Tijs Ketelaar

Unstable F-Actin Specifies the Area and Microtubule Direction of Cell Expansion in Arabidopsis Root Hairs

Plant cells expand by exocytosis of wall material contained in Golgi-derived vesicles. We examined the role of local instability of the actin cytoskeleton in specifying the exocytosis site in Arabidopsis root hairs. During root hair growth, a specific actin cytoskeleton configuration is present in the cells subapex, which consists of fine bundles of actin filaments that become more and more fine toward the apex, where they may be absent. Pulse application of low concentrations of the actin-depolymerizing drugs cytochalasin D and latrunculin A broadened growing root hair tips (i.e., they increased the area of cell expansion). Interestingly, recovery from cytochalasin D led to new growth in the original growth direction, whereas in the presence of oryzalin, a microtubule-depolymerizing drug, this direction was altered. Oryzalin alone, at the same concentration, had no influence on root hair elongation. These results represent an important step toward understanding the spatial and directional regulation of root hair growth.

The Actin-Interacting Protein AIP1 Is Essential for Actin Organization and Plant Development

Cell division, growth, and cytoplasmic organization require a dynamic actin cytoskeleton. The filamentous actin (F-actin) network is regulated by actin binding proteins that modulate actin dynamics. These actin binding proteins often have cooperative interactions. In particular, actin interacting protein 1 (AIP1) is capable of capping F-actin and enhancing the activity of the small actin modulating protein, actin depolymerising factor (ADF) in vitro. Here, we analyze the effect of the inducible expression of AIP1 RNAi in Arabidopsis plants to assess AIP1s role in vivo. In intercalary growing cells, the normal actin organization is disrupted, and thick bundles of actin appear in the cytoplasm. Moreover, in root hairs, there is the unusual appearance of actin cables ramifying the root hair tip. We suggest that the reduction in AIP1 results in a decrease in F-actin turnover and the promotion of actin bundling. This distortion of the actin cytoskeleton causes severe plant developmental abnormalities. After induction of the Arabidopis RNAi lines, the cells in the leaves, roots, and shoots fail to expand normally, and in the severest phenotypes, the plants die. Our data suggest that AIP1 is essential for the normal functioning of the actin cytoskeleton in plant development.

A Mechanism for Reorientation of Cortical Microtubule Arrays Driven by Microtubule Severing

Introduction Organization of the cortical cytoskeleton guides the growth and morphogenesis of organisms, from bacteria to higher plants, that depend on cell walls. By positioning wall-building enzymes, the cytoskeleton acts as an interior scaffold to direct construction of the cell’s exterior. In plants, environmental and hormonal signals that modulate cell growth cause reorganization of cortical microtubule arrays. These arrays do not appear to be remodeled by moving individual microtubules, but rather by rules that govern how microtubules are assembled or disassembled. In this Research Article, we investigate the mechanism by which blue light, an important signal from the environment, causes a rapid 90° reorientation of cortical arrays in growing cells of the plant axis. Blue light perception stimulates generation of a cascade of newly oriented microtubules by katanin severing. A confocal microscopy time series of the cortical microtubule array (white, preexisting; blue, newly assembled) in an Arabidopsis epidermal cell is shown. Perception of blue light by phototropin receptors has stimulated severing at microtubule intersections. Growth of the new ends creates new and co-oriented microtubules. Together, the organization of the preexisting array and the statistical behavior of severing favor the growth of longitudinal microtubules, driving array reorientation. Methods We used spinning-disk confocal microscopy to image the reorganization of cortical microtubule arrays in real time and visualize functional proteins tagged with fluorescent proteins. We developed image-analysis methods to measure changes in array organization and behaviors of individual microtubules during array reorientation. To test hypotheses about signaling and reorganizational mechanisms, we analyzed mutants in light-perception pathways and in activity of the microtubule-severing protein katanin. Finally, we conducted photomorphogenesis assays in plant seedlings to place our findings in a physiological context. Results We discovered a mechanism, based on microtubule severing by the protein katanin, that reorients cortical microtubule arrays in response to perception of blue light. Specifically, we observed that katanin localized to microtubule crossovers, where it was required to preferentially catalyze the severing of the newer microtubule, an activity that was stimulated by the function of phototropin blue light receptors. New plus ends created by severing were stabilized and immediately grew at a high frequency, resulting in the effective creation of new microtubules. Most microtubules generated during reorientation were created by this mechanism, producing ~83% of new longitudinal microtubules. Cortical arrays failed to reorient in a mutant lacking the katanin protein. Microtubules produced by severing at crossovers can make new crossovers and, thus, opportunities for further rounds of severing and initiation, constituting a molecular amplifier that rapidly builds a new population of microtubules orthogonal to the initial array. Further experiments put this mechanism in a physiological context by revealing that katanin function is required for directional blue light to stimulate bending of the plant axis toward the light source. Discussion Cortical microtubule arrays in higher plants are being recognized as systems with self-organizing properties arising from rules governing the outcomes of microtubule interactions. In this Research Article, we present evidence that one outcome of microtubule interaction, katanin-mediated severing at crossover sites, is regulated by light perception and acts to reorient the array. Severing is thought to help build microtubule arrays in neurons and meiocytes, but it has been difficult to test this idea directly because of imaging limitations. With live imaging of plant cell cortical arrays, we have been able to investigate the cellular function of severing at the level of individual molecular events, revealing how generation of microtubules by severing is used to construct a new array. Light Turns the Array The organization of cortical microtubule arrays in higher plant cells is essential for organizing cell and tissue morphogenesis, but it is not clear how specific architectures are acquired and reconfigured in response to environmental cues. Lindeboom et al. (10.1126/science.1245533, published online 7 November; see the Perspective by Roll-Mecak) used live-cell imaging and genetic studies to show that the microtubule-severing protein, katanin, plays a crucial role in reorienting cortical arrays from transverse to longitudinal in Arabidopsis seedlings in response to blue light perception. Katanin localized to microtubule intersections where, stimulated by blue light receptors, it preferentially catalyzed the severing of the newer microtubule. The microtubule “plus” end created by severing were observed to grow preferentially, effectively building a new population of microtubules orthogonal to the initial array. The net effect of this process steers the growing seedling toward light. A self-organizing system makes the microtubule array in plants rearrange in order for the shoot to turn toward blue light. 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.

