK) and S-LOCUS PROTEIN11 (SP11). SP11 binding to SRK induces SRK autophosphorylation and initiates a signaling cascade leading to the rejection of self pollen. However, the mechanism controlling hydration and germination arrest during self-pollination is unclear. In this study, we examined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination in the papilla cell during pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization, whereas self-pollination induced actin reorganization and likely depolymerization. By monitoring transiently expressed green fluorescent protein fused to the actin-binding domain of mouse talin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization and likely depolymerization at the self-pollen attachment site; the results correspond to those obtained by rhodamine-phalloidin staining. We further showed that the coat of self pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Self-pollination disrupted the vacuole network, whereas crosspollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that self-and cross-pollination differentially affect the dynamics of the actin cytoskeleton, leading to changes in vacuolar structure associated with hydration and germination.

Memory processing in nonhuman animals has been typically tested in situations where the animals are repeatedly trained to retrieve their memory trace, such as delayed matching to sample, serial probe recognition, etc. In contrast, how they utilize incidentally formed memory traces is not well investigated except in rodents. We examined whether domestic dogs could solve an unexpected test based on a single past experience. In Experiment 1, leashed dogs were led to 4 open, baited containers and allowed to eat from 2 of them (Exposure phase). After a walk outside for more than 10 min, during which time the containers were replaced with new identical ones, the dogs were unexpectedly returned to the site and unleashed for free exploration (test phase). Eleven out of 12 dogs first visited one of the containers from which they had not eaten. In Experiment 2, two containers had food in them, one had a nonedible object, and the last one was empty. Dogs visited all 4 containers and were allowed to eat one of the food rewards in the Exposure phase. In the test phase, unleashed dogs first visited the previously baited container from which they had not eaten significantly more often than chance. These results demonstrate that in an unexpected, test dogs may retrieve "what" and "where" information about seen (now invisible) items from incidental memory formed during a single past experience.