Volume 39, Issue 6, Pages 975-987 (September 2010) Imaging-Based Identification of a Critical Regulator of FtsZ Protofilament Curvature in Caulobacter  Erin D. Goley, Natalie A. Dye, John N. Werner, Zemer Gitai, Lucy Shapiro  Molecular Cell  Volume 39, Issue 6, Pages 975-987 (September 2010) DOI: 10.1016/j.molcel.2010.08.027 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 A Microscopy-Based Assay Distinguishes Division Proteins that Bind to FtsZ from Those that Do Not (A) DIC images of Caulobacter bearing a plasmid with PxylX driving expression of ftsZ∗ grown for 0 hr or 1.5 hr. (B) Localization of indicated fluorescent fusions to Caulobacter cell division proteins in otherwise wild-type cells. Scale bar in B represents 2 μm and applies to (B)–(E). (C and D) Localization of the indicated fluorescent fusions in cells overexpressing ftsZ∗. (E) Localization of Venus-FtsX or Venus-FtsW in cells overexpressing only ftsZ∗ (left) or ftsE and ftsZ∗ (right). (F) Localization of mCherry-MipZ in cells overexpressing ftsZ∗. In (B)–(F), fluorescence (red) overlaid on phase contrast (PC) (top) and fluorescence (bottom) is shown. Scale bar represents 2 μm. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 FzlA and FzlC Are Uncharacterized FtsZ-Binding Proteins (A) Localization of mCherry fusions to class I candidate proteins in cells overexpressing ftsZ∗. Fluorescence (red) overlaid on PC (top) and fluorescence (bottom) is shown. Scale bar represents 2 μm. (B) Localization of mCherry fusions to class II proteins in cells overexpressing ftsZ∗. Scale bar represents 2 μm. See also Figure S1. (C) Colocalization of mCherry fusions to FzlA and FzlC (red) with FtsZ-CFP (green) in wild-type cells. In the merged image, fluorescence is overlaid on phase contrast. Scale bar represents 2 μm. (D) Localization of mCherry fusions to FzlA and FzlC in cells depleted of FtsZ (−FtsZ) and after FtsZ depletion followed by repletion of FtsZ for 1 hr (FtsZ repletion). Fluorescence and PC images are shown. Scale bar represents 2 μm. (E) Coomassie-stained SDS-PAGE of supernatant (right) and pellet (left) from cosedimentation reactions containing 3 μM of the candidate protein ± 3 μM FtsZ. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 FzlA Is Critical for Cell Division and Is Cell Cycle Regulated (A) Genomic context of fzlA (WT) and construction of a xylose-dependent FzlA depletion strain (EG312). (B) Immunoblots of cell lysates from wild-type (WT) cells or cells from strain EG312 grown in the absence of xylose for the indicated times (hr). Equivalent total OD units of lysates were probed with antibodies recognizing the indicated proteins. (C) Phase-contrast images of cells of strain EG312 grown in the absence of xylose (FzlA depleted) for the indicated times. Scale bar represents 2 μm. (D) PC and mCherry-FzlA fluorescence images showing FzlA localization over the cell cycle. Synchronized swarmer cells were placed on M2G-agarose pads and imaged every 30 min. Scale bar represents 2 μm. (E) Normalized abundance of ftsZ and fzlA transcript levels over the cell cycle. (F) Immunoblots using antibodies against the indicated proteins of cell lysates from synchronized wild-type cells grown in M2G (top) or PYE (bottom). See also Figure S2. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 FzlA Affects FtsZ Structure In Vivo and Promotes Formation of Higher-Order FtsZ Structures In Vitro (A) Localization of FtsZ-CFP in cells from FzlA depletion strain EG316 grown in xylose (fzlA induced) or glucose (FzlA depleted) for 24 hr. Arrows indicate morphologically normal FtsZ rings. Scale bar represents 2 μm. (B) Localization of FtsZ-CFP in Caulobacter bearing a plasmid with PxylX driving overexpression of fzlA. Cells were grown for 23 hr in glucose (uninduced) or xylose (FzlA overproduced). Arrows indicate FtsZ-CFP foci associated with the sides of the cell. Hatched arrows indicate multiple mislocalized FtsZ-CFP foci. In (A) and (B), fluorescence (red) overlaid on phase contrast (PC) (left) and fluorescence (right) are shown. Scale bar represents 2 μm. (C) Left: Coomassie-stained SDS-PAGE of pellet fractions after high-speed centrifugation of FtsZ polymerization reactions containing the indicated concentrations of FzlA and 2 μM FtsZ. Right: bar graph depicting relative amount of FtsZ in the pellet at each FzlA concentration. Graph is normalized relative to FtsZ in the pellet with 0 μM FzlA and mean and SEM are shown (n = 3). (D) Coomassie-stained SDS-PAGE of pellet fractions after low-speed centrifugation of FtsZ polymerization reactions containing 2 μM FtsZ ± 4 μM FzlA. (E) Right-angle light scattering over time of FtsZ in the presence of the indicated concentrations of FzlA. Arrow indicates addition of FzlA or buffer control and hatched arrow indicates addition of GTP. (F) Right-angle light scattering reactions as in (E) but containing 0.1 mM of the indicated nucleotide. Four micromolar FzlA is included where indicated. Inset graph is an expanded view of the bottom of the graph at left. (G) Inorganic phosphate (Pi) concentration in solution over time in the presence of 2 μM FtsZ, 2 mM GTP, and the indicated concentrations of FzlA. GTPase rates (Pi released per FtsZ molecule per min) for each reaction are indicated. See also Figure S3. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 FzlA Stabilizes FtsZ In Vitro and In Vivo (A) Illustration of the localization of MipZ (yellow) and FtsZ (cyan) in swarmer cells (0 min after synchrony) and stalked cells (25 min after synchrony in PYE). (B) Bottom: Coomassie-stained SDS-PAGE of pellet fractions after high-speed centrifugation of FtsZ polymerization reactions containing the indicated concentrations of FzlA and 2 μM FtsZ ± 2 μM MipZ. Top: bar graph depicting relative amount of FtsZ in the pellet at each FzlA concentration. Bar graph is normalized relative to FtsZ in the pellet with 0 μM FzlA and 0 μM MipZ. Mean and SEM are shown (n = 3). (C) Bar graph depicting percentages of cells of EG496 and EG499 grown for 24 hr with xylose with the indicated localization of FtsZ and MipZ at 25 min after synchrony. Mean and SEM are shown. (D) Representative images of cells from (C). Arrows indicate polar foci of FtsZ-CFP that overlap with MipZ-YFP. Scale bar represents 2 μm. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Helical Bundles of FtsZ Form in the Presence of FzlA (A) Negative-stain EM of 2 μM FtsZ under polymerizing conditions in the absence (left) or presence (right) of 4 μM FzlA at the indicated magnifications. Scale bars represent 100 nm. (B) Negative-stain EM of 2 μM FtsZ with 4 μM FzlA applied to grids at the indicated times after addition of GTP. Scale bars represent 100 nm. (C) Negative-stain EM of 2 μM FtsZ with the indicated concentrations of FzlA. Scale bars represent 50 nm. See also Figure S4. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 The Effect of FzlA on FtsZ Polymerization Is Nucleotide Dependent (A) Coomassie-stained SDS-PAGE of pellet fractions after high-speed centrifugation of FtsZ polymerization reactions containing 2 μM FtsZ, ± 4 μM FzlA and 0.2 mM of the indicated nucleotide. (B) Right-angle light scattering of FtsZ polymerization reactions ± 4 μM FzlA and the indicated nucleotides (GTP and GDP were used at 2 mM and GMP-CPP was used at 0.2 mM). (C) Negative-stain EM of FtsZ polymerization reactions containing 2 μM FtsZ, ± 4 μM FzlA, and 0.2 mM of the indicated nucleotide. Mean diameter ± standard deviation is indicated for each. Scale bars represent 50 nm. See also Figure S5. (D) Speculative model for formation of FzlA-FtsZ helical bundles in the presence of GTP. FzlA binds to monomers or short oligomers of FtsZ and promotes elongation of paired, curved FtsZ protofilaments with the indicated dimensions. Molecular Cell 2010 39, 975-987DOI: (10.1016/j.molcel.2010.08.027) Copyright © 2010 Elsevier Inc. Terms and Conditions