Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes

Transcription

1 ... Cellulose II: , 2004., KlulVer Academic Publishers. Printed in the.velherlands. 385 Theoretical considerations of immunogold labeling of cellulose synthesizing terminal complexes Takao Itoh l,*, Satoshi Kimura 2 and R. Malcolm Brown, Jr. 3 I Wood Research Inslitute, Kyoto University. Uji, Kyoto I, Japan; 2Forestry and Forest Products Research Institute, Tsukuba. Ibaraki Japan; 3Section of _Wolecular Genetics and Microbiology. School of Biological Sciences. University of Texas at Austin, Austin. TX 78712, USA; *Author for correspondence ( _ titoh@rish.kyoto-u.ac.jp; fax: _c ) Received 25 November 2000; accepted in revised form 26 April 2004 Key words: c-di-gmp binding protein, Cellulose synthase, Freeze-fracture labeling, Linear TC, Rosette, Terminal complex Abstract The sodium dodecyl sulfate (SDS)-solubilized freeze fracture replica labeling (SDS-FRL) technique has been successfully applied to a vascular plant, Vigna angularis [Kimura et a!. 1999, Plant Cell II: ] and a bacterium, Acetobaeter xylinum [Kimura et al. 2001, J. Bacteriol. 183: ] for understanding cellulose biosynthesis. However, we do not know yet how many gold particles can be bound with antibodies that label a single rosette or linear TC. We analyzed these techniques from a theoretical background by considering the size of gold particle, primary and secondary antibody, and the rosette and linear TC. The 10 nm gold particles coated with primary and secondary antibodies result in a 30 nm diameter for the gold-conjugated antibody that binds to a rosette TC. Therefore, a rosette TC theoretically could be labeled with, at most, four gold particles and commonly with 1-3 gold particles after the topological consideration of the immunogold labeling of the rosette Te. We interpreted an actual image of freeze-fracture labeling of TCs by comparison with a scale model of the antibody-labeling to a rosette Te. The labeling of linear TCs with the gold-conjugated antibody of c-di-gmp binding protein was quite stable, and 75% of the 277 gold particles were found within 20 nm of the linear row. Labeled TCs with depressions and indistinct particle arrays and non-labeled TCs with distinct particle arrays were observed on the P-fracture face of the outer membrane, suggesting that TCs in A. xylinum are composed of two types of subunits. Introduction More than 20 years have passed since the first demonstration of the existence of cellulose synthesizing terminal complexes in the plasma membrane of a fresh water alga, Ooeystis apiculata, which came to be known as linear terminal complexes (Brown and Montezinos 1976). Traced back to the history of terminal complexes, Roelofsen (1958) first predicted that cellulose could be assembled by a large enzyme complex at the growing tip. Preston (1964) presented a model in which three-layered particle complexes in the plasma membrane are involved in the synthesis of cellulose microfibrils. The deposition of cellulose among a variety of cellulosic organisms is most abundant in higher plant cells. Mueller and Brown (1980) found another type of terminal complex,

2 386 the so-called rosette TC, associated with cellulose microfibril imprints on the P-fracture face of the plasma membrane of corn root cells during the assembly of cellulose microfibrils. Only indirect evidence has been available for the involvement of the TC in cellulose biosynthesis. First, the TC can be observed at the terminus of cellulose microfibril imprints. Second, the linear TCs in O. apiculata can be observed as a pair in different developmental stages of TC growth, suggesting that an individual TC in a pair may move in opposite directions by producing cellulose microfibrils (Brown 1978). Third, the length of linear TCs increases during the transition from primary to secondary walls in Valonia and Boer[<esenia (Hoh and Brown 1988). There has been no direct evidence to prove that cellulose synthesis-related enzymes are localized in the TC on the plasma membrane. Recently, we demonstrated that the rosette TC in a vascular plant and a linear TC in a bacterium are labeled by the antibodies against the CesA cellulose synthase protein (Kimura et al. 1999) and the c-di-gmp binding protein (Kimura et al. 2001) using the SDS-FRL freeze-fracture replica labeling technique first developed by Fujimoto (1995). The dimension of the component membrane particle is approximately 8 nm, while the diameter of the gold particle used in the present experiment is 10 nm. When gold particles are treated with antibodies, the final diameter of the gold-conjugated antibodies should be much larger than 20 nm. Therefore, the labeling of rosette and linear TCs with antibodies must be quite