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, 23 (9), 1131-44

Bacterial Intermediate Filaments: In Vivo Assembly, Organization, and Dynamics of Crescentin

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Bacterial Intermediate Filaments: In Vivo Assembly, Organization, and Dynamics of Crescentin

Godefroid Charbon et al. Genes Dev.

Abstract

Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin also shares in vivo properties of assembly and dynamics with IF proteins by forming stable filamentous structures that continuously incorporate subunits along their length and that grow in a nonpolar fashion. De novo assembly of crescentin is biphasic and involves a cell size-dependent mechanism that controls the length of the structure by favoring lateral insertion of crescentin subunits over bipolar longitudinal extension when the structure ends reach the cell poles. The crescentin structure is stably anchored to the cell envelope, and this cellular organization requires MreB function, identifying a new function for MreB and providing a parallel to the role of actin in IF assembly and organization in metazoan cells. Additionally, analysis of an MreB localization mutant suggests that cell wall insertion during cell elongation normally occurs along two helices of opposite handedness, each counterbalancing the other's torque.

Figures

Figure 1.
Figure 1.
Crescentin forms a stable and cohesive filamentous structure. (A) Western blot of crescentin protein levels during the cell cycle of wild-type CB15N cells. Samples from synchronized cell cycle populations were sonicated, and 20 μg of total protein were loaded. Anti-CtrA and anti-MreB antibodies were used to assess synchrony quality and sample loading, respectively. (SW) Swarmer cell; (ST) stalked cell; (PD) predivisional cell. (B) Time-lapse series of the GFP-labeled crescentin structure (red, overlaid over phase images) during the course of the cell cycle, starting with synchronized CJW815 (CB15N creS∷pBGST18creS-gfp∷pBGENTcreS) swarmer cells. The cell shown represents a common example in which the expansion of the crescentin structure was delayed, resulting in crescentin-free regions (asterisk). Elongation of the crescentin structure toward the stalked pole later resumed (>>>). Bar, 1 μm. (C) Photobleaching analysis of crescentin structure dynamics. After pretreatment with chloramphenicol (300 μg/mL) for 30 min to block protein synthesis, CJW815 cells were placed on an agarose pad containing 300 μg/mL chloramphenicol for 10 min before photobleaching. The panel shows a representative cell in which the central part of the GFP-labeled crescentin structure was bleached. Bar, 1 μm. (D) Quantitative analysis of time-lapse recordings of 32 photobleached crescentin structures treated as described in C. Blue diamonds and green squares show the relative fluorescence intensities of bleached and unbleached regions of crescentin structures, respectively (as illustrated in the schematic). The data were corrected for background fluorescence and for photobleaching associated with image acquisition. (E) CJW2208 cells (CB15N Pvan∷pBGENTPvancreS-tc) grown in the presence of 5 μM vanillic acid to induce the synthesis of crescentin-TC. These cells were labeled with FlAsH then washed and placed on an agarose-padded slide devoid of vanillic acid to stop further synthesis of crescentin-TC while endogenous crescentin was still being produced (from its native promoter).
Figure 2.
Figure 2.
After initial pole-to-pole expansion along a straight line, the crescentin structure thickens through lateral association of crescentin anywhere along the existing structure. (A) CJW815 (CB15N creS∷pBGST18creS-gfp∷pBGENTcreS) cells expressing both creS and creS-gfp from the native promoter were placed on an agarose pad and whole cells were photobleached. Accumulation of newly synthesized GFP-crescentin was followed over time by time-lapse fluorescence microscopy as cells grew and produced new crescentin-GFP molecules. (B) Three-dimensional fluorescence intensity plot of the cell shown in A. (C, left) CJW1782 cells (CB15N ΔcreS Pxyl∷pBGENTPxylcreS-tc) grown in the presence of glucose (instead of xylose) to repress crescentin-TC synthesis (panel i) and CJW1647 cells (CB15N ΔcreS pMR10PxylcreS) producing untagged crescentin in xylose-containing medium (panel ii) were stained with FlAsH and used here as controls to show the background fluorescence due to FlAsH staining. (Right) Crescentin-TC synthesis in CJW1782 cells was induced by adding xylose in the culture medium. (Panels iiivii) Cell samples were stained with FlAsH to visualize crescentin-TC at indicated times following xylose addition. (D) Synthesis of a GFP-labeled crescentin structure in CJW2207 cells (CB15N ΔcreS Pvan∷pBGENTPvancreS-tc∷pHL32PvancreS-gfp) was induced on an agarose-padded slide containing vanillic acid (5 μM). DIC image and corresponding crescentin-GFP signal were recorded by time-lapse microscopy. (E) Linescans of crescentin-GFP signal intensity across the length of the cell represented in D.
