Figure 4.

Actinosomes as protein-producing bioreactors. (a) Encapsulation of Cy5-labeled RNA (1.25 mM, polyU 20-mer) encapsulated inside the actinosomes. (b) Line graph corresponding to the dotted line in panel (a) showing the localization of polyU-RNA (solid line, blue) near the actinosome border. Actin (dotted line, red) and polylysine (dashed line, green) profiles are also shown. (c) Schematic illustrating actinosomes encapsulating an in vitro translation machinery along with GFP-mRNA (left). Upon incubation, GFP-mRNA inside the actinosomes produces active GFP protein that remains encapsulated. (d) Expression of GFP inside actinosomes by encapsulation of GFP-encoding mRNA and a cell-free in vitro translation machinery (IVT). As can be seen, GFP fluorescence (green) increases over the course of an hour inside actinosomes, while the background remains dark, indicating the protein expression is carried out predominantly inside the containers. (e) Negative control (no GFP-mRNA but translation machinery is still present) showing no increase in fluorescence over the same duration. (f) Analysis of GFP expression inside actinosomes (n = 11) showed an initial steep increase before gradually reaching a plateau over the course of an hour. Analysis of actinosomes (n = 6) lacking GFP-encoding RNA but with encapsulated IVT had a significantly lower and a relatively constant signal intensity over the same period. We note that the t = 0 min corresponds to roughly 5 min after the reaction had started; the delay is caused due to technical limitations like adjusting the focus and selecting an optimal field-of-view. Error bars indicate standard deviations. Images were acquired in epifluorescence microscopy.