difficult. Thus, we need to know how many gold particles could be labeled to a single rosette TC or a linear TC. Materials and methods Plant materials Cotton plants (Gossypium hirsutum) were grown in the greenhouse under 14 h of light at 32C and 10 h of darkness at 20 C. Vigna radiata seedlings were germinated in darkness for 3-5 days at 25 0c. Isolation of cotton fiber RNA, construction of a cotton cdna library, and cloning of the cotton cellulose synthase are referred to Kimura et al. (1999). Bacterial materials Acetobacter xylinum NQ5 (ATCC 53582) was grown statically in SH medium at 27 cc. The culture and isolation of the cells is referred to Kimura et al. (2001). Antibody production of cotton cellulose synthase The antibody production of cotton cellulose synthase is referred to Kimura et al. (1999). Antibody production of c-di-gmp bindin[< protein The antibody production of c-di-gmp binding protein is referred to Chen and Brown (1999). Freeze-fracture labeling We pioneered the fracture labeling technique, known as the so-called SDS-FRL, with plant cells for the first time. The technique was initially developed by Fujimoto (1995) for animal cells. The application of the technique to plant cells has been difficult because the cell wall is not removed after SDS treatment and obscures evaluation of the replicas of the fractured face of the membrane. This difficulty has been overcome by treating the tissue attached to the replicas with a cellulase mixture. Replicas obtained in this manner appear similar to those obtained by using conventional freeze-fracture techniques, in which harsh acid treatments customarily have been used to remove cell and tissue components. The freeze fracture and immunogold labeling for the rosette TC in V. radiata are referred to Kimura et al. (1999), while those for linear TC in A. xylinum are referred to Kimura et al. (200 I). Results and discussion Localization of the catalytic subunit protein of cellulose synthase in the rosette TC Figure I shows a model of CesA protein redrawn by referring to the report of Pear et al. (1996). We have isolated GhCesA gene, which is one of the

3 386 the so-called rosette TC, associated with cellulose microfibril imprints on the P-fracture face of the plasma membrane of corn root cells during the assembly of cellulose microfibrils. Bacterial materials Acetobacter xylinum NQ5 (ATCC 53582) was grown statically in SH medium at 27 cc. The culture and isolation of the cells is referred to Kimura et al. (2001). Antibody production of cotton cellulose synthase The antibody production of cotton cellulose synthase is referred to Kimura et al. (1999). Antibody production of c-di-gmp bindin[< protein The antibody production of c-di-gmp binding protein is referred to Chen and Brown (1999). Freeze-fracture labeling le is approximately 8 nm, while the diameter of the gold particle used in the present experiment is 10 nm. When gold particles are treated with antibodies, the final diameter of the gold-conjugated antibodies should be much larger than 20 nm. Therefore, the labeling of rosette and linear TCs with antibodies must be quite difficult. Thus, we need to know how many gold particles could be labeled to a single rosette TC or a linear TC. Materials and methods Plant materials Cotton plants (Gossypium hirsutum) were grown in the greenhouse under 14 h of light at 32C and 10 h of darkness at 20 C. Vigna radiata seedlings were germinated in darkness for 3-5 days at 25 0c. Isolation of cotton fiber RNA, construction of a cotton cdna library, and cloning of the cotton cellulose synthase are referred to Kimura et al. (1999). We pioneered the fracture labeling technique, known as the so-called SDS-FRL, with plant cells for the first time. The technique was initially developed by Fujimoto (1995) for animal cells. The application of the technique to plant cells has been difficult because the cell wall is not removed after SDS treatment and obscures evaluation of the replicas of the fractured face of the membrane. This difficulty has been overcome by treating the tissue attached to the replicas with a cellulase mixture. Replicas obtained in this manner appear similar to those obtained by using conventional freeze-fracture techniques, in which harsh acid treatments customarily have been used to remove cell and tissue components. The freeze fracture and immunogold labeling for the rosette TC in V. radiata are referred to Kimura et al. (1999), while those for linear TC in A. xylinum are referred to Kimura et al. (200 I). Results and discussion Localization of the catalytic subunit protein of cellulose synthase in the rosette TC Figure I shows a model of CesA protein redrawn by referring to the report of Pear et al. (1996). We have isolated GhCesA gene, which is one of the

