In situ structure of FtsZ mini-rings in Arabidopsis chloroplasts
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Chloroplasts are essential plant organelles that divide by binary fission through a coordinated ring-shaped division machinery located both on the outside and inside of the chloroplast. The first step in chloroplast division is the assembly of an internal division ring (Z-ring) that is composed of the key filamentous chloroplast division proteins FtsZ1 and FtsZ2. How the individual FtsZ filaments assemble into higher-order structures to form the dividing Z-ring is not well understood and the most detailed insights have so far been gleaned from prokaryotic FtsZ. Here, we present in situ data of chloroplast FtsZ making use of a smaller ring-like FtsZ assembly termed mini-rings that form under well-defined conditions. Structured illumination microscopy (SIM) permitted their mean diameter to be determined as 208 nm and also showed that 68 % of these rings are terminally attached to linear FtsZ filaments. A correlative microscopy-compatible specimen preparation based on freeze substitution after high-pressure freezing is presented addressing the challenges such as autofluorescence and specific fluorescence attenuation. Transmission electron microscopy (TEM) and scanning TEM (STEM) imaging of thin sections exhibited ring-like densities that matched in size with the SIM data, and TEM tomography revealed insights into the molecular architecture of mini-rings demonstrating the following key features: (1) overall, a roughly bipartite split into a more ordered/curved and less ordered/curved half is readily discernible; (2) the density distribution in individual strands matches with the X-ray data, suggesting they constitute FtsZ protofilaments; (3) in the less ordered half of the ring, the protofilaments are able to assemble into higher-order structures such as double helices and supercoiled structures. Taken together, the data suggest that the state of existence of mini-rings could be described as metastable and their possible involvement in filament storage and Z-ring assembly is discussed.
KeywordsChloroplast division Correlative imaging FtsZ mini-rings Fluorescence microscopy Transmission electron microscopy Electron tomography Structured illumination microscopy High-pressure freezing Freeze substitution
Chloroplasts are essential plant organelles that arose from cyanobacterial ancestors by the process of endosymbiosis. Chloroplast division is required to maintain both the size and number of chloroplasts, which in turn affect photosynthetic performance of the plant via the control of surface-to-volume ratio with larger ratios facilitating the exchange of metabolites between the chloroplast and the cytosol. The size of starch-storing plastids (amyloplasts) affects the properties of starch granules. Controlling the latter in crops is of interest to industries concerned with the applications of starch [1, 2], and to this end it is critical to understand by what mechanism plastid/chloroplast size is controlled. Basically, both bacteria and chloroplasts divide by the process of binary fission. Following on from that, the division process is initiated by assembly of the cytoskeletal protein FtsZ into filaments that form a ring structure, the Z-ring, at the division site [3, 4]. Plants and algae encode two conserved FtsZ families, FtsZ1 and FtsZ2 [5, 6, 7], which are functionally different and are both required for plastid division and plastid size control. Changes in FtsZ expression levels or assembly lead to dramatic phenotypes such as single giant chloroplasts covering entire mesophyll cells [8, 9, 10]. Z-ring formation/assembly depends on the balance between FtsZ and accessory chloroplast division proteins, which either stabilize (ARC6 [11, 12], MinE [3, 13, 14]) or destabilize (ARC3 , PARC6 ) FtsZ assemblies.
