In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice

Parts used

Key findings

• In EGFP, adding the sperm acrosin N-terminal signal sequence (MVEMLPTVAVLVLAVSVVA) at the N terminus and a Thy-1-derived C-terminal GPI-anchor sequence (KDNTTLQEFATLAN) at the C terminus yields GFP-GPI, which labels the plasma membrane of mouse ES cells expressing it from the CAG promoter, and also labels Golgi membranes and some secretory vesicles in live cells.
• Adding the N-terminal nine amino acids of avian c-Src (MGSSKSKPK, which provide a glycine at position 2 for myristoylation) to the N terminus of EGFP or Venus yields myr-GFP and myr-Venus, which show identical subcellular localization in transiently transfected Cos-7 cells (data not shown in the paper).
• In live mouse ES cells expressing it from the CAG promoter, myr-Venus is concentrated at the plasma membrane, the Golgi, endosomes of the secretory pathway and perinuclear regions, and also appears in vesicular structures inside the nucleus, with a higher density of labeled cytoplasmic structures than GFP-GPI.
• Hemizygous and homozygous mice carrying CAG::GFP-GPI or CAG::myr-Venus transgenes are viable, fertile and indistinguishable from wildtype littermates, show sustained widespread fluorescence into adulthood, and transmit the transgene to F1 progeny in a Mendelian fashion, so widespread lipid-modified GFP or Venus expression is compatible with normal development. Homozygous animals have the same subcellular distribution but higher fluorescence than hemizygotes.
• In tetraploid (4n) chimeras made from three independent ES cell lines per construct and examined at E9.5, all CAG::GFP-GPI lines gave widespread GFP-GPI expression with identical localization, whereas of the three CAG::myr-Venus lines two gave widespread myr-Venus expression and one gave mosaic expression that became more widespread at later stages.
• Zygotic expression of CAG::GFP-GPI and CAG::myr-Venus begins in early uncompacted mouse morulae, and both fusions label the plasma membrane with a high signal-to-noise ratio, which allows the inner cells of the morula to be distinguished from the outer cells and the blastocoelic cavity to be seen at E3.5.
• GFP-GPI is enriched at apical plasma membranes of specific epithelial cells in mice, for example the endoderm and gut at E9.5, the developing lung bronchioles at E13.5 and arterioles of the P4 kidney, whereas myr-Venus is not polarized and is present throughout the plasma membrane and cytosol while excluded from the nucleus.
• The Thy-1-derived GPI-anchor sequence in GFP-GPI carries QLEN in place of the KLEN used in an earlier GFP-GPI mouse, where N is the omega site, because the lysine is sensitive to some proteases such as kallikrein that could release GFP-GPI from the membrane. GFP-GPI with the QLEN sequence shows basolateral as well as apical localization, whereas the KLEN version showed apical distribution, and the paper favors this sequence difference as the explanation.
• In mouse tissues, the distributions of GFP-GPI and myr-Venus depend on cell type and developmental stage. In P4 liver hepatocytes neither fusion concentrates at cell boundaries and both are diffuse in the cytoplasm, whereas in P4 small intestinal mucosal epithelium myr-Venus is present in lateral membranes and apical brush borders but GFP-GPI signal is diffuse.
• GFP and Venus fluorescence from CAG::GFP-GPI and CAG::myr-Venus double transgenic E9.5 embryos can be separated in live tissue by linear unmixing, which shows both distinct and overlapping domains of the two fusions, for example distinct myr-Venus domains in somite epithelial cells, apical-only GFP-GPI enrichment in gut, and overlap on plasma membranes of intermediate mesoderm.
• GFP-GPI and myr-Venus colocalize with an anti-Golgi 58K antibody in ES cells induced to differentiate, confirming Golgi localization, but colocalization with secretory pathway markers in undifferentiated ES cells could not be established unequivocally because of their large nucleus, minimal cytoplasm and clumped growth.
• In ES cells and E9.5 somites incubated with Alexa-Fluor 568-conjugated 10K dextran, GFP-GPI and myr-Venus localize to vesicles that overlap with dextran-positive endosomes and also to vesicles that do not, which suggests that the two fusions occupy distinct vesicles in addition to endosomes of the secretory pathway.
• GFP-GPI and myr-Venus retain both fluorescence and subcellular localization after paraformaldehyde fixation of specimens, and fixed samples show the same localization as live samples, although fixation followed by detergent permeabilization for immunofluorescence gives a more diffuse localization than in live cells.

Abstract

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.