4 Cell Wall Plasma membrane Cytoplasm :'-l-ter... { ~..' / ::;D: : : : :: : ;:::W: "::: n D~:(... '-' (;..' I C-Ier Figure J. Proposed model of plant CesA protein. Cytoplasmic domain of GhCesA protein (dotted area) was used as a specific antigen for the preparation of the CesA antibody. homologs of cotton CesA. The recombinant proteins of the catalytic site (dotted region in Figure 1) exposed to the cytoplasmic side were transformed into Escherichia coli and used as an antigen of CesA antibody. The antigen region includes the D,D,D,QxxRW motif which is the amino acid sequence prerequisite for the activation of CesA. As the antibody is specific for the cytoplasmic region of CesA protein, it was expected that the antibody would specifically recognize the rosette on the P-fracture face of the plasma membrane when subjected to SDS-FRL techniques. The actively elongating region of the seedlings in Azuki bean (V. radiata) was used as the 387 experimental material. The elongating region has some advantages in the present study; first, cellulose is actively synthesized in this region; second, the plasma membrane will be exposed more frequently during the freeze-fracturing because of the small size of the tissue cells; third, the disintegration of cell walls by cellulase treatment is easy because the cell wall is not fully thickened and without lignification. In practice, the cellulase treatment was not effective for disintegrating thickened walls. The remnants of cell walls were often attached to the replica membrane, thus making it difficult to secure a clean replica. Figure 2 shows a rosette TC labeled with CesA antibody. The gold particles indicating the presence of secondary antibodies were observed on the rosette TC or close to the structure. This figure shows rosettes labeled with gold. Nine rosettes among 10 are labeled with gold and one rosette is not labeled in the upper left figure. Eight rosettes among 12 are labeled with gold and four are not labeled in the lower left figure. The figures on the right are a higher magnification of the labeled rosette. The figure on the upper right shows a single rosette labeled with three gold particles. The figure on the middle right shows two rosettes, each labeled with a gold particle. The figure on the lower right shows a single rosette labeled with a gold particle. Most of the rosettes were labeled Figure 2. SDS-FRL image of the P-fractllre face of a Vigna cell. Rosette TCs labeled with gold particles are shown by black solid circles and non-labeled TC is shown by a black dotted circle (upper and lower left). A rosette labeled with three gold particles, two rosettes each labeled with a single particle, and a rosette labeled with a single particle are shown in the upper, middle and lower right of the figure, respectively.

5 </> B" 60 i 50 '- 0 G 40 c: g 30 J: The number of gold particles associated with a single rosette 1 Figure 3. SDS-FRL image of the E-fracture face of a Vigna cell. Two terminal globules surrounded by white dotted circles were never labeled with CesA antibodies. Furthermore, no labeling was detected in the E-fracture face of replicas. with one or two gold particles and often with three gold particles. The terminal globules on the E fracture face complementary to rosettes were not labeled with the antibody (Figure 3). These findings demonstrate that the CesA is indeed a component protein of a rosette TC, and the catalytic site of CesA is localized on the cytoplasmic side, that is, the P-fracture face of a plasma membrane. Examining Figure 2 closer, the distance between any two individual gold particles labeled to rosette TCs is almost the same among the three photos. The individual rosette has a potentiality to produce cellulose microfibrils approximately 4 nm in diameter, calculated to be composed of 36 glucan chains. It is suggested that a single particle subunit among 6 subunits of rosette TC is composed of 6 CesA proteins (Doblin et al. 2002). We predicted that a rosette TC will be labeled with a variable number of particles at the beginning of our experiment. However, this was nol the case. It is valuable, therefore, to analyze how many gold particles can be labeled to a single rosette TC In order to measure the frequency of the number of gold particles per rosette, we re-examined electron micrographs from 17 different cells that include 119 rosettes. Twelve rosettes had no labeling of gold particles. We made a histogram of the number of gold particles labeled to the other 107 rosettes (Figure 4). As shown in the figure, 70 rosettes were labeled with a single gold particle, 28 with two gold particles, and 8 with three gold Figure 4. The histogram shows the frequency of the number of gold