The properties and the assembly mechanism of plant FtsZ1 and FtsZ2 have been explored in vitro. FtsZ1 and FtsZ2 have differential guanosine triphosphatase (GTPase) activity [17, 18] and are capable of linear co-assembly to form heteropolymers . FtsZ1 and FtsZ2 exhibit dynamic turnover in vivo  that is promoted by the ARC3 protein . How the individual FtsZ filaments assemble into higher-order structures to form the chloroplast dividing Z-ring is not well understood. Some insight has been gained from studies with FtsZ from prokaryotes. Cryo-TEM tomography revealed that the Z-ring is composed of short, partially overlapping protofilaments . In contrast, in vitro reconstitution with only FtsZ and FtsA showed a continuous protofilament encircling the constricted liposomes several times . The constriction force is thought to result from either bending of FtsZ protofilaments upon guanosine triphosphate (GTP) hydrolysis [23, 24] or, as was proposed recently, may involve protofilament sliding that is independent of GTP hydrolysis . GTP hydrolysis and dynamic turnover are nevertheless essential for spatial regulation of Z-rings by accessory proteins that also mediate remodeling of FtsZ filaments into highly curved bundles and vortices [25, 26].
In contrast to bacterial FtsZ, the in vitro assembled plant FtsZ1 and FtsZ2 proteins did not show highly curved filaments [17, 27], hinting that in chloroplast the curvature and force for constriction could be generated by other components of the division machinery, such as the dynamin-related protein ARC5 residing on the outside of the chloroplast envelope [28, 29]. The arrangement of FtsZ protofilaments in the Z-ring in chloroplasts and how they bend and proceed in chloroplast division remains unknown. Highly curved, small ring-like FtsZ assemblies, termed mini-rings, have been reported in plant chloroplasts and studied by optical microscopy [3, 15, 30, 31]. It is hypothesized that the structural signature principles identified in these highly curved small assemblies may also play a role in the actual Z-ring. In this study, a detailed analysis of FtsZ mini-ring structure was conducted using SIM and electron tomography in Arabidopsis thaliana chloroplasts.
Arabidopsis thaliana was chosen for this study as it is an established model organism. The A. thaliana ecotype Columbia (Col-0) and the mutants used in this study were obtained from the Arabidopsis Biological Resource Center (ABRC; http://www.arabidopsis.org): arc6 (AT5G42480, CS286) and arc12 (At1G69390, CS16472). Negative control plants were untransformed ftsZ2-1/2-2 double knockout plants . The AtFtsZ2–mYFP construct has been described previously . Plants were grown in Redi-Earth (SunGro Horticulture, Bellevue, WA, USA) substrate in a rooftop greenhouse at a temperature of 72 °C and a relative humidity of 62 %. Agrobacterium-mediated transformation of Arabidopsis wild-type Col-0 plants and selection of the herbicide-resistant seedlings were performed as described [4, 32].
Immunofluorescence labeling and optical microscopy
Leaf and shoot meristematic tissue was fixed, embedded in Steedman’s wax, immunolabeled with anti-FtsZ2 antibody and documented by wide-field fluorescence microscopy as described previously . Live imaging was performed using an Olympus FV1000 confocal microscope (Olympus Scientific Solutions America, Waltham, MA, USA) equipped with a 60×/1.2 water immersion objective and a 515-nm laser for excitation. Prior to imaging, a drop of perfluorodecalin  was placed on the leaf tissue to remove air pockets from intercellular spaces and ensure good optical quality. YFP fluorescence was detected through a 535- to 575-nm bandpass filter, while the chlorophyll autofluorescence was collected in the 670- to 700-nm range. Sections from resin-embedded tissue after HPF–FS were mounted in immersion oil and imaged using a 100×/1.4 oil immersion objective.
