The Stability and Expression Level of Bok Are Governed by Binding to Inositol 1,4,5-Trisphosphate Receptors.

Bok is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, although the role that Bok plays in this pathway is unclear. We have shown previously in cultured cell lines that Bok interacts strongly with inositol 1,4,5-trisphosphate receptors (IP3Rs), suggesting that it may contribute to the structural integrity or stability of IP3R tetramers. Here we report that Bok is similarly IP3R-assocated in mouse tissues, that essentially all cellular Bok is IP3R bound, that it is the helical nature of the Bok BH4 domain, rather than specific amino acids, that mediates binding to IP3Rs, that Bok is dramatically stabilized by binding to IP3Rs, that unbound Bok is ubiquitinated and degraded by the proteasome, and that binding to IP3Rs limits the pro-apoptotic effect of overexpressed Bok. Agents that stimulate IP3R activity, apoptosis, phosphorylation, and endoplasmic reticulum stress did not trigger the dissociation of mature Bok from IP3Rs or Bok degradation, indicating that the role of proteasome-mediated Bok degradation is to destroy newly synthesized Bok that is not IP3R associated. The existence of this unexpected proteolytic mechanism that is geared toward restricting Bok to that which is bound to IP3Rs, implies that unbound Bok is deleterious to cell viability and helps explain the current uncertainty regarding the cellular role of Bok.

Reelin Counteracts Chondroitin Sulfate Proteoglycan-Mediated Cortical Dendrite Growth Inhibition.

Disruptions in neuronal dendrite development alter brain circuitry and are associated with debilitating neurological disorders. Nascent apical dendrites of cortical excitatory neurons project into the marginal zone (MZ), a cell-sparse layer characterized by intense chondroitin sulfate proteoglycan (CSPG) expression. Paradoxically, CSPGs are known to broadly inhibit neurite growth and regeneration. This raises the possibility that the growing apical dendrite is somehow insensitive to CSPG-mediated neurite growth inhibition. To test this, developing cortical neurons were challenged with both soluble CSPGs and CSPG-positive stripe substrates in vitro Soluble CSPGs inhibited dendritic growth and cortical dendrites respected CSPG stripe boundaries, effects that could be counteracted by prior CSPG inactivation by chondroitinase. Importantly, addition of Reelin, an extracellular signaling protein highly expressed in the MZ, partially rescued dendritic growth in the presence of CSPGs. High-resolution confocal imaging revealed that the CSPG-enriched areas of the MZ spatially correspond with the areas of reduced dendritic density in the Reelin null (reeler) cortex compared with controls. Chondroitinase injections into reeler explants resulted in increased dendritic growth into the MZ, recovering to near wild-type levels. Activation of the serine threonine kinase Akt is required for Reelin-dependent dendritic growth and we find that CSPGs induce Akt dephosphorylation, an effect that can be counteracted by Reelin addition. In contrast, CSPG application had no effect on the cytoplasmic adaptor Dab1, which is rapidly phosphorylated in response to Reelin and is upstream of Akt. These findings suggest CSPGs do inhibit cortical dendritic growth, but this effect can be counteracted by Reelin signaling.

Automating hESC differentiation with 3D printing and legacy liquid handling solutions.

Historically, the routine use of laboratory automation solutions has been prohibitively expensive for many laboratories. As legacy hardware has begun to emerge on the secondary market, automation is becoming an increasingly affordable option to augment workflow in virtually any laboratory. To assess the utility of legacy liquid handling in stem cell differentiation, a used liquid handling robot was purchased at auction to automate a stem cell differentiation protocol that gives rise to CD14 + CD45+ mononuclear cells. To maintain sterility, the automated liquid handling robot was housed in a custom constructed HEPA filtered enclosure. A custom cell scraper and a disposable filter box were designed and 3D printed to permit the robot intricate cell culture actions required by the protocol. All files for the 3D printed labware are uploaded and are freely available. •A used liquid handling robot was used to automate an hESC to monocyte differentiation protocol.•The robot-performed protocol induced monocytes as effectively as human technicians.•Custom 3D printed labware was made to permit certain cell culture actions and are uploaded for free access.