Figure 3.
Figure 3.
Crescentin assembly is biphasic. (A) CJW2209 cells (CB15N ftsZ∷pBJM1 ΔcreS Pvan∷pBGENTPvancreS-tc∷pHL32PvancreS-gfp) from a PYE xylose culture (allowing ftsZ expression and cell division) were placed on an agarose-padded slide with vanillic acid but without xylose to induce crescentin-TC and crescentin-GFP while depleting FtsZ and blocking cell division. Time-lapse imaging followed the assembly of the GFP-labeled crescentin structure during cell filamentation. Selected images are shown. Colored dashed lines represent regions used for the linescans shown in B. (B) Linescans of crescentin-GFP signal intensity along the length of the cell shown in A. (C) The length of the cell (blue) shown in A and the length of its GFP-labeled crescentin structure (red) were plotted over time. (D) YB1585 cells (CB15N ftsZ∷pBJM1) carrying ftsZ under the xylose-inducible promoter were grown without xylose for 6 h to deplete FtsZ, then imaged in liquid (i.e., without cell immobilization) using phase contrast microscopy and 1 msec exposure time. (E) After growth in PYE xylose medium, CJW2281 cells (CB15N ftsZ∷pBJM1 ΔcreS Pvan∷pBGENTPvancreS-tc) were transferred to liquid PYE medium containing 50 μM vanillic acid but no xylose in order to simultaneously induce crescentin-TC synthesis while depleting FtsZ. After 4 h of growth, the cells were stained with FlAsH and mounted on an agarose-padded slide to visualize the crescentin-TC structure.
Figure 4.
Figure 4.
Elongation of the crescentin structure is determined by cell length. (A) Cells of different length were generated by depleting CJW2209 cells (CB15N ftsZ∷pBJM1 ΔcreS Pvan∷pBGENTPvancreS-tc∷pHL32PvancreS-gfp) of FtsZ for 0–150 min. Synthesis and assembly of a GFP-labeled crescentin structure was then induced on agarose-padded slides containing vanillic acid (to induce creS-tc and creS-gfp) and lacking xylose (to maintain repression of ftsZ expression). Ensuing cell growth and extension of the crescentin structure were then immediately recorded by time-lapse microscopy. The length of the crescentin structure after 300 min of growth was then plotted as a function of cell length at t = 0 (n = 66). (B) Same as A except that the growth and extension of the crescentin structure recorded during time-lapse microscopy was plotted over time. The top and bottom plots show examples of a “short” cell (∼3 μm) and a “long” cell (∼10 μm), respectively, at the start of the time lapse (t = 0). (C) Same as B except that the length of the crescentin structure was plotted as a function of cell elongation (i.e., cell length increment) rather than time. Shown are examples of cells ranging from 2.7 μm to 11.5 μm at the time of initiation of crescentin-GFP synthesis. (D) Same as A except that shown are DIC and fluorescence micrographs of a short cell (left) and long cell (right) at t = 0 and after t = 300 min. The long cell was generated by depletion of FtsZ in PYE glucose liquid medium for 150 min prior to induction of GFP-crescentin synthesis on the agarose-padded slide containing vanillic acid and lacking xylose.
Figure 5.
Figure 5.
De novo assembly of the crescentin structure can occur and generate cell curvature on any side of the cell regardless of pre-existing cell curvature constraints. (A) CJW2046 cells (CB15N ftsZ∷pBJM1 creS∷pBGST18creS-gfp∷pBGENTcreS) producing crescentin and crescentin-GFP from the endogenous promoter were depleted of FtsZ for 4 h, then placed on an agarose pad containing xylose to replete FtsZ and subjected to time-lapse microscopy. Selected images at indicated times (minutes) are shown. Fluorescent images (top row) show the GFP-labeled crescentin signal, which was overlaid (red) with the corresponding DIC images (bottom row). The arrow indicates a crescentin structure assembling on the outer curvature of the cell. Red dotted lines show cell outlines obtained from the DIC images. (B) Same as in A with top images showing DIC and fluorescent images at t = 0 min and bottom images showing ensuing time-lapse images of the cell region defined by the white box in the top DIC image. Arrows indicate a division site.