particles per rosette. It is based on the re-examination of electron micrographs that cover 17 cells including [19 rosettes. ft should be noted that 12 rosettes were not labeled with gold particles. The other l07 rosettes were labeled with I 4 gold particles. particles. Only one among 107 rosettes was labeled with four gold particles. Figure 5 shows a top view of a rosette TC and a side view of a rosette TC perpendicular to the plasma membrane. The diameter of the rosette TC is approximately 25 nm. At the same time, immunoglobulin G, which is the primary component of CesA, secondary antibody, and 10 nm gold particles are all illustrated at the same scale showing the rosette TC Based on previous research on the analysis of molecular weight and X-ray diffraction for the antibody, the IgG shows an Y shape with 10 nm total length. The gold-labeled IgG which is used as the secondary antibody has a structure attached with a number of IgG molecules surrounding the gold particles, as shown in Figure 5. In short, the gold particles under electron microscopic observation are surrounded by a number of 10 nm antibodies which are not visible. The size of both primary and secondary antibodies is 27.2 nm (Sarma et al. 1971). The actual distance of the gold particle from the antigen, cellulose synthase, could be >27 nm because the proteins in the rosette TC are denatured and extended by SDS treatment. Nevertheless, we selected 20 nm or less as a conservative distance for a 'labeled' rosette TC in our qualitative analyses. Considering these situations, the gold-labeled secondary antibody is approximately 30 nm in diameter, which is larger than the individual rosette TC Figure 6 is a hypothetical illustration showing the labeling of antibody to rosette TC, that is

6 389 Top view ad ~~~ Clue 25 nm ~ ~ Primary -< antibody Secondary antibody =< GO.1e Side view 25 run ~ 10 nm gold-conjugated secondary antibody with 30 nm Figure 5. Scale model of the roselle TC, antibodies and gold particles. The diameter of a roselle is 25 nm. The diameter of the primary antibody, secondary antibody and gold particles is 10 om. The diameter of an antibody conjugated with a gold particle is approximately 30 nm. based on the scale model of Figure 5. A rosette TC labeled with three gold particles is shown. The rosette TC is redrawn by a solid line in the top view model to make the rosette more distinct. The rosette TC is suggested to be roughly covered with gold-conjugated antibodies. Two gold-conjugated antibodies are closely associated with the rosette TC, and one gold-conjugated antibody facing the cytoplasmic side is drawn in the side view model. An alternative positioning of gold particles can be seen for the labeling of antibodies, but it will be easy to understand that there is no room for the labeling of more than four gold particles. The individual gold particle is coated by the antibody, which is 10 nm long. Therefore, it is clear that usually I -3 and at most 4 gold particles can be labeled to a single rosette TC. The antibody labeling to the rosette TC as shown in Figure 3 makes sense from the scale model analysis and, at the same time, supports the successful immunogold labeling experiments. Cellulose synthase is primarily important for the biosynthesis of cellulose in vascular plants. However, Korrigan cellulase and the other types of cellulose synthase are also thought to be involved in the biosynthesis of cellulose and suggested to be components of the rosette TC (Doblin et al. 2002). One of the best ways to prove this concept is to apply SDS-FRL to visualize these proteins localized in the rosette TC. The visualization of Korrigan cellulase could be done by a single labeling of its antibody. However, visualization of different types of cellulose synthases could be done by a double or triple labeling of gold particles. We do not know yet whether there are two or more CesA proteins in a single rosette TC or whether the individual rosette TC is composed of different CesA proteins. Thus, independent immunogold labeling for different CesA antibodies will not solve these questions. We do need double or triple labeling of CesA antibody of the rosette TC. According to our scale model explaining the relation between rosette TC and antibody mentioned above, a rosette TC will be labeled at most with two or three gold particles. We should not expect Original image Top view Side view Figure 6. Scale model for an immunogold-labeled rosette TC. The original image of a TC labeled with three gold particles is shown on the left. The top view in the middle shows a rosette TC redrawn by a solid line. The side view on the right shows two gold-conjugated antibodies closely associated with a rosette and one facing the cytoplasmic side.

