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Intracellular Dynamics of Bacterial Proteins Are Revealed by Super-resolution Microscopy
Biophysical Journal Volume 105 October 2013 1547–1548
1547
New and Notable Intracellular Dynamics of Bacterial Proteins Are Revealed by Super-resolution Microscopy Julie S. Biteen* Department of Chemistry, University of Michigan, Ann Arbor, Michigan
Fluorescence imaging has been uncovering subcellular details in eukaryotic cells for many decades. However, prokaryotes, with their small size, have more typically been characterized and defined only by the absence of intracellular structures like the nucleus and organelles. It is now obvious that very careful, precise protein localization and dynamics must be at play to ensure the proper behavior, division, and reproduction of prokaryotes. Accordingly, an explosion of interest in bacterial cell biology began ~15 years ago (1), and over the past five years, the application of superresolution imaging techniques to bacteria has allowed the inner workings of live bacteria cells to be directly probed, further uncovering the mysteries of subcellular microbiology (2). The organization of proteins in bacteria cells is only just beginning to be understood. The actin homolog MreB has been at the center of many investigations. In particular, an open question in microbiology remains: how does MreB direct and maintain bacterial shape? In vitro, this protein can assemble into various filamentous structures, but the in vivo dynamics of MreB, and its association with peptidoglycan (PG) in the cell wall, remain unclear. With the advent of high-resolution optical microscopy, the dynamics of MreB molecules and assemblies have been studied extensively in live cells of the model
Submitted July 31, 2013, and accepted for publication August 19, 2013. *Correspondence: [email protected] Editor: David Odde. Ó 2013 by the Biophysical Society 0006-3495/13/10/1547/2 $2.00
organisms Caulobacter crescentus, Escherichia coli, and Bacillus subtilis, using epi-fluorescence microscopy (3–7), spinning disk confocal microscopy (8), single-molecule-based approaches (9,10), and total internal reflection fluorescence (TIRF) microscopy (11). Taken together with biochemical data, this leads to an emerging consensus that MreB forms short cytoplasmic filaments, which interact with inner-membrane proteins MreC, MreD, and RodZ and lipid II synthesis enzymes MraY and MurG to spatially direct PG precursor synthesis proteins to insertion points along the cell wall (12). In this issue of the Biophysical Journal, von Olshausen et al. (13) couple structured illumination microscopy (SIM) to TIRF microscopy to achieve 120-nm resolution images of MreB in live B. subtilis (14). With TIRF-SIM, the authors detect filaments of lengths up to ~1 mm in the cells. These MreB strands are transported as a whole, and move perpendicular to the long axis of the cell. Importantly, relative to other superresolution studies in live bacterial cells, the TIRF-SIM technique is very fast, here allowing frame rates up to 0.8 Hz. This enables von Olshausen et al. (13) to study MreB filament dynamics with high temporal resolution and to propose a model of filament transport by coupled PG synthesis motors that bind and unbind stochastically via a transmembrane complex to MreB. The authors find a nonmonotonic filament length-dependent translation speed: short filaments move faster as they increase in length whereas longer filaments move slower as they increase further in length. This length dependence, coupled with the observation that the filaments move equally in both directions, is explained by a model that evokes the application of force by motors near the cell membrane: the average velocity decreases linearly by opposing forces, i.e., the friction generated by the bound motors. In analogy
with models for eukaryotic systems (15), this mechanistic multiple-motor model is consistent with MreB filament motion driven by the addition of PG monomers to the ends of PG strands, and is corroborated by the authors’ observations in protoplasts, where MreB filaments remain stationary in the absence of a cell wall. By addressing the problem of MreB filament motion with TIRF-SIM, von Olshausen et al. not only improve our understanding of MreB dynamics, but also demonstrate that the low photobleaching and fast imaging speeds afforded by this technique should be widely applicable to other questions in microbiology. REFERENCES 1. Callaway, E. 2008. Cell biology. Bacteria’s new bones. Nature. 451:124–126. 2. Biteen, J. S., and W. E. Moerner. 2010. Single-molecule and superresolution imaging in live bacteria cells. Cold Spring Harb. Perspect. Biol. 2:a000448. 3. Gitai, Z., N. Dye, and L. Shapiro. 2004. An actin-like gene can determine cell polarity in bacteria. Proc. Natl. Acad. Sci. USA. 101:8643–8648. 4. Swulius, M. T., and G. J. Jensen. 2012. The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-terminal yellow fluorescent protein tag. J. Bacteriol. 194:6382–6386. 5. Jones, L. J. F., R. Carballido-Lo´pez, and J. Errington. 2001. Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell. 104:913–922. 6. Figge, R. M., A. V. Divakaruni, and J. W. Gober. 2004. MreB, the cell shapedetermining bacterial actin homologue, coordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 51:1321–1332. 7. van Teeffelen, S., S. Wang, ., Z. Gitai. 2011. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc. Natl. Acad. Sci. USA. 108:15822– 15827. 8. Garner, E. C., R. Bernard, ., T. Mitchison. 2011. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science. 333:222–225. 9. Kim, S. Y., Z. Gitai, ., W. E. Moerner. 2006. Single molecules of the bacterial actin
http://dx.doi.org/10.1016/j.bpj.2013.08.022
1548
Biteen
MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc. Natl. Acad. Sci. USA. 103:10929–10934.