Figure 6.
Figure 6.
Proper organization of the crescentin structure in the cell requires MreB function. (A) Images of CJW815 cells (CB15N creS∷pBGST18creS-gfp∷pBGENTcreS) producing crescentin and crescentin-GFP from the native promoter and treated with 50 μM A22 for 3 h prior to imaging. Asterisk shows an example of two crescentin structures within a single cell. (B) Time-lapse images of CJW2046 cells (CB15N ftsZ∷pBJM1 creS∷pBGST18creS-gfp∷pBGENTcreS) producing crescentin and crescentin-GFP from the native promoter. Cells were grown in absence of xylose to deplete FtsZ for 2 h and then placed on an agarose pad with xylose and 25 μM A22 to disrupt MreB localization while restoring ftsZ expression. Restoring FtsZ synthesis was necessary to prevent growth arrest, which occurs when both FtsZ and MreB functions are disrupted. Selected images are shown; see Supplemental Movie S4 for the whole sequence. (C) Time-lapse sequence of CJW2207 cells (CB15N ΔcreS Pvan∷pBGENTPvancreS-tc∷pHL32PvancreS-gfp) grown in PYE liquid medium containing 50 μM A22 for 330 min prior to simultaneous induction of crescentin-TC and crescentin-GFP synthesis on an agarose-padded slide containing 50 μM vanillic acid and 25 μM A22 to disrupt MreB localization. Imaging shows the localization of the GFP-labeled crescentin structure. (D) Fluorescence and DIC micrographs of CJW1790 cells (CB15N mreBG165D Pxyl∷pXGFP4-C1mreBG165D) producing GFP-MreBG165D. (E) DIC images of CB15N cells (wt), CJW763 cells (CB15N creS∷Tn5), CJW1789 cells (CB15N mreBG165D), and CJW1788 cells (CB15N mreBG165D creS∷Tn5). (F) Scanning electron micrographs of CJW1789 cells (CB15N mreBG165D). (G) Straight CJW1791 cells (CB15N mreBG165D creS∷pBGST18creS-gfp∷pBGENTcreS) with a detached GFP-labeled crescentin structure. (H) Crescentin-Flag coimmunoprecipitation with MreB using anti-Flag antibodies. Protein extracts from wild-type CB15N C. crescentus cells were prepared (negative control as they lack Flag-tagged proteins) along with extracts from CJW932 cells (CB15N creS∷pJM21creS-flag) producing crescentin-Flag at endogenous levels in place of crescentin. After immunoprecipitation (using anti-Flag antibody) and elution with Flag peptide, each eluate was loaded on a SDS-PAGE gel and analyzed by Western blotting using anti-Flag and anti-MreB antibodies. Also shown is a corresponding Western blot of cell lysates (input; 20 μg of total protein).
Figure 7.
Figure 7.
Aberrant growth of the mreBG165D mutant causes cell twisting on solid medium or when an internal crescentin structure is present. (A) Localization of the GFP-labeled crescentin structure in a sigmoid mreBG165D CJW1791 cell (CB15N mreBG165D creS∷pBGST18creS-gfp∷pBGENTcreS). (B, left) FlAsH-stained CJW1785 cells (CB15N mreBG165D creS∷Tn5 Pxyl∷pBGENTPxylcreS-tc) grown in PYE glucose liquid medium to show background fluorescence due to FlAsH staining. (Right) Induction of crescentin-TC synthesis in CJW1785 cells was achieved by adding xylose to the liquid culture. Cells were sampled at indicated times and stained with FlAsH to visualize crescentin-TC. (C) Time-lapse images of CJW2883 cells (CB15N mreBG165D creS∷Tn5 Pvan∷pBGENTPvancreS-tc∷pHL32PvancreS-gfp ftsZ∷pXMCS7ftsZ) carrying the mreBG165D mutation. Cells were first grown in absence of xylose to deplete FtsZ for 1 h, after which synthesis of a GFP-labeled crescentin structure was induced on an agarose-padded slide containing vanillic acid. Selected images of the GFP-labeled crescentin structure (in red in the overlays) at indicated times (minutes) are shown. (D) DIC micrographs of FtsZ-depleted CJW2882 cells (CB15N mreBG165D creS∷Tn5 ftsZ∷pXMCS7ftsZ) after growth and filamentation in PYE liquid culture (left) or on an agarose pad (right). Arrows indicate regions of cell twisting. (E) Cartoons illustrating how cells carrying the mreBG165D mutation grow in the absence (left) or presence (right) of a crescentin structure. Cell elongation causes the two poles to rotate in opposite directions relative to each other. Thus, growth causes fixed points on the cell wall to move relative to each other according to the pole rotation pattern. A crescentin structure that is connected to the cell wall will be twisted by growth, thereby modifying the curvature of the cell.

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