7 390 to obtain clear results after the double labeling of the antibodies by the application of present experimental methods. Labeling could be improved with more than double the number of gold particles. This can be successfully accomplished by using antibodies with a lower molecular weight as well as gold particles. The primary and secondary antibodies could be partially hydrolyzed and some parts of hydrolysates necessary for the labeling could be used, thus reducing the diameter of gold particles to a smaller size. It is worth while to note the reason why we used 10 nm gold particles. The reason is that membrane proteins observed by freeze-fracturing are mostly 5-10 nm in diameter, and it is difficult to know the difference between the shadowed membrane proteins and gold particles. However, small gold particles can be differentiated from membrane proteins by controlling the shadowing strength and image processing. It could be beneficial to prepare gold-conjugated primary antibodies for the direct labeling of a rosette TC. An alternative way is to express recombinant CesA with some tag and visualize the tag with fluorescein. For example, the histidine tag often is used. In conclusion, we need to improve a number of techniques and approaches to overcome some of the difficulties in visualizing directly the component proteins of the rosette TC by the application of SDS-FRL. Localization of c-di-gmp binding protein in linear TC We have also applied SDS-FRL to visualize one of the component proteins of cellulose synthases in A. xylinum. The antibody used for the present experiment in Acetobacter is the anti-c-di-gmp binding protein prepared by Chen and Brown (J 999). The antibody is purified against a full length of 93 kda protein by product entrapment. As the c-di-gmp binding protein is suggested to be a transmembrane protein, it was thought that its antibody would recognize TC particles on the P-fracture face of outer membrane or plasma membrane in A. xylinum. When the antibody of c di-gmp binding protein was used for SDS-FRL of A. xylinum, the gold particles specific to the labeling of the antibody were observed in a single row of linear TCs on the P-fracture face of the outer membrane (Figure 7A). It is worthwhile to note that the bacterial cells will be fractured in high frequency at the junction of the outer membrane. When we took electron micrographs of fractured membranes of 300 Acetobacter cells at random, only two showed a fractured plasma membrane. We only selected images that show gold labeled TCs. Usually TC can be observed in every 10 cells. This means that the actual frequency to show the fractured plasma membrane is less than one tenth of the above frequency. Figure 7A D shows all the fractured outer membranes. Figure 7A, B, D shows the P-fracture face of the outer membrane from different bacterial cells after labeling with antibodies against the c-di-gmp binding protein. It should be noted that Figure 7B does not show any reaction of the antibody in spite of the evidence of a distinct row of TC particles. Whenever we had a positive reaction of the antibody to TCs as shown in Figure 7A, there is no distinct row of TC particles but a line of depressions or pits with an indistinct particle array. Thus, the TC structure on the P-fracture face of the outer membrane often showed depressions after freezefracturing. To the contrary, a distinct row of TC particles on the E-fracture face of the outer membrane was often observed as shown in Figure 7C. However, positive labeling of linear TCs was not observed on the E-fracture face. A very rare case of freeze-fracturing showed a distinct row of TC particles and depressions or pits in a single line on the same P-fracture face of the outer membrane (Figure 7D). The TCs in the region of the depressions or pits were exclusively labeled by the antibodies, while the distinct row of TC particles was never labeled by the antibodies. These findings strongly support that two types of TC structures can be seen on the P-fracture face of the outer membrane, depending on the presence or absence of any remaining distinct low of particles after freeze-fracturing of the outer membrane. A scale model showing the close relation between TCs and antibodies on the outer membrane of the bacterium is shown in Figure 8A, B. Compared to the labeling of the rosette TC in Azuki beans (Vigna angularis) with the antibody ofcesa, the number of gold-labeling to the outer membrane of the bacterium was fewer; however, the antibodies against the c-di-gmp binding protein were specifically labeled on and along the row of TCs. The distance between the linear TCs and gold particles was calculated by measuring the vertical