For structured illumination microscopy, leaf tissue was formaldehyde fixed under microwave irradiation, a process that preserves both the structural detail and the fluorescent protein signal [20, 34]. The tissue was vacuum infiltrated with 3 % formaldehyde in phosphate-buffered saline (PBS, 0.14 M NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 5 mM KH2PO4, 3.0 mM NaN3; pH = 7.3) and incubated at room temperature for 30 min. It was then fixed in a Pelco Biowave (Ted Pella, Inc., Redding, CA, USA) laboratory microwave processor equipped with a ColdSpot® temperature control system, with power set to 250 W and a 6-min cycle (2 min on, 2 min off, 2 min on). The temperature cutoff was set to 37 °C. The tissue was then rinsed in PBS three times, with microwave irradiation 1 min at 250 W power in each rinse and then gradually infiltrated with 30, 50, 80 % v/v Glycerol in PBS mixture, with microwave irradiation of 1 min at 250 W power in each step. The tissue was then left in fresh 80 % v/v glycerol/PBS at 4 °C overnight and then either sent for imaging on the OMX microscope to GE Healthcare (formerly Applied Precision, Inc. Issaquah, WA, USA) or imaged on a Zeiss Elyra-S system. OMX imaging was performed using a 100×/1.4 oil immersion objective, excitation 488 nm, emission centered at 525 nm, and voxel size 40 × 40 × 120 nm. Elyra-S imaging was performed with a 63×/1.4 oil immersion objective and voxel size 40 × 40 × 110 nm. Mini-ring diameter was measured using ImageJ software (http://imagej.nih.gov/ij/) for mini-rings that were parallel to the XY plane. A line intensity profile across the mini-ring was plotted and the peak-to-peak distance, which corresponds to mini-ring diameter, was determined from these plots.
Extracts from expanding leaves from approximately 5-week old plants were prepared, separated by SDS-PAGE and analyzed by immunoblotting as described . Extract from approximately 1-mg fresh tissue was loaded per lane. Immunodetection with affinity-purified goat anti-peptide antibody, recognizing the residues 168 through 184 in AtFtsZ2-1  was performed as described in Johnson et al. . Equal loading was confirmed by Ponceau S staining of the RuBisCO band on membranes (0.1 % (w/v) Ponceau S in 5 % (v/v) acetic acid).
High-pressure freezing (HPF)–freeze substitution (FS)
Conventional FS and modified QFS procedures
FS (4 % UA/acetone)
FS (4 % UA/acetone)
−90 °C (76 h)
–169 to 23 °C over the course of 5 h
−45 °C (30 h)
Infiltration schedule (−25 °C)
Infiltration schedule (25 °C)
10 % (6 h)
10 % (13 h)
25 % (16 h)
20 % (3 h)
50 % (7.5 h)
30 % (2 h)
75 % (14.5 h)
40 % (19 h)
100 % (6.5, 19, 7 h)
50 % (2 h)
60 % (2 h)
70 % (3 h)
80 % (1 h)
90 % (1 h)
100 % (15, 2, 2 h)
−45 °C (48 h)
60 °C (24 h)
20 °C (48 h)
Microtomy and immunogold labeling
Semi-thin (300 nm) sections were cut on a Reichert-Jung Ultracut E microtome equipped with a diamond knife and mounted onto silane-coated microscope slides for light microscopy. Thin sections (80–100 nm) were cut in the same manner and picked up on uncoated nickel grids. Thin sections were blocked with 4 % (v/v) cold water fish gelatin (Sigma) in PBS in the Biowave microwave processor with power set to 250 W and a 2–2–2 cycle (2 min on, 2 min off, 2 min on). The temperature cutoff was set to 37 °C. The grids were reacted with monoclonal mouse anti-GFP antibody (Millipore, Temecula, CA, USA) diluted 1:500 in the PBS blocker, using the 2–2–2 cycle. Grids were washed 2 × 1 min with PBS and 2 × 1 min with Tris-buffered saline (TBS; 0.15 M NaCl, 20 mM Tris–HCl pH 7.4), then blocked with 4 % (v/v) cold water fish gelatin in TBS at 250 W using the 2–2–2 cycle. Grids were incubated in a 1:30 dilution of donkey anti-mouse IgG conjugated to 12-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA, USA) at 250 W with a 2–2–2 cycle. Grids were washed in TBS 3 × 1 min followed by 3 × 1 min with deionized water, then reacted with 1 % (v/v) glutaraldehyde for 5 min on the bench before being washed for 3 × 1 min with deionized water and dried on a slide warmer.
