Pattern formation of cortical microtubules and cellulose microfibrils

In this thesis we study the roles of microtubules at the plasma membrane and the cellulose microfibrils in the cell wall and how they are organized. This topic is introduces in chapter 1. In chapter 2 we study the formation of the transverse cortical microtubule array that is characteristic for elongating plant cells. We found that the cortical microtubule array starts ordered, and that the first direction of microtubule order is not transverse to the axis of cell elongation but have a diagonal bias. Quantification of the orientation of microtubule nucleations revealed a significant diagonal bias, which we confirmed by simulations to be sufficient to explain the initial diagonal order. We found that during disassembly the microtubules also showed a diagonal bias and a significant amount of early microtubule nucleations were not generated from γ-TuRC microtubule nucleating complexes. This led to the idea that a proportion of the initial nucleations stem from small microtubule fragments of preexisting microtubules that remained at the cell cortex during cytokinesis or drug induced microtubule disassembly. We showed with simulations that this type of nucleation has the capacity to increase the speed with which the cortical microtubule array is reformed. In chapter 3 we investigate the trafficking of cellulose synthase complexes from assembly in the Golgi system to their insertion into the plasma membrane. We find that the actin cytoskeleton is important for the global distribution of Golgi bodies, which in turn is important for the global distribution of cellulose synthase complexes in the plasma membrane. Cellulose synthase complexes were inserted into the membrane preferentially at locations where cortical microtubules were present. We showed that osmotic stress and a number of cellulose synthesis inhibitors blocked cellulose synthase insertion into the plasma membrane. The cellulose synthase complex containing compartments were seemingly still being delivered to the cortical microtubules where they accumulated. These compartments tracked depolymerizing microtubule ends. When the osmotic stress was relieved, cellulose sythase complex insertion was resumed from these compartments. Rapid movement of proteins, organelles and metabolites in the cytoplasm of plant cells depends on the actin cytoskeleton, whereas microtubules are important in regulating the location of proteins and cellular processes. In chapter 4 we found physical interactions between cortical microtubules and actin. We also found that the formation of the actin cytoskeleton after washing out the actin depolymerizing drug latrunculin B was dependent on the presence of microtubules. In the presence of cortical microtubules, new actin filaments initiated on and in the direction of cortical microtubules. In chapter 5 we investigate the mechanism reorientation of the cortical microtubule array from transverse to longitudinal in response to light signaling. We found that cortical microtubule array reorientation in dark grown hypocotyl cells was regulated by phototropin a blue light photoreceptor. We found that microtubule reorientation was delayed in phot1 phot2 mutants. We also found that ktn1-1, a null mutant of KATANIN P60, and spr3, a GCP2 allele with impaired function, severely retarded microtubule array reorientation in response to light. spry has altered angles of microtubule nucleation relative to the mother polymer, and ktn1-1 abolishes liberation of microtubules form their nucleation complexes to yield treadmilling polymers and microtubule severing at microtubule crossovers. We found that in response to blue light, the proportion of microtubule nucleations branching at 40 degrees from the mother microtubule to nucleation at 0 degrees from the mother microtubule was higher in wild type plant than in the phot1 phot2 mutant. We also found that the chance of microtubule severing at microtubule crossovers was significanlty higher in wild type than in the phot1 phot2 mutant. We propose that upregulation of the branching nucleations is needed to create a number of longitudinally oriented microtubules. These microtubules make a large number of crossovers with the existing transverse array and have an increased chance of being severed. Severing events that result in a stable new tip contribute to the increase in longitudinal microtubule order and ultimately lead to complete reorientation from transverse to longitudinal. The spatial organization of cortical microtubules and cellulose microfibrils are essential for plant morphogenesis, but the mechanism by which is unclear. Chapter 6 discusses the process of cell elongation and offers possible additional roles for cortical microtubules beyond guiding cellulose microfibril deposition in this process.</p

Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-¿-tubulin complex protein2-tagged ¿-nucleation complexes (¿-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving ¿-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation

Shared Decision Making in the Heart Team

This heart team review gives an overview of the current status of SDM in heart teams, and investigates the perceived needs for implementation of a SDM approach in clinical practice through an exploratory cross-sectional survey (N=101) and in-depth interviews (N=9) among an international community of heart team physicians specialized in HVD. Although heart team physicians agree on the importance of involving patients in heart team treatment decisions, half leaned toward the heart team making final decisions. In addition, limited understanding of the concept of SDM poses another barrier for physicians in involving patients in their own clinical practice. Finally, limited knowledge of and experience with the use of evidence-based decision aids is hampering wider implementation of SDM in clinical practice. The perceived needs and requirements for implementation of SDM according to heart team physicians forecast a long and winding road forward to sustainable implementation of SDM in heart teams. However, directly addressing attitudes, skills and tools may pave the way to effective implementation of SDM in heart teams. In conclusion, SDM is a means to improve care delivery for patients with HVD. Barriers exist for successful implementation by heart teams, yet opportunities arise as the culture shifts to physicians supporting patient engagement in decision making

Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is to drive organization of cellulose microfibrils by guiding the trajectories of active cellulose synthase (CESA) complexes in the plasma membrane, thus orienting nascent microfibrils. Here we provide evidence that cortical microtubules also position the delivery of CESA complexes to the plasma membrane and interact with small CESA-containing compartments by a mechanism that permits motility driven by microtubule depolymerization. The association of CESA compartments with cortical microtubules was greatly enhanced during osmotic stress and other treatments that limit cellulose synthesis. On recovery from osmotic stress, delivery of CESA complexes to the plasma membrane was observed in association with microtubule-tethered compartments. These results reveal multiple functions for the microtubule cortical array in organizing CESA in the cell corte

Cellulose microfibril deposition: coordinated activity at the plant plasma membrane

Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose synthase complexes into the plasma membrane. These complexes, the nanomachines that produce the cellulose microfibrils, move inside the plasma membrane leaving the cellulose microfibrils in their wake. Cellulose microfibril angle is an important determinant of cell development and of tissue properties and as such relevant for the industrial use of plant material. Here, we provide an integrated view of the events taking place in the not more than 100 nm deep area in and around the plasma membrane, correlating recent results provided by the distinct field of plant cell biology. We discuss the coordinated activities of exocytosis, endocytosis, and movement of cellulose synthase complexes while producing cellulose microfibrils and the link of these processes to the cortical microtubule

A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing

Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array

The Microtubule Plus-End Tracking Proteins SPR1 and EB1b Interact to Maintain Polar Cell Elongation and Directional Organ Growth in Arabidopsis

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues

Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesi