A Glorious Half‐Century of Microtubules

The 2013 special issue of The Plant Journal commemorates the discovery of microtubules in plant cells a half-century ago (Ledbetter and Porter, 1963). In October 1963, Myron Ledbetter and Keith Porter, while working at Harvard University, published an article in The Journal of Cell Biology with the title A “microtubule” in plant cell fine structure. This article is a literature classic. Working with the newly developed fixative glutaraldehyde, Ledbetter and Porter were able to identify with clarity the tubular nature of 25 nm diameter fiber-like structures in plant cells, which they described as microtubules. They immediately recognized the ubiquitous nature of these tubular elements, which they correctly predicted to be the same structures found in mitotic spindles and flagella. With these first clear images of microtubules in plant cells, Ledbetter and Porter noted the close coalignment of microtubules with wall microfibrils, which were transverse to the long axis of expanding cells. This finding elucidated previous observations by Paul Green, another microtubule pioneer, that colchicine, a known disruptor of the mitotic spindle, could also cause cells to swell radially instead of to elongate (Green, 1962). At that point in history, the verdict seemed clear: microtubules were responsible for determining the direction in which cellulose microfibrils were deposited. Yet the question as to how this is achieved and, indeed, how microtubule orientation is controlled has been slow to answer, despite 50 years of investigations. Even today, there remains controversy, incomplete evidence and exceptions to apparent rules in a field that has become even more competitive with the drive to harness cellulosic biofuels. Although the major observations of Ledbetter and Porter highlighted the cortical microtubule array and its likely role in directional cell expansion, subsequent microtubule research has gone in many directions, some of which are encapsulated in the 12 review articles of this special issue. The early work also stimulated others to use glutaraldehyde in a variety of cell types, and they were quickly joined by other researchers whose research benefited from the improvements accorded by improved fixation methods for electron microscopy. Several talented young researchers consolidated their careers and reputations by entering the microtubule research field. We are very fortunate to introduce this special issue with “Some Retrospectives on Early Studies of Plant Microtubules” by three of these eminent scientists, Peter Hepler, Jeremy Pickett-Heaps and Brian Gunning (Hepler et al. 2013). Their article provides a fascinating glimpse of the events surrounding the discovery of microtubules 50 years ago, and the exciting directions in which the field moved as their careers progressed. Keith Porter set the atmosphere in our laboratory at the Biological Laboratories in Harvard as one of cooperation and sharing of experiences. We had three new electron microscopes, giving us easy access to time on those instruments. This was a period when the techniques for examining biological specimens were improving at a remarkable pace. Added to this was Keith's magic in handling the bulk of administrative duties himself at a time when the paper work was less demanding, leaving us more time for research and discovery! Shortly after the introduction of aldehyde fixation by David Sabatini, Keith encouraged us to explore its use. He alerted me to be on the lookout for slender tubular structures which were beginning to be seen in various eucaryotic cells fixed in this way. When found, they became the focus of interest. These slender structures were particularly abundant in the cortical cytoplasm, adjacent to the growing cell wall. Their orientation was of particular interest being paralleled to that of the adjacent cellulosic fibers. Keith was, at first, skeptical of this coincidence, but once convinced, the writing began. We tried to create some descriptive term to relate these structures specifically to plant cytology, but eventually used “microtubule,” already in the literature. After our manuscript had been submitted to and reviewed by The Journal of Cell Biology, Eldon Newcomb arrived at Keith's invitation as a visiting scientist from Wisconsin. It was enjoyable to have a fellow botanist in our group. Shortly after Eldon's arrival, we had a chat about a discovery he and one of his students, Peter Hepler, had made in plant cells and a manuscript they had prepared. They too had seen microtubules, and though their interpretation of function was quite different from ours, they had seen them. Keith immediately showed Eldon our manuscript. Concern about being “scooped” had made me a frequent visitor to our library in pursuit of the latest botanical reports. Now, I was on hand every Thursday morning when our librarian set out the new journal issues. Neither before nor since have I been so abreast of the latest advances in plant sciences! Mild by today's standards, the competition surrounding the discovery of microtubules was not without controversy (Wasteneys and Lechner 2013). An article by DB Slautterback titled “Cytoplasmic microtubules I. Hydra” also appeared in the Journal of Cell Biology in 1963, just one issue prior to the Ledbetter and Porter article (Slautterback, 1963). Slautterback speculated that microtubules comprised phospholipid-protein membranes and were involved in ion or water transport, in contrast to Ledbetter and Porter's correct prediction that microtubules were composed of protein. The identification of this protein soon followed. Recognizing colchicine's disruptive effects on mitotic spindle fibers, Taylor and his colleagues used tritiated colchicine to purify “colchicine-binding protein” (Borisy and Taylor, 1967; Shelanski and Taylor, 1967), which was eventually determined to comprise a dimer of two similar but non-identical subunits (Adelman et al., 1968; Mohri, 1968; Shelanski and Taylor, 1968), which soon thereafter were renamed alpha and beta tubulin to reflect the tubular nature of the macromolecular into which tubulins assemble (Adelman et al., 1968; Mohri, 1968). The evolution of tubulins, microtubule-associated proteins, and the microtubule-organizing centres is the subject of three of our special issue contributions. As ubiquitous eukaryotic structures, it is not surprising that microtubules are on the one hand one of the most highly conserved features while on the other hand, their diversification parallels that of the tree of life. Diego Breviario, Silvia Giani and Laura Morello tackle the subject of tubulin evolution, revisiting the multi-tubulin hypothesis and highlighting the importance of tubulin diversification in many aspects of plant development (Breviario et al. 2013). In his review, John Gardiner analyzes the evolution of microtubule-associated proteins (MAPs) in plant lineages using the genomic information available for algal and plant models including Arabidopsis thaliana, Oryza sativa, Selaginella