8 391 Figure 7. SDS-FRL images of Ace/abae/er cells. (A) P-fracture face of TC on the outer-membrane with numerous gold particjes (attows) along depressions or pits of the linear TC; (B) P-fracture face of the TC on the ollter-membrane with a distinct row of membrane particles that were never labeled with gold particles; (C) L-fracture face of the TC on the outer-membrane with a distinct row of membrane particles that were never labeled with gold particles; (D) P-fracture face of TC on the outer-membrane with two different particle arrays. Distinct TC particles were not labeled (right half of D), however, a single row of TCs with depressions or pits (left half of D) was labeled with gold particles (arrows). distance between the edge of gold particles and a findings suggested that c-di-gmp binding protein linear row of TC particles. For frequency analysis, is one of the major components of linear TCs in 277 gold particles were randomly sampled from 30 the bacterium, and the principal domain of the different cells that have a single row oftcs. In this protein is localized on the cytoplasmic side. A case, 75% of the 277 gold particles were found number of gold-labeling regions on the outer within 20 nm of the linear row. Most gold particles membrane suggests that the transmembrane rewere found within nm, with a median dis gion of the protein extends not only across the tance of 9.3 nm from the linear row of TCs. These plasma membrane and periplasmic regions, but

9 392 Figure 8. (A) SDS-FRL image showing the distance between gold particles and a linear TC (dotted line). (B) Scale model redrawn from the actual image of (A). Gold particles are all drawn with a coating by the antibodies. also across the outer membrane. It is not realistic to imagine that the c-di-gmp binding protein passes through the outer membrane; however, part of the c-di-gmp binding protein may have extended to the outer membrane. Localization of c di-gmp binding protein in the outer membrane and the methods how we recognize the protein by the labeling of its antibody will be discussed later. The results obtained from the labeling of TCs in the bacterium by antibodies against the c-di-gmp binding protein can be explained as follows: (1) During freeze-fracturing of bacterial cells, almost au fractured planes occurred through the region occupied by c-di-gmp binding protein on the outer membrane. (2) The linear TCs of bacterial cells observed on the outer membrane show depressions or pits on the P-fracture face and distinct membrane particles on the E-fracture face. These are complementary to a single row of TCs on the outer membrane. (3) The TC labeled by antibodies against the c-di GMP binding protein can be observed on the P-fracture face, and the gold particles can be seen only in the depressions or pits. (4) The antibody does not recognize the distinct mem brane particles of TCs on both the P-fracture and E-fracture faces. These findings primarily support the hypothesis that the distinct membrane particles of TCs on both P- and E-fracture faces of the outer membrane are not composed of c-di-gmp binding proteins but rather, other component proteins of TCs. Second, the depressions or pits of TC on the P-fracture face of the outer membrane are caused by a peeling of membrane particles towards the E fracture face due to the complementary structure between the membrane particles of TC on the E fracture face and depressions or pits of TC on the P-fracture face. These membrane particles often are observed on the E-fracture face, but are rarely found on the P-fracture face. Figure 7D shows such a structure found by chance. The c-di-gmp binding protein is held by the replica membrane on the depressions or pits oftc on the P-fracture face of the outer membrane. The membrane particles on TCs not recognized by the antibody are presumed to be 'C' or 'D' proteins of the bacterial cellulose synthase operon as hypothesized by Wong et al. (1990). These proteins are localized in the outer membrane and appear to be involved in the secretion and crystallization of cellulose microfibrils. We summarize the present results using a model illustration (Figure 9). The transmembrane domain of c-di-gmp binding proteins passes through not only the plasma membrane, but also the peripjasmic space and outer membranes (Figure 9A). The 'C' or 'D' proteins in the series of the cellulose synthetic operon are localized in the outer membrane and attached to the c-di-gmp binding proteins. When the outer membrane is fractured, 'C' or 'D' proteins will remain on either the P- (left half of Figure 9B) or the E-fracture face (right half of Figure 9B). In practice, these membrane proteins mostly are fractured away from the FAracture face. When 'C' or 'D' proteins of the TCs in the outer membrane are fractured away, depressions or pits are made in the membrane on the P fracture face, and parts of c-di-gmp binding proteins will be exposed. The c-di-gmp binding proteins will be held by the replica membrane at the Pt-shadowing and carbon-backing (right half of Figure 9C). On the contrary, when 'C' or 'D' proteins remain on top of the c-di-gmp binding proteins, they will not be fixed by the replica membrane (left half of Figure 9C). The plasma membrane and periplasmic space in the bacterium not fixed by replica membrane, and c-di-gmp binding proteins not fixed directly by the replica will be washed away after SDS treatment

