A New View of Cellulose Synthase

My graduate student, Andy Bowling, has been working very hard during the past several years to find a method which will allow observation of the cytoplasmic domain of cellulose synthesizing complexes while they are still embedded in the plasma membrane of a plant cell. Since our first discovery of the vascular plant cellulose synthase known as the "rosette TC" in 1980 (see: Mueller, S.C. and R.M. Brown, Jr. 1980. Evidence for an intramembranous component associated with a cellulose microfibril synthesizing complex in higher plants. J. Cell Biol. 84:315-326.), everyone has assumed that the entire TC is the rosette! This is not so! The rosette TC which we aptly named in 1980 is a membrane-spanning complex of 6 subunits. We do not believe that this transmembrane domain contains the catalytic subunits for cellulose synthase. Since we know that the cellulose microfibril that it generates has approximately 36 glucan chains, it has been often cited that each subunit is responsible for 6 of those 36 glucan chains.

In 1993, my colleagues Dr. Krystyna Kudlicka, Professor Alan Wardrop and I found in sectioned material of the giant algal cell Boergesenia, that the linear TC has a rather massive domain projecting deeply into the cytoplasm immediately adjacent to the transmembrane linear array of subunits (see:Kudlicka, K., A. Wardrop, T. Itoh, and R.M. Brown, Jr. 1987. Further evidence from sectioned material in support of a linear terminal complex in cellulose synthesis. Protoplasma 136:96-103.).

Below is a photo from this work. Note that the TC extends deeply into the cytoplasm and is much larger dimensionally that the bimolecular leaflet dimensions of the plasma membrane and is slightly larger than the microtubule, which is reported to be 24 nm in diameter.

Extending this idea to the rosette TC, Inder Saxena and I have recently published a review which takes into account our new concept of the structure of the TC. It is shown diagrammatically below.

Thus, based on this model, we expect to actually image a cytoplasmic structure which is considerably larger than the rosette portion of the TC. This has recently been accomplished by Andy Bowling. How did he accomplish this extraordinary feat? First, it was not easy, as even Andy will tell you! The idea is rather simple, but the execution is very difficult. Andy has had his best success with tobacco BY-2 cells grown in suspension culture. First, he removes the cell wall using cellulose- and pectin-degrading enzymes. Then he allows these protoplasts to just start generating a new wall of cellulose microfibrils. Then he attaches these young cells to a glass coverslip coated with poly-l-lysine. The cells are then vigorously disrupted, and what remains attached to the slide is a "footprint" of the plasma membrane with nascent cellulose trapped between the membrane and the glass. These fragments are then rotary shadowed using platinum-carbon evaporation very similar to the methods employed with freeze fracture. The replicas are removed from the glass by floating onto HF which dissolves the glass, then they are cleaned and examined by TEM. The results are nothing short of remarkable! All of the cytoplasmic components that are associated with the plasma membrane are easily visualized. For example, the cortical microtubules attached to the plasma membrane with cross bridges are very common. Clathrin coated vesicles are also observed. Most importantly for us, torn through the cytoplasmic side of the plasma membrane are windows where the nascent cellulose microfibrils can be viewed. In rare instances, these plasma membrane impressions can be traced back to a terminus which is the TC!

Examine enlarged views of the image thumbnails below by clicking on the thumbnails

A membrane sheet from tobacco BY-2 suspension culture cells. Parallel bundles of cortical microtubules can be seen as well as extensive plasma membrane-associated rough ER. A huge number of ribosomes can be seen stuck to the PLL-coated substrate just outside the boundaries of the membrane sheet itself. click on photo for an enlarged view

A closer view of a membrane sheet. Again, a large number of parallel cortical microtubules are tightly associated with the plasma membrane. Parts of the ER are clearly visible. Clathrin-coated pits can just be seen between microtubles budding off of the membrane. click on photo for an enlarged view

An anaglyph showing a bundle of cortical microtubules, some rough ER, as well as many clathrin-coated vesicles. click on photo for an enlarged view

An anaglyph showing a row of clathrin-coated vesicles near the edge of a membrane sheet. Cellulose microfibrils can also be seen here. click on photo for an enlarged view

A few microtubles can be seen along with a small piece of the ER, however, notice the cellulose microfibrils which can be seen passing under the microtubules. click on photo for an enlarged view

Four images of truly "terminal" complexes. Particles of sizes ranging from approx. 40 to 60 nm in diameter can be seen associated with the termini of cellulose microfibrils. The actual "rosette" component that we first discovered using freeze fracture in 1980 is only 25 nm in diameter. Thus, the 'business end' of the TC is nearly twice the diameter of the trans-membrane rosette component. We believe this cytoplasmic part contains the domains for the catalytic sites where some 36 glucan chains are simultaneously polymerized to form the cellulose microfibril. click on individual photos for enlarged views

These results clearly show that the vascular plant TC with is multi-enzyme complex has a cytoplasmic component deeply embedded within the cytoplasm and that the rosette component may be the channel through which the nascent glucan chain complex is directed for crystallization upon exit to the cell's exterior. This material is now being prepared for publication, and Andy will receive his doctorate by the end of this summer. For anyone out there looking for a top notch post-doctoral, here is a superbly trained candidate! If you are interested, please send an email to me at: rmbrown@mail.utexas.edu

This work was supported by grants from the Energy Biosciences Division of the Department of Energy (DE-FG03-94ER20145) and the Welch Foundation (F-1217) to RMB.

If you would like to communicate directly with Andy about this new technique or to review the results herein, please email Andy at the following: abowling@mail.utexas.edu