moellendorffii, Physcomitrella patens, Volvox carteri, and Chlamydomonas reinhardti. Interesting ideas and hypotheses are proposed that explore how selection pressure may have led to acquisition of functional and structural features of MAPs to sustain unique roles of microtubules in the complexity in multicellularity, sessility and hetero-gametophytic reproduction (Gardiner 2013). As a result of their interaction with various MAPs and motors, microtubules organize into some of the most exquisite nanomachines found in nature. These include the flagellar apparatus, which emerged early in eukaryotic evolution and is derived from the centrosome, a microtubule organizing center (MTOC) characterized by a conspicuous centriole pair surrounded by proteins that promote microtubule nucleation. In their contribution, Naoji Yobuki and Brian Leander (2013) review the history of the discovery of the flagellar apparatus and the challenges for its discovery and characterization. Through a multiphyletic analysis, the authors also bring to light common features of this structure throughout eukaryotic lineages. Plant microtubule arrays are highly specialized. Centrosomes, which form the basis of both interphase arrays and spindle poles in animal cells, are gone altogether from most vascular plants, and only found in the male gametes of plant and algal lineages that do not use pollen to transmit sperm. How microtubules are nucleated and assembled into the mitotic spindles of acentrosomal plant cells is the subject of the review by Kinda Masoud, Etienne Herzog, Marie-Edith Chabouté and Anne-Catherine Schmit (2013), which brings to light many conserved eukaryotic features. The turgor pressure that plant cells generate through passive uptake of fresh water drives cell expansion but renders useless the force that can be generated by the cytoskeletal constriction rings that divide other eukaryotic cells. Instead, plant cells partition by forming cell plates. The preprophase bands that determine division planes and the phragmoplasts that construct the cell plates once mitosis is complete is described in the article of Carolyn Rasmussen, Amanda Wright and Sabine Mueller (Rasmussen et al. 2013). They tackle the key questions of how plant cells first determine the appropriate division plane through the positioning of preprophase bands, and how the information for precise insertion of the cell plate is maintained after the disappearance of the preprophase band. Organization of the cortical microtubule array first described by Ledbetter and Porter (Ledbetter and Porter, 1963) is the subject of Erica Fishel and Ram Dixit's exploration of the role that gamma tubulin complexes play in nucleating microtubules from existing microtubules. In their review they bring together ideas generated from genetic strategies, live cell imaging and mathematical modelling to conclude that this highly dispersed form of microtubule nucleation not only supplies new microtubules but also contributes to array orientation, which is critical for determining cell shape and other features of differentiating cells (Fishel and Dixit 2013). The absence of centrosomes is once again a hallmark of plant cell specialization. As sessile organisms, plants have evolved complex and efficient suites for responding to biotic and abiotic stresses as well as for discriminating useful from wasteful interactions. Fascinating roles of microtubules in the establishment of symbioses and in plant-pathogen responses are extensively reviewed in the contribution from Adrienne Hardham (2013). It is apparent that in plant interactions with other organisms, microtubules have important roles to establish and sustain a defense layer of response against pathogens, as well as to facilitate the establishment of positive symbioses. Paradoxically, both events are based on invasion of the plant cell with either negative or positive consequences to the plant, and the article discusses evidence for mechanisms that are at the basis for the distinction of the nature of the biotic invasion, which may include a crosstalk of the actin cytoskeleton with microtubules in endomembrane traffic. An excellent companion contribution by Niehl et al. (2013) discusses the amazing scavenging action of viruses on the plant cell upon the hijacking of the microtubule cytoskeleton for the formation of unusual cellular structures with pivotal roles as hubs of virus reproduction. Microtubules not only have key roles in biotic responses, a role for microtubules in anticipating, sensing, integrating and responding to abiotic stresses is discussed in the contribution from Peter Nick (Nick 2013). In this context, the mechanisms for a role of the microtubule cytoskeleton as mechanosensorial structures for plant cells appear to be intertwined for discriminating internal turgor and external pressure, a relationship that needs to take into account the structural support of the cell wall. Microtubules are of course critical for determining the mechanical properties of the cell wall but their spatial organization is, in turn, responsive to the mechanical stress caused by isotropic turgor pressure but the directional nature of which is specified by cellular, tissue and organ geometries. The role that mechanical stress plays in the organization of microtubule arrays is the subject of Benoît Landrein and Olivier Hamant's review. They bring together over 50 years of research in this field and propose a model to explain how mechanical stress in the cell wall can be translated into microtubule organization and morphological outcomes (Lendrein and Hamant 2013). In the final contribution to this special issue we highlight recent work that brings to light a role for microtubules in endomembrane trafficking and organization (Brandizzi and Wasteneys 2013). One feature distinguishing plant cells from their animal counterparts is the predominance of actin filament-based motility via myosin motor proteins, which control the movement of components of the plant cell secretory pathway along with other major organelles. What role microtubules play has remained obscure but recent discoveries show that microtubules are indeed important for organizing endomembrane compartments, including those carrying cellulose synthase complexes and those involved in the recycling or degradation of the auxin efflux carrier PIN2. Since the early glimpses of microtubules in plant cells 50 years ago, a vast wealth of research has been directed at testing numerous models, using strategies ranging from electron microscopy to molecular genetics to explore the relationship described by Ledbetter and Porter between cortical microtubules and cellulose microfibrils. The specialist reviews that comprise this special issue extend far beyond the role that microtubules have in plant cell wall formation and morphogenesis to encompass the diverse fields for which microtubules are central players. It will be interesting to see what another half-century of research will yield.

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