ment of MreB-associated cell wall biosynthetic complexes in bacteria. Science. 333:225–228.
10. Biteen, J. S., M. A. Thompson, ., W. E. Moerner. 2008. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat. Methods. 5:947–949. 11. Domı´nguez-Escobar, J., A. Chastanet, ., R. Carballido-Lo´pez. 2011. Processive move-
12. Typas, A., M. Banzhaf, ., W. Vollmer. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10:123–136.
Biophysical Journal 105(7) 1547–1548
13. Cragg, G. E., and P. T. C. So. 2000. Lateral resolution enhancement with standing evanescent waves. Opt. Lett. 25:46–48.
14. von Olshausen, P., H. J. D. Soufo, ., A. Rohrbach. 2013. Super-resolution imaging of dynamic MreB filaments in B. subtilis— a multiple motor-driven transport? Biophys. J. 105:1171–1181. 15. Klumpp, S., and R. Lipowsky. 2005. Cooperative cargo transport by several molecular motors. Proc. Natl. Acad. Sci. USA. 102:17284–17289.
1547
New and Notable Intracellular Dynamics of Bacterial Proteins Are Revealed by Super-resolution Microscopy Julie S. Biteen* Department of Chemistry, University of Michigan, Ann Arbor, Michigan
Fluorescence imaging has been uncovering subcellular details in eukaryotic cells for many decades. However, prokaryotes, with their small size, have more typically been characterized and defined only by the absence of intracellular structures like the nucleus and organelles. It is now obvious that very careful, precise protein localization and dynamics must be at play to ensure the proper behavior, division, and reproduction of prokaryotes. Accordingly, an explosion of interest in bacterial cell biology began ~15 years ago (1), and over the past five years, the application of superresolution imaging techniques to bacteria has allowed the inner workings of live bacteria cells to be directly probed, further uncovering the mysteries of subcellular microbiology (2). The organization of proteins in bacteria cells is only just beginning to be understood. The actin homolog MreB has been at the center of many investigations. In particular, an open question in microbiology remains: how does MreB direct and maintain bacterial shape? In vitro, this protein can assemble into various filamentous structures, but the in vivo dynamics of MreB, and its association with peptidoglycan (PG) in the cell wall, remain unclear. With the advent of high-resolution optical microscopy, the dynamics of MreB molecules and assemblies have been studied extensively in live cells of the model
Submitted July 31, 2013, and accepted for publication August 19, 2013. *Correspondence: [email protected] Editor: David Odde. Ó 2013 by the Biophysical Society 0006-3495/13/10/1547/2 $2.00
organisms Caulobacter crescentus, Escherichia coli, and Bacillus subtilis, using epi-fluorescence microscopy (3–7), spinning disk confocal microscopy (8), single-molecule-based approaches (9,10), and total internal reflection fluorescence (TIRF) microscopy (11). Taken together with biochemical data, this leads to an emerging consensus that MreB forms short cytoplasmic filaments, which interact with inner-membrane proteins MreC, MreD, and RodZ and lipid II synthesis enzymes MraY and MurG to spatially direct PG precursor synthesis proteins to insertion points along the cell wall (12). In this issue of the Biophysical Journal, von Olshausen et al. (13) couple structured illumination microscopy (SIM) to TIRF microscopy to achieve 120-nm resolution images of MreB in live B. subtilis (14). With TIRF-SIM, the authors detect filaments of lengths up to ~1 mm in the cells. These MreB strands are transported as a whole, and move perpendicular to the long axis of the cell. Importantly, relative to other superresolution studies in live bacterial cells, the TIRF-SIM technique is very fast, here