Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Three-dimensional arrangements of ribosomes inside fast growing E. coli cells
Translating bacterial ribosomes can form structurally ordered polysomes. The three-dimensional (3D) organization of such polysomes has been described in solution using cryoelectron tomography (CET) and template matching (Brandt et al., 2009). Also a characteristic arrangement of non-translating ribosome pairs has been described in starved E. coli cells (Ortiz et al., 2010). However, little is known about how polysomes are organized in their native cellular space when translating cytosolic or membrane proteins during the exponential phase of growth. We propose to examine the 3D arrangement of membrane associated ribosomes in situ, for an optimal preservation of polysomes architecture.
To enrich the population of ribosomes specially associated to the inner membrane, we expressed a transmembrane protein fused to a SecM-stalling sequence in slow- and fast-growing E. coli cells. The intact cells were rapidly frozen. These vitrified fast-growing cells (>800 nm in diameter) were physically sectioned using focused ion beam (FIB) technology to reduce the sample thickness (<400 nm) (Rigort et al., 2012). We applied dual-axis tilt CET to the FIB-milled samples. In order to localize 70S ribosomes inside tomograms we used a 3-D pattern recognition algorithm.
Advanced 3D imaging techniques of FIB-milled fast growing E. coli cells allowed a novel view of cellular ribosomes. The analysis of cytosolic ribosomes reveals a fraction of particles in similar 3D organization to the previously described for dense polysomes in vitro (Brandt et al., 2009). While in vitro studies allowed the visualization of translocating ribosomes bound to isolated membrane vesicles, those vesicles do not resemble the natural curvature of the inner membrane. Here, the in situ visualization of translocating ribosome was feasible in slow- and fast-growing cells.
Polysomal organizations, previously described only in vitro, are shown here to occur also in situ. The described approaches are also adequate to reveal structures of macromolecular complexes inside cells with unprecedented resolutions.
Ortiz, J. O., Brandt, F., Matias, V. R., Sennels, L., Rappsilber, J., Scheres, S. H., Eibauer, M., Hartl, F. U., Baumeister, W. (2010). J Cell Biology 190:613-621
Rigort, A., BÃ¤uerlein, F. J., Villa, E., Eibauer, M., Laugks, T., Baumeister, W., Plitzko, J. M. (2012) Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc Natl Acad Sci U S A. 109:4449-4454
Thomas Hoffmann 1, 2
1 Department of Structural Biology