Polymeric crowding agents improve passive biomacromolecule encapsulation in lipid vesicles.

Large solutes such as high molecular weight proteins can be difficult to encapsulate in lipid vesicles. Passive trapping of these macromolecular solutes during vesicle formation typically results in concentrations inside the vesicles that are much lower than in the external solution. Here, we investigated the effect of macromolecular crowding on passive encapsulation of biological macromolecules with molecular weights ranging from 52 kDa to 660 kDa within both individual giant lipid vesicles (GVs, > 3 microm diameter) and populations of 200 nm diameter large unilamellar vesicles (LUVs). Fluorescently labeled biomacromolecules were encapsulated during vesicle formation in the presence or absence of three weight percent poly(ethylene glycol) (PEG; 8 kDa) or dextran 500 kDa, which served as crowding agents. Encapsulation efficiency of the labeled biomolecules was higher for the lower molecular weight solutes, with internal concentrations essentially equal to external concentrations for labeled biomacromolecules with hydrodynamic radii (r(h)) less than 10 nm. In contrast, internal concentrations were reduced markedly for larger solutes with r(h) > or = 10 nm. Addition of PEG or dextran during vesicle formation improved encapsulation of these larger proteins up to the same levels as observed for the smaller proteins, such that internal and external concentrations were equal. This observation is consistent with PEG and dextran acting as volume excluders, reducing the hydrodynamic radius of the biomacromolecules and increasing their encapsulation. This work demonstrates a simple and general route to improved encapsulation of otherwise poorly encapsulated macromolecular solutes in both GV and LUVs up to their concentration in the solution present during vesicle formation.

Microcompartmentation in artificial cells: pH-induced conformational changes alter protein localization.

We report artificial cells in which protein localization in a primitive synthetic model for the cytoplasm is controlled by pH. Our model cells are giant lipid vesicles (GVs, ca. 5-30 microm diameter) with two coexisting aqueous compartments generated by phase separation of an encapsulated poly(ethylene glycol) (PEG) and dextran solution. Proteins are localized to a microcompartment by partitioning between the phases. We quantified the local concentration of fluorescently labeled human serum albumin (HSA) via confocal fluorescence microscopy. At pH 6.5, the labeled HSA was more concentrated in the dextran-rich phase, but at partially/fully denaturing pH (4.1 or 12) it was localized in the PEG-rich phase. This partitioning behavior is consistent with a more expanded, hydrophobic conformation at low and high pH. Labeled HSA could be relocalized from the PEG-rich into the dextran-rich phase domain by increasing the pH from 4.1 to 6.5 to renature the protein. This approach to controlling protein localization does not require extensive reorganization of the vesicle interior; coexisting PEG-rich and dextran-rich compartments are maintained throughout the experiments. It is also quite general; we demonstrated that several other proteins varying in size and isoelectric point also relocalized within compartmentalized artificial cells in response to external pH change. This work presents stimulus-responsive protein relocalization between compartments in an artificial cell; such experimental models can provide a framework for investigating the consequences of protein localization in cell biology.

FoxO3a gene is a target of deletion in mouse lung adenocarcinoma.

Lung adenocarcinomas (LAC) of smokers and never-smokers differ from one another in epidemiology, and clinical and molecular characteristics. The pathogenetic differences between these tumors are potential biomarkers and therapeutic targets. Mouse carcinogenesis models of human LAC are proven tools applicable for the identification of these molecular changes. Allelic loss frequency on human chromosome 6q is higher in LAC of smokers compared with never smokers. We analyzed the orthologous region on mouse chromosome 10 and found this region similarly was a more frequent site of allelic loss in carcinogen-induced LAC compared with non-induced or spontaneous LAC. We then conducted high resolution quantitative PCR-based deletion mapping of this region and identified the FoxO3a gene as the focus of bi-allelic or homozygous deletion (HD). HDs were detected in 5 out of 15 (33.3%) LAC cell lines and in 6 out of 75 (8%) carcinogen-induced primary LAC. FoxO3a was exclusively affected by HD in 7 of the samples examined, as loss of both alleles did extend to the nearest flanking genes of FoxO3a. Deletion of FoxO3a, either by HD or subclonal loss was detected in 38 out of 75 (50.7%) of carcinogen-induced LAC in contrast to only 1 out of 10 (10%) of LAC of untreated mice. Several of the samples also were subjected to direct sequence analysis; however, no intragenic mutations were detected. These results implicate FoxO3a as a selective target of deletion in mouse LAC. Significant association with carcinogenic induction suggests that deletion of FoxO3a contributes to the development of carcinogen-initiated tumors.