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Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy.

Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS, Betzig E, Liphardt J - PLoS Biol. (2009)

Bottom Line: One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters.Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly.The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California Berkeley, Berkeley, California, United States of America.

ABSTRACT
The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

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PALM images of single cells reveal small chemotaxis clusters.Single-cell PALM images containing 3,000–13,000 labeled chemotaxis proteins per cell. Largest chemotaxis clusters are found at the poles, small lateral clusters are found in all cells. DIC images (inset) correspond to cell outlines (dashed lines). (A and B) Two representative Δtar cells with pALM6001 (Tar-mEos). (C and D) Two representative ΔcheW cells with pALM 5001 (tdEos-CheW). (E and F) Two representative ΔcheY cells with pALM5003 (CheY-tdEos). Although CheY-tdEos does not support chemotaxis, its abundance in polar regions suggests it retains functional interactions with chemotaxis clusters. (G) Fluorescent reporter tdEos (pALM5000) does not form clusters without fusion to chemotaxis proteins. (H) tdEos-CheW does not form clusters in a receptor knockout strain. Scale bar in (A–H) is 1 µm. (I and J) Histograms of the number of small clusters (10–100 proteins) of Tar-mEos ([I] n = 84 cells) or tdEos-CheW ([J] n = 130 cells). (K) Percentage of proteins that are found in small clusters (<100 proteins) or as solitary receptors. Error bars indicate the standard error of the mean.
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pbio-1000137-g003: PALM images of single cells reveal small chemotaxis clusters.Single-cell PALM images containing 3,000–13,000 labeled chemotaxis proteins per cell. Largest chemotaxis clusters are found at the poles, small lateral clusters are found in all cells. DIC images (inset) correspond to cell outlines (dashed lines). (A and B) Two representative Δtar cells with pALM6001 (Tar-mEos). (C and D) Two representative ΔcheW cells with pALM 5001 (tdEos-CheW). (E and F) Two representative ΔcheY cells with pALM5003 (CheY-tdEos). Although CheY-tdEos does not support chemotaxis, its abundance in polar regions suggests it retains functional interactions with chemotaxis clusters. (G) Fluorescent reporter tdEos (pALM5000) does not form clusters without fusion to chemotaxis proteins. (H) tdEos-CheW does not form clusters in a receptor knockout strain. Scale bar in (A–H) is 1 µm. (I and J) Histograms of the number of small clusters (10–100 proteins) of Tar-mEos ([I] n = 84 cells) or tdEos-CheW ([J] n = 130 cells). (K) Percentage of proteins that are found in small clusters (<100 proteins) or as solitary receptors. Error bars indicate the standard error of the mean.

Mentions: Strikingly, PALM images of all three strains (Tar, CheW, and CheY) revealed small lateral clusters and solitary receptors (Figure 3A–3F) not previously observed. All cells contained a significant fraction of receptors within small clusters or as solitary receptors (Figure 3K). For example, 38% of labeled Tar receptors were found outside of large clusters (>100 proteins). Most cells (∼95%) contained between one and 48 small clusters (<100 proteins) (Figure 3I and 3J). Small lateral clusters and solitary Tar receptors were observed in all expression conditions tested. When Tar-mEos was expressed at higher levels (1 mM IPTG induction), we saw banded patterns spanning the cell length (Figure S6) that may be helical structures reflecting the organization of the general protein translocation machinery, as previously observed [16]. Many small clusters and solitary receptors were present even at this higher level of expression.


Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy.

Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS, Betzig E, Liphardt J - PLoS Biol. (2009)

PALM images of single cells reveal small chemotaxis clusters.Single-cell PALM images containing 3,000–13,000 labeled chemotaxis proteins per cell. Largest chemotaxis clusters are found at the poles, small lateral clusters are found in all cells. DIC images (inset) correspond to cell outlines (dashed lines). (A and B) Two representative Δtar cells with pALM6001 (Tar-mEos). (C and D) Two representative ΔcheW cells with pALM 5001 (tdEos-CheW). (E and F) Two representative ΔcheY cells with pALM5003 (CheY-tdEos). Although CheY-tdEos does not support chemotaxis, its abundance in polar regions suggests it retains functional interactions with chemotaxis clusters. (G) Fluorescent reporter tdEos (pALM5000) does not form clusters without fusion to chemotaxis proteins. (H) tdEos-CheW does not form clusters in a receptor knockout strain. Scale bar in (A–H) is 1 µm. (I and J) Histograms of the number of small clusters (10–100 proteins) of Tar-mEos ([I] n = 84 cells) or tdEos-CheW ([J] n = 130 cells). (K) Percentage of proteins that are found in small clusters (<100 proteins) or as solitary receptors. Error bars indicate the standard error of the mean.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691949&req=5

pbio-1000137-g003: PALM images of single cells reveal small chemotaxis clusters.Single-cell PALM images containing 3,000–13,000 labeled chemotaxis proteins per cell. Largest chemotaxis clusters are found at the poles, small lateral clusters are found in all cells. DIC images (inset) correspond to cell outlines (dashed lines). (A and B) Two representative Δtar cells with pALM6001 (Tar-mEos). (C and D) Two representative ΔcheW cells with pALM 5001 (tdEos-CheW). (E and F) Two representative ΔcheY cells with pALM5003 (CheY-tdEos). Although CheY-tdEos does not support chemotaxis, its abundance in polar regions suggests it retains functional interactions with chemotaxis clusters. (G) Fluorescent reporter tdEos (pALM5000) does not form clusters without fusion to chemotaxis proteins. (H) tdEos-CheW does not form clusters in a receptor knockout strain. Scale bar in (A–H) is 1 µm. (I and J) Histograms of the number of small clusters (10–100 proteins) of Tar-mEos ([I] n = 84 cells) or tdEos-CheW ([J] n = 130 cells). (K) Percentage of proteins that are found in small clusters (<100 proteins) or as solitary receptors. Error bars indicate the standard error of the mean.
Mentions: Strikingly, PALM images of all three strains (Tar, CheW, and CheY) revealed small lateral clusters and solitary receptors (Figure 3A–3F) not previously observed. All cells contained a significant fraction of receptors within small clusters or as solitary receptors (Figure 3K). For example, 38% of labeled Tar receptors were found outside of large clusters (>100 proteins). Most cells (∼95%) contained between one and 48 small clusters (<100 proteins) (Figure 3I and 3J). Small lateral clusters and solitary Tar receptors were observed in all expression conditions tested. When Tar-mEos was expressed at higher levels (1 mM IPTG induction), we saw banded patterns spanning the cell length (Figure S6) that may be helical structures reflecting the organization of the general protein translocation machinery, as previously observed [16]. Many small clusters and solitary receptors were present even at this higher level of expression.

Bottom Line: One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters.Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly.The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California Berkeley, Berkeley, California, United States of America.

ABSTRACT
The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

Show MeSH
Related in: MedlinePlus