Transmission electron microscopy and tomography
Grids were viewed in an FEI (Hillsboro, OR, USA) Tecnai™ G2 F20 operated at an acceleration voltage of 200 kV and equipped with a dedicated Fischione (Export, PA, USA) high-angle annular dark-field (HAADF) STEM detector, a GATAN (Pleasanton, CA, USA) Tridiem energy filter and a 2 k × 2 k GATAN Ultrascan 1000 CCD camera. Micrographs were recorded at calibrated magnifications, leading to a sampling of 1.1 nm/pixel in STEM and 0.39 nm/pixel in energy-filtered TEM mode. Tilt series were acquired from −75° to +73° in 1° increments using the FEI Xplore3D™ software. Datasets were reviewed using FEI Inspect 3D.
Reconstruction, segmentation and 3D rendering
All processing was performed in EM3D [37, 38]. Micrographs from −50° to 50° tilt could effectively be used yielding a total of 101 projections. Where gold fiducials were not present, alignment was done using statistically consistent features in the background as fiducial markers. Aligned projections were reconstructed into a 3D volume with 72 slices. The object edge on each slice was defined by dots, and the spline option provided a smooth edge for the connected dots. With filaments extending over 8–20 slices and with stacked regions, sudden changes in features were defined as edges between filaments. Segmented protofilaments were then colored and rendered with 40 % saturation of isosurface value so as not to smooth out surface modulations. Curvature measurements were performed in ImageJ with the three-point circular ROI plugin. The curvature was calculated as the inverse of the circle radius (1/r).
Results and discussion
FtsZ rings and mini-rings
Characterization of mini-rings by superresolution light microscopy
HPF–FS for correlative imaging of plant chloroplasts
The HPF approach offers to preserve organelles and other cellular components by instantaneous stabilization  in a manner superior to chemical fixatives . Both acrylic resins used, Lowicryl HM20 and LR White, proved capable of maintaining the fluorescent protein signal. This is in agreement with previous reports where methacrylates, LR White or Lowicryl have been employed in conjunction with plant and animal tissue and yeast cells [45, 46, 47, 48].
In situ electron microscopy/tomography of chloroplast mini-ring assemblies
Prior studies with prokaryotic FtsZ, which is closely related to the plant FtsZ2 protein [5, 6, 7], showed the presence of small circular assemblies under certain in vitro assembly conditions. Some were very small, measuring approximately 25 nm in diameter and were termed mini-rings [49, 50]. Under conditions of molecular crowding, larger circular assemblies (~200 nm) termed mini-rings or toroids were reported [51, 52]. The relevance of these in vitro assemblies to the assembly of FtsZ in vivo is being debated.
On comparing the left-hand side (LHS) with the right-hand side (RHS) of the ring, different degrees of orders are observed echoing the trend already observed before segmentation and rendering (Figs. 4, 5). The RHS shows a higher degree of unraveling and entanglement, while the LHS is characterized by one curvilinear bundle consisting of parallel protofilaments surrounded by loosely interwoven filaments. Another distinct difference between the LHS and RHS relates to overall filament curvature. While the RHS of the ring showed only a slight degree of curvature, the LHS’ curvature was on average threefold higher (Fig. 6b). This suggests a correlation between protofilament bundling and curvature. Upon closer inspection of the 3D reconstruction, it appears that the protofilaments can participate in higher-order structures at three levels. (1) The primary level is characterized by the aforementioned protofilaments. Distinct grooves delineate the long axis of the protofilaments at around 4-nm intervals and these filaments measure approximately 3 nm across (Fig. 6c). (2) Secondary-level structures are represented by two protofilaments together twisting around a central axis, thus creating quasi-double helical structures. These structures have a pitch of between 10 and 15 nm and a width of 7.6 nm (Fig. 6d). These data are in good agreement with the 13.25-nm pitch observed with double-stranded FtsZ protofilaments from Mycobacterium tuberculosis . (3) A tertiary level of hierarchical structures is observed when double helical structures intertwine into superhelical assemblies, the example of which is depicted in Fig. 6e. As a general trend for this particular mini-ring, the higher-order structures dominate the RHS of the ring while the more orderly structured LHS appears to be more populated by protofilaments. Such a dichotomy points at a dynamics not reported previously. In essence, the structure looks as if it presents a balance between two states: a state of disassembly and one of assembly. The latter could lead from an interwoven and entangled arrangement to orderly layered structures. We speculate that if the tightly arranged filament bundles are disturbed, individual filaments may be able to assume different higher-order structures such as the ones described above.