allowing frame rates up to 0.8 Hz. This enables von Olshausen et al. (13) to study MreB filament dynamics with high temporal resolution and to propose a model of filament transport by coupled PG synthesis motors that bind and unbind stochastically via a transmembrane complex to MreB. The authors find a nonmonotonic filament length-dependent translation speed: short filaments move faster as they increase in length whereas longer filaments move slower as they increase further in length. This length dependence, coupled with the observation that the filaments move equally in both directions, is explained by a model that evokes the application of force by motors near the cell membrane: the average velocity decreases linearly by opposing forces, i.e., the friction generated by the bound motors. In analogy
with models for eukaryotic systems (15), this mechanistic multiple-motor model is consistent with MreB filament motion driven by the addition of PG monomers to the ends of PG strands, and is corroborated by the authors’ observations in protoplasts, where MreB filaments remain stationary in the absence of a cell wall. By addressing the problem of MreB filament motion with TIRF-SIM, von Olshausen et al. not only improve our understanding of MreB dynamics, but also demonstrate that the low photobleaching and fast imaging speeds afforded by this technique should be widely applicable to other questions in microbiology. REFERENCES 1. Callaway, E. 2008. Cell biology. Bacteria’s new bones. Nature. 451:124–126. 2. Biteen, J. S., and W. E. Moerner. 2010. Single-molecule and superresolution imaging in live bacteria cells. Cold Spring Harb. Perspect. Biol. 2:a000448. 3. Gitai, Z., N. Dye, and L. Shapiro. 2004. An actin-like gene can determine cell polarity in bacteria. Proc. Natl. Acad. Sci. USA. 101:8643–8648. 4. Swulius, M. T., and G. J. Jensen. 2012. The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-terminal yellow fluorescent protein tag. J. Bacteriol. 194:6382–6386. 5. Jones, L. J. F., R. Carballido-Lo´pez, and J. Errington. 2001. Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell. 104:913–922. 6. Figge, R. M., A. V. Divakaruni, and J. W. Gober. 2004. MreB, the cell shapedetermining bacterial actin homologue, coordinates cell wall morphogenesis in Caulobacter crescentus. Mol. Microbiol. 51:1321–1332. 7. van Teeffelen, S., S. Wang, ., Z. Gitai. 2011. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc. Natl. Acad. Sci. USA. 108:15822– 15827. 8. Garner, E. C., R. Bernard, ., T. Mitchison. 2011. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science. 333:222–225. 9. Kim, S. Y., Z. Gitai, ., W. E. Moerner. 2006. Single molecules of the bacterial actin
http://dx.doi.org/10.1016/j.bpj.2013.08.022
1548
Biteen
MreB undergo directed treadmilling motion in Caulobacter crescentus. Proc. Natl. Acad. Sci. USA. 103:10929–10934.
ment of MreB-associated cell wall biosynthetic complexes in bacteria. Science. 333:225–228.
10. Biteen, J. S., M. A. Thompson, ., W. E. Moerner. 2008. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat. Methods. 5:947–949. 11. Domı´nguez-Escobar, J., A. Chastanet, ., R. Carballido-Lo´pez. 2011. Processive move-
12. Typas, A., M. Banzhaf, ., W. Vollmer. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 10:123–136.
Biophysical Journal 105(7) 1547–1548
13. Cragg, G. E., and P. T. C. So. 2000. Lateral resolution enhancement with standing evanescent waves. Opt. Lett. 25:46–48.
14. von Olshausen, P., H. J. D. Soufo, ., A. Rohrbach. 2013. Super-resolution imaging of dynamic MreB filaments in B. subtilis— a multiple motor-driven transport? Biophys. J. 105:1171–1181. 15. Klumpp, S., and R. Lipowsky. 2005. Cooperative cargo transport by several molecular motors. Proc. Natl. Acad. Sci. USA. 102:17284–17289.