Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissuewide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.EMT | growth regulation | mechanics | mitosis

We evaluated the o-nitrobenzyl platform for designing photolabile protecting groups with red-shifted absorption that could be photolyzed upon one- and two-photon excitation. Several synthetic pathways to build different conjugated o-nitrobenzyl backbones, as well as to vary the benzylic position, are reported. Relative to the reference 4,5-dimethoxy-2-nitrobenzyl group, several o-nitrobenzyl derivatives exhibit a large and red-shifted one-photon absorption within the near-UV range. Uncaging after one-photon excitation was studied by measuring UV-visible absorption and steady-state fluorescence emission on model caged ethers and esters. In the whole series investigated, the caged substrates were released cleanly upon photolysis. Quantum yields of uncaging after one-photon absorption lie within the 0.1-1 % range. We observed that these drop as the maximum wavelength absorption of the o-nitrobenzyl protecting group is increased. A new method based on fluorescence correlation spectroscopy (FCS) after two-photon excitation was used to measure the action uncaging cross section for two-photon excitation. The series of o-nitrobenzyl caged fluorescent coumarins investigated exhibit values within the 0.1-0.01 Goeppert-Mayer (GM) range. Such results are in line with the low quantum yields of uncaging associated with cross-sections of 1-50 GM for two-photon absorption. Although the cross-sections for one- and two-photon absorption of o-nitrobenzyl photolabile protecting groups can be readily improved, we emphasize the difficulty in enlarging the corresponding action uncaging cross-sections in view of the observed trend of their quantum yield of uncaging.

Persistent changes in synaptic strength are locally regulated by both protein degradation and synthesis; however, the coordination of these opposing limbs is poorly understood. Here, we found that the RISC protein MOV10 was present at synapses and was rapidly degraded by the proteasome in an NMDA-receptor-mediated activity-dependent manner. We designed a translational trap to capture those mRNAs whose spatiotemporal translation is regulated by MOV10. When MOV10 was suppressed, a set of mRNAs--including alpha-CaMKII, Limk1, and the depalmitoylating enzyme lysophospholipase1 (Lypla1)--selectively entered the polysome compartment. We also observed that Lypla1 mRNA is associated with the brain-enriched microRNA miR-138. Using a photoconvertible translation reporter, Kaede, we analyzed the activity-dependent protein synthesis driven by Lypla1 and alpha-CaMKII 3'UTRs. We established this protein synthesis to be MOV10 and proteasome dependent. These results suggest a unifying picture of a local translational regulatory mechanism during synaptic plasticity.

Engineered fluorogenic dyes enable the dynamic switching of the fluorescence color of Fluorescence-Activating and absorption-Shifting Tag (FAST).

MicroRNAs are a class of small RNA regulators that are involved in numerous cellular processes, including development, proliferation, differentiation, and plasticity. The emerging concept is that microRNAs play a central role in controlling the balance between stem cell self-renewal and fate determination by regulating the expression of stem cell regulators. This review will highlight recent advances in the regulation of neural stem cell self-renewal and neurogenesis by microRNAs. It will cover microRNA functions during the entire process of neurogenesis, from neural stem cell self-renewal and fate determination to neuronal maturation, synaptic formation, and plasticity. The interplay between microRNAs and both cell-intrinsic and -extrinsic stem cell players, including transcription factors, epigenetic regulators, and extrinsic signaling molecules will be discussed. This is a summary of the topics covered in the mini-symposium on microRNA regulation of neural stem cells and neurogenesis in SFN 2010 and is not meant to be a comprehensive review of the subject.