While the mini-rings could be considered metastable as per the earlier reasoning, they may also serve in this capacity as an energetically favorable way of protofilament storage. This would be particularly important in times of high abundance of FtsZ such as that observed in young, metabolically active cells. By virtue of their curvature, they could also bestow the assembly with inherent energy leading to pre-energized, ‘ready-to-assemble’ protofilaments for a more responsive supply of Z-ring building blocks. The relatively high percentage of mini-rings found attached to linear filaments corroborate such an idea.
In a broader context, the presented data provide first insights into the molecular architecture of plant FtsZ assemblies in situ featuring bundling of filaments, a multilayered structure, differential protofilament curvature as well as higher-order structures such as superhelices. While the curvature of plant FtsZ protofilaments was not observed in vitro [17, 27], clearly the mini-rings show that FtsZ is able to form curved assemblies without depending on cytosolic components such as ARC5 [28, 29]. Perhaps, the propensity of FtsZ to curve is predicated on the features summarized above. On the other hand, these features could also echo the dynamic remodeling that FtsZ assemblies are constantly undergoing [19, 20].
Chloroplast division is highly complex and of societal importance as the mechanisms underlying this process also control the starch granule size.
FtsZ is a key protein of chloroplast division and this report presents the first electron tomographic 3D structure of plant FtsZ assembly in situ. The assembly form presented here are the FtsZ mini-rings which occur under defined physiological conditions.
Comparative SIM and EM imaging yielded matching mini-ring diameters. Combining light microscopy screening with a detailed EM analysis via tomography laid the foundations for the development of correlative microscopy-compliant protocols involving HPF and FS.
Assessment of resolution for the tomographic data set according to the Crowther theorem  only suggests 4 to 5-nm resolution in the 3D reconstruction; however, the resolution is much better in projection as demonstrated by a comparison with X-ray data. The latter led to the identification of protofilaments in the tomogram. These protofilaments can also assemble into higher-order structures such as double helices and superhelical arrangements.
The data provide a glimpse into the dynamic life of FtsZ assemblies. Electron microscopy and tomography readily revealed a very dynamic picture of the mini-rings highlighted by a bipartite structure splitting the ring into an ordered and less ordered half. This implies that the rings may undergo dynamic remodeling by providing FtsZ building blocks for Z-ring assembly or a form of storage of protofilaments.
CBJ carried out the cloning, plant transformation and sample preparation for TEM, participated in the design of experiments and drafted the manuscript. ZLo performed the reconstruction and segmentation of the EM tomograms. ZLu performed the STEM and EFTEM imaging. RS carried out the immunoassays and MWS the fitting of/comparison with X-ray data. SV performed the confocal microscopy and carried out sample preparation for SIM. AH conceived the study and participated in EM data analysis. AH and SV participated in the design of experiments and carried out the SIM data analysis. AH and SV co-wrote the manuscript. All authors read and approved the final manuscript.
The authors thank the Office of the Vice President for Research at Texas A&M University for the continued support of the Microscopy and Imaging Center. SIM was performed by GE Healthcare Life Sciences (formerly Applied Precision; Issaquah, WA, USA) and Zeiss Microimaging (Thornwood, NJ, USA). The ftsZ2-1/2-2 double knockout plants were obtained from Dr. Katherine Osteryoung (Michigan State University).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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