Tip growth in neuronal cells, plant cells, and fungal hyphae is known to require tip-localized Rho GTPase, calcium, and filamentous actin (F-actin), but how they interact with each other is unclear. The pollen tube is an exciting model to study spatiotemporal regulation of tip growth and F-actin dynamics. An Arabidopsis thaliana Rho family GTPase, ROP1, controls pollen tube growth by regulating apical F-actin dynamics. This paper shows that ROP1 activates two counteracting pathways involving the direct targets of tip-localized ROP1: RIC3 and RIC4. RIC4 promotes F-actin assembly, whereas RIC3 activates Ca2+ signaling that leads to F-actin disassembly. Overproduction or depletion of either RIC4 or RIC3 causes tip growth defects that are rescued by overproduction or depletion of RIC3 or RIC4, respectively. Thus, ROP1 controls actin dynamics and tip growth through a check and balance between the two pathways. The dual and antagonistic roles of this GTPase may provide a unifying mechanism by which Rho modulates various processes dependent on actin dynamics in eukaryotic cells.
Rho family small GTPases are signaling switches controlling many eukaryotic cellular processes. Conversion from the GDPto GTP-bound form is catalyzed by guanine nucleotide exchange factors (GEFs). Rho GEFs in animals fall into two structurally distinct classes containing DH and DOCKER catalytic domains. Using a plant Rho GTPase (ROP1) as bait in yeast two-hybrid screens, we identified a family of Rho GEFs, named RopGEFs. The Arabidopsis thaliana RopGEF family of 14 members contains a conserved central domain, the domain of unknown function 315 (DUF315), and variable N-and C-terminal regions. In vitro GEF assays show that DUF315 but not the full-length version of RopGEF1 has high GEF activity toward ROP1. Our data suggest that the variable regions of RopGEF1 are involved in regulation of RopGEF through an autoinhibitory mechanism. RopGEF1 overexpression in pollen tubes produced growth depolarization, as does a constitutively active ROP1 mutant. The RopGEF1 overexpression phenotype was suppressed by expression of a dominant-negative mutant of ROP1, probably by trapping RopGEF1. Deletion mutant analysis suggested a requirement of RopGEF activity for the function of RopGEF1 in polar growth. Green fluorescent protein-tagged RopGEF1 was localized to the tip of pollen tubes where ROP1 is activated. These results provide strong evidence that RopGEF1 activates ROP1 in control of polar growth in pollen tubes.
Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes.
Cellulose synthase (CESA) complexes can be observed by live-cell imaging to move with trajectories that parallel the underlying cortical microtubules. Here we report that CESA interactive protein 1 (CSI1) is a microtubule-associated protein that bridges CESA complexes and cortical microtubules. Simultaneous in vivo imaging of CSI1, CESA complexes, and microtubules demonstrates that the association of CESA complexes and cortical microtubules is dependent on CSI1. CSI1 directly binds to microtubules as demonstrated by in vitro microtubule-binding assay.cell expansion | cellulose | cell walls T he control of plant cell shape, and ultimately morphology, is achieved mostly by anisotropic expansion that results from the combined effects of uniform outward turgor pressure and nonuniform counteracting resistance exerted by cell walls. Cellulose microfibrils, as the major load-bearing polymers in cell walls, are the predominant component enforcing the asymmetric cell expansion (1). In growing cells, cellulose microfibrils are laid down transversely to the axis of elongation, thus forming a spring-like structure that reinforcing the cell laterally and favoring longitudinal expansion. The predominant theory of how plant cells establish cellulose microfibril orientation has implicated the cortical microtubules (1-7). Cortical microtubules were reported to be oriented in parallel to the cellulose microfibrils during cellulose synthesis in many different cell types and organisms (2, 4), and disruption of cortical microtubules using various microtubule inhibitors disorganizes the pattern of cellulose microfibril deposition (8-11).A recent advance in testing the role of microtubules in cellulose synthesis was made by visualizing cortical microtubule and cellulose synthase (CESA) complexes simultaneously (12). CESA complexes can be directly observed by live-cell imaging moving through the plasma membrane on trajectories that parallel the underlying cortical microtubules. When the microtubule array is disorganized by exposure to oryzalin, a microtubuledisrupting herbicide, the trajectories of the CESA particles change accordingly (12). These experiments provide convincing evidence to support the idea that the orientation of cortical microtubules specifies the spatial orientation in which cellulose microfibrils are deposited. However, this concept is an oversimplification because there are circumstances where the alignment of cellulose microfibrils apparently occurs independently of microtubules and the mechanism of interaction between microtubules and cellulose synthase complexes has not been described (2). Here, we report that the recently identified CESA interactive protein 1 (CSI1) mediates an interaction between microtubules and cellulose synthase. Results CSI1Colocalizes with Cortical Microtubules. CESA complexes move along trajectories that closely parallel microtubules (12-15). We examined whether CSI1 coaligns with microtubules in a transgenic line bearing both YFP-TUA5 (an α-tubulin) and red fluorescent protein (RFP)-CSI...
The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.
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