10 393 (6) (e) Outer membrane Plasma membrane Freeze fracture ~PoreofPF Knife~--UU~faceon the ~ ~/ outer- 11fiJ.:or-<fffl.l.'lr<(i'JjJ~~Wl:~Bmembrane (D) Lysozyme & SDS-treatment EF-face~ & (E) Antibody labeling EF-face~ Figure 9. Schematic diagram showing c-di-gmp binding proteins revealed in freeze-fractured images. (A) c-di-gmp binding proteins span both the outer membranes and the cytoplasmic membranes. (B) After freeze-fracture, the outer membrane proteins remain attached to the c-di-gmp binding proteins or separate from the latter. (C) After shadowing, the outer membrane proteins are fixed on the P-fraclUre face or the E-fracture face of the outer membrane. In the latter case, the c-di-gmp binding proteins are fixed onto the P fracture face of the outer membrane. (D) After lysozyme and SDS trealment, the c-di-gmp binding proteins will be removed in the case of the left half of the illustration, or they will not be removed in the case of the right half of the illustration. (E) After labeling with antibody, the distinct particle row as shown in 'a' on the P-fraclure face and 'b' on the F-fracture face will not be labeled by the antibody. The depression in the TC structure where c-di-gmp binding proteins are fixed on the P-fraclUre face is labeled by antibodies, as shown in 'c' on the P-fracture face. (Figure 9D). When the replica is labeled by antibodies against the c-di-gmp binding proteins, gold particles are observed on the depressions or pits of the TCs on the outer membrane (inset 'c' in Figure 9E). However, the distinct membrane particles of TC on both the P- (inset 'a' in Figure 9E) and E-fracture faces (inset 'b' in Figure 9E) were not labeled at all. These considerations make reasonable sense to explain the relationship between TC structure and gold-labeling in Acetobacter. We visualized the reaction of a single antibody of a c-di-gmp binding protein; however, the present investigation suggests that a linear TC in Acetobacter could be made of two different membrane proteins. The SDS-FRL appears to be very useful to clarify the component proteins in TCs that are composed of multimeric enzyme complexes, allowing an analysis of the topology of TC structure. Acetobacter xylinum is a relatively easy model system to apply the freeze-fracture technique. It is also a model organism for an understanding of cellulose biosynthesis from the aspects of molecular genetics and biochemistry. A more general understanding of Acetobacter TCs will be clarified in the future through the application of SDS-FRL with the major goal to prepare antibodies against the component proteins of linear TCs such as 'C' or 'D' proteins of cejlulose synthase operon as well as glucanase (Doblin et a ), which is increasingly important for an understanding of cellulose biosynthesis.

11 394 References Sci. USA 73: Brown R.M. Jr Biogenesis of natural polymer systems with special reference to cellulose assembly and deposition. In: The Third Philip Morris Science Symposium, pp Chen H.P. and Brown R.M. Jr Thermal stability of the cellulose synthase complex of AcelObacler xylinwl1. Cellulose 6: Doblin M.S., Hurek I., Jacob-Wilk D. and Delmer D.P Cellulose biosynthesis in plants: from genes to rosettes. Plant Cell Physiol. 43(12): Fujimoto K Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. J. Cell Sci. 108: Itoh T. and Brown R.M. Jr Development of cellulose synthesizing complexes in Borgesenia and Valonia. Protoplasma 144: Kimura S., Chen H.P., Saxena I.M., Brown R.M. Jr. and Itoh T Localization of c-di-gmp-binding protein with the linear terminal complexes of Acetobacter xylinul11. J. Bacteriol. 183: Kimura S., Laosinchai W., Itoh T., Cui X., Linder C.R. and Brown R.M. Jr Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant, Vigna angularis. Plant Cell II: Mueller S.c. and Brown R.M. Jr Evidence for an intramembranous component associated with a cellulose microfibril synthesizing complex in higher plants. J. Cell BioI. 84: Pear J.R., Kawagoe Y., Schreckengost W.E., Delmer D.P. and Stalker D.M Higher plants contain homologs of the bacterial CesA genes encoding the catalytic subunit of cellulose synthase. Proc. Natl. Acad. Sci. USA 93: Preston R.D Structural and mechanical aspects of plant cell walls with particular reference to synthesis and growth. In: Zimmerman M.H. (ed.), Formation of Wood in Forest Trees. Academic Press, New York, pp Roelofsen A Cell wall structure as related to surface growth. Acta Bot. ~eerl. 7: n 89. Sarma V.R., Silverton E.W., Davies D.R. and Terry W.D. 197/. The three-dimensional structure at 6 Aresolution of a human yg I immunoglobulin molecule. J. BioI. Chern. 216: Wong H.C., Fear A.L., Calhoon R.D., Eichinger G.H., Ylayer R., Amikam D., Benziman M., Gelfand D.H., Meade l.h., Emerick A.W., Bruner R., Ben-Bassat A. and Tal R Genetic organization of the cellulose synthase operon in AcelObacter xylintlm. Proc. Natl. Acad. Sci. USA 87: lll