Viktor Kirik

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.

CLASP localizes in two discrete patterns on cortical microtubules and is required for cell morphogenesis and cell division in Arabidopsis

In animals and yeast, CLASP proteins are microtubule plus-end tracking proteins (+TIPS) involved in the regulation of microtubule plus-end dynamics and stabilization. Here we show that mutations in the Arabidopsis CLASP homolog result in various plant growth reductions, cell form defects and reduced mitotic activity. Analysis of Arabidopsis plants that carry a YFP:AtCLASP fusion construct regulated by the AtCLASP native promoter showed similarities to the described localization of the animal CLASP proteins, but also prominent differences including punctate and preferential localization along cortical microtubules. Colocalization studies of YFP:AtCLASP and CFP:EB1b also showed that AtCLASP is enriched at the plus ends of microtubules where it localizes behind the AtEB1b protein. Moreover, AtCLASP overexpression causes abnormal cortical microtubule bundling and array organization. Cortical microtubule arrays have evolved to be prominent in plants, and our findings suggest that plant CLASP proteins may have adopted specific functions in regulating cortical microtubule properties and cell growth.

TONNEAU2/FASS Regulates the Geometry of Microtubule Nucleation and Cortical Array Organization in Interphase Arabidopsis Cells

Presented data demonstrate that TON2/FASS, which encodes the B′′ regulatory subunit of the protein phosphatase PP2A, regulates microtubule nucleation in interphase Arabidopsis cells. The key finding is that TON2 function regulates the balance of branched versus parallel microtubule-associated microtubule nucleation, thus revealing regulation of nucleation geometry. Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B′′ subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation.

CLASP interacts with sorting nexin 1 to link microtubules and auxin transport via PIN2 recycling in Arabidopsis thaliana.

Polarized movement of auxin generates concentration gradients within plant tissues to control cell division patterns and growth direction by modulating microtubule organization. In this study, we identify a reverse mechanism, wherein microtubules influence polar auxin transport. We show that the microtubule-associated protein CLASP interacts with the retromer component sorting nexin 1 (SNX1) to mediate an association between endosomes and microtubules. clasp-1 null mutants display aberrant SNX1 endosomes, as do wild-type plants treated with microtubule-depolymerizing drugs. Consistent with SNX1s role in trafficking of the auxin efflux carrier PIN-FORMED2 (PIN2), clasp-1 mutant plants have enhanced PIN2 degradation, and PIN2 movement to lytic vacuoles is rapidly induced by depolymerization of microtubules. clasp-1 mutants display aberrant auxin distribution and exhibit numerous auxin-related phenotypes. In addition to mechanistically linking auxin transport and microtubules, our data identify a ubiquitous endosome-microtubule association in plants.

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1s defense function, which is linked to the activation of ETI.

The Phragmoplast-Orienting Kinesin-12 Class Proteins Translate the Positional Information of the Preprophase Band to Establish the Cortical Division Zone in Arabidopsis thaliana

Kinesin-12 class motor proteins at the cortical division site maintain cortical division site identity proteins for efficient guidance of the phragmoplast in late cytokinesis. The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers.

Quantitative changes in microtubule distribution correlate with guard cell function in Arabidopsis.

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.

GCP-WD Mediates γ-TuRC Recruitment and the Geometry of Microtubule Nucleation in Interphase Arrays of Arabidopsis

Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis, but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed γ-tubulin ring complexes (γ-TuRCs) at the cell cortex that are associated primarily with existing microtubules and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40° to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis, we found that a conserved peripheral protein of the γ-TuRC, GCP-WD/NEDD1, associated with motile γ-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of γ-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls γ-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.

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

This work shows that Arabidopsis EB1b and SPR1, two microtubule plus-end tracking proteins, interact both genetically and biochemically. These proteins have complex interactions as they load onto microtubules and direct polar cell expansion and chiral organ growth in response to directional cues. 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.

Arabidopsis GLASSY HAIR genes promote trichome papillae development

Specialized plant cells form cell walls with distinct composition and properties pertinent to their function. Leaf trichomes in Arabidopsis form thick cell walls that support the upright growth of these large cells and, curiously, have strong light-reflective properties. To understand the process of trichome cell-wall maturation and the molecular origins of this optical property, mutants affected in trichome light reflection were isolated and characterized. It was found that GLASSY HAIR (GLH) genes are required for the formation of surface papillae structures at late stages of trichome development. Trichomes in these mutants appeared transparent due to unobstructed light transmission. Genetic analysis of the isolated mutants revealed seven different gene loci. Two—TRICHOME BIREFRINGENCE (TBR) and NOK (Noeck)—have been reported previously to have the glassy trichome mutant phenotype. The other five glh mutants were analysed for cell-wall-related phenotypes. A significant reduction was found in cellulose content in glh2 and glh4 mutant trichomes. In addition to the glassy trichome phenotype, the glh6 mutants showed defects in leaf cuticular wax, and glh6 was found to represent a new allele of the eceriferum 10 (cer10) mutation. Trichomes of the glh1 and glh3 mutants did not show any other phenotypes beside reduced papillae formation. These data suggest that the GLH1 and GLH3 genes may have specific functions in trichome papillae formation, whereas GLH2, GLH4, and GLH6 genes are also involved in deposition of other cell-wall components.