Inner Drive

Conventional wisdom says the most profound changes come from within. Based on work newly published in Biomaterials from Carpenedo et al., this insight may also apply to differentiation of embryonic stem cells within the spheroid cell clusters known as embryoid bodies (EB). Attempts to control differentiation of EB cells by adjusting culture conditions have been disappointing, likely because the EB shell blocks diffusion of morphogenic factors. To overcome this barrier, Carpenedo et al. tried working from the inside out—that is, forming EBs in the presence of gelatin-coated microspheres containing small molecules. Preliminary studies with fluorophore-loaded micro-spheres confirmed that the microparticles distributed throughout the interior of EBs and that small molecule release occurred over a several-day period. Subsequent experiments examined the effect of microspheres loaded with retinoic acid, a morphogen that has been used to induce neural differentiation of embryonic stem cells. In this system, the EBs formed large cystic structures, of which a significant subset were spheroids fringed with an epiblast-like layer enveloped by visceral endoderm. The authors observed a dose-dependent effect of the microspheres; by contrast, culture-provided retinoic acid did not lead to cystic regions. Transcriptional profiling of EBs formed with retinoic acid micro-spheres revealed a pattern reminiscent of early post-implantation mouse embryos. These structures may prove indispensable for in-depth study of embryogenesis; in addition, the basic approach may produce differentiated cell types more efficiently by spatially controlled exposure to morphogenic factors.

Click Here

Click chemistry has become a robust tool for tagging target molecules. In proteomics, particularly entrancing targets are membrane proteins, which are often missed in traditional protein isolations due to their poor solubility. Rather than hoping for membrane proteins to come to them, Gubbens et al. synthesized a click chemistry–ready lipid analog designed to retrieve proteins with affinity for phospholipid headgroups. This new phosphatidylcholine analog, described in a recent edition of Chemistry & Biology, comprises a photoactivatable headgroup and lipid acyl chains that terminate in azide groups for subsequent click chemistry. The compounds are incorporated into a membrane, crosslinked to adjacent proteins, and then unbound factors are washed away. Afterwards, click chemistry labels the lipid analog–protein conjugate with a fluorescent marker or a biotin tag for pull-down by avidin beads. SDS-PAGE of fluorescently labeled proteins showed multiple bands, with slightly different protein sets depending on the photoactivatable moiety used. For identification of proteins, the biotin tag was used for purification and these samples were then separated by SDS-PAGE for subsequent LC-MS/MS. In the authors’ chosen system—inner mitochondrial membrane vesicles from yeast—many recognized components of the inner membrane proteome were detected, as well as several factors not yet known to be present in this area. These studies show the potential of these lipid analogs for completing membrane proteomes.

Scratching the Surface

Using electron microscopy (EM) to reconstruct a three-dimensional (3-D) view of a cell's internal structure has meant the preparation of serial thin sections followed by transmission EM. Though spatial resolution is 200 nm or better, this procedure is relatively slow. Enter ion-abrasion scanning EM (IA-SEM), a technique which cycles between SEM and focused ion-beam removal of surface material. Previously this method has been applied to yeast cells and fixed tissue; Heymann et al., writing in the Journal of Structural Biology, extend to visualizing subcellular organization of metazoan cells. The result is a set of spectacular images with a resolution of ∼20 nm in the direction of milling and ∼6 nm in the viewing plane. Working with melanoma cells, the authors show the images are comparable to those from the more laborious transmission electron micrographic method. The authors also successfully detect 10- and 15-nm gold particles, as well as quantum dots (the latter hinting at the option of dual fluorescence and EM-based imaging). Future applications for IA-SEM include mapping mitochondrial perturbations in cancer cells, and imaging nanoparticles in tissue biopsies to track their localization and potential toxicity when employed in drug delivery. Although x-ray tomography and optical microscopy show increasing promise for looking inside live cells, imaging plastic-embedded cells will continue to be an important complementary need, particularly for translational research, and these new data show the power of IA-SEM for this application.