Green Fluorescent Protein-mTalin Causes Defects in Actin Organization and Cell Expansion in Arabidopsis and Inhibits Actin Depolymerizing Factor's Actin Depolymerizing Activity in Vitro

Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-mTalin colocalizes with the actin cytoskeleton, its effect on actin dynamics and cell expansion has not been studied in detail. We created Arabidopsis (Arabidopsis thaliana) plants harboring alcohol inducible GFP-mTalin constructs to assess the effect of GFP-mTalin expression in vivo. We focused on the growing root hair as this is a model cell for studying cell expansion and root hair tip growth that requires a highly dynamic and polar actin cytoskeleton. We show that alcohol inducible expression of GFP-mTalin in root hairs causes severe defects in actin organization, resulting in either the termination of growth, cell death, and/or changes in cell shape. Fluorescence recovery after photobleaching experiments demonstrate that the interaction of GFP-mTalin and actin filaments is highly dynamic. To assess how GFP-mTalin affects actin dynamics we performed cosedimentation assays of GFP-mTalin with actin on its own or in the presence of the actin modulating protein, actin depolymerizing factor. We show that that GFP-mTalin does not affect actin polymerization but that it does inhibit the actin depolymerizing activity of actin depolymerizing factor. These observations demonstrate that GFP-mTalin can affect cell expansion, actin organization, and the interaction of actin binding proteins with actin.

Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis

This work investigates coordinated actin filament and microtubule activities. It shows that actin filaments and microtubules interact dynamically and that actin filaments depend on microtubules to recover following drug-induced depolymerization events. In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

Grab a Golgi: Laser Trapping of Golgi Bodies Reveals in vivo Interactions with the Endoplasmic Reticulum

In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached‐ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are ‘sticky’ and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.

The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants

Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

Patterning and Lifetime of Plasma Membrane-Localized Cellulose Synthase Is Dependent on Actin Organization in Arabidopsis Interphase Cells

The rate of insertion and lifetime of cellulose-synthesizing complexes at the plasma membrane is dependent on the organization of the actin cytoskeleton. 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 biogenesis.

BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways

Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a post-translationally regulated BBM:GR protein and cycloheximide to identify target genes that are directly activated by BBM expression in Arabidopsis seedlings. We show that BBM activated the expression of a largely uncharacterized set of genes encoding proteins with potential roles in transcription, cellular signaling, cell wall biosynthesis and targeted protein turnover. A number of the target genes have been shown to be expressed in meristems or to be involved in cell wall modifications associated with dividing/growing cells. One of the BBM target genes encodes an ADF/cofilin protein, ACTIN DEPOLYMERIZING FACTOR9 (ADF9). The consequences of BBM:GR activation on the actin cytoskeleton were followed using the GFP:FIMBRIN ACTIN BINDING DOMAIN2 (GFP:FABD) actin marker. Dexamethasone-mediated BBM:GR activation induced dramatic changes in actin organization resulting in the formation of dense actin networks with high turnover rates, a phenotype that is consistent with cells that are rapidly undergoing cytoplasmic reorganization. Together the data suggest that the BBM transcription factor activates a complex network of developmental pathways associated with cell proliferation and growth.

Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins

Autophagy is the non‐selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains. The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function. Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two‐hybrid assay. Interestingly, Atg8 shows significant homology with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 binds microtubules. Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process.