Nick T. Peters

The Microtubule Plus-End Binding Protein EB1 Functions in Root Responses to Touch and Gravity Signals in Arabidopsis

Microtubules function in concert with associated proteins that modify microtubule behavior and/or transmit signals that effect changes in growth. To better understand how microtubules and their associated proteins influence growth, we analyzed one family of microtubule-associated proteins, the END BINDING1 (EB1) proteins, in Arabidopsis thaliana (EB1a, EB1b, and EB1c). We find that antibodies directed against EB1 proteins colocalize with microtubules in roots, an observation that confirms previous reports using EB1-GFP fusions. We also find that T-DNA insertion mutants with reduced expression from EB1 genes have roots that deviate toward the left on vertical or inclined plates. Mutant roots also exhibit extended horizontal growth before they bend downward after tracking around an obstacle or after a 90° clockwise reorientation of the root. These observations suggest that leftward deviations in root growth may be the result of delayed responses to touch and/or gravity signals. Root lengths and widths are normal, indicating that the delay in bend formation is not due to changes in the overall rate of growth. In addition, the genotype with the most severe defects responds to low doses of microtubule inhibitors in a manner indistinguishable from the wild type, indicating that microtubule integrity is not a major contributor to the leftward deviations in mutant root growth.

Ectocarpus: A Model Organism for the Brown Algae

The brown algae are an interesting group of organisms from several points of view. They are the dominant organisms in many coastal ecosystems, where they often form large, underwater forests. They also have an unusual evolutionary history, being members of the stramenopiles, which are very distantly related to well-studied animal and green plant models. As a consequence of this history, brown algae have evolved many novel features, for example in terms of their cell biology and metabolic pathways. They are also one of only a small number of eukaryotic groups to have independently evolved complex multicellularity. Despite these interesting features, the brown algae have remained a relatively poorly studied group. This situation has started to change over the last few years, however, with the emergence of the filamentous brown alga Ectocarpus as a model system that is amenable to the genomic and genetic approaches that have proved to be so powerful in more classical model organisms such as Drosophila and Arabidopsis.

How to Cultivate Ectocarpus

This article describes the standard procedure for growing Ectocarpus in the laboratory. The culture is started with partheno-sporophyte (or sporophyte) filaments because this is the stage that is usually maintained in strain collections. The standard medium is Provasoli-enriched natural seawater (PES), but Ectocarpus can also be grown in artificial seawater, which allows more precise control over the culture conditions. The algae can be cultivated either in plastic Petri dishes or in 10-L bottles with bubbling, if large amounts of biomass are required. Standard growth conditions are 13°C with a 12h/12h d/night cycle and 20 µmol photons m(-2) s(-1) irradiance using daylight-type fluorescent tubes. All manipulations of Ectocarpus cultures should be performed in a clean environment (if possible, under a laminar flow hood). Forceps should be dipped in ethanol and allowed to dry under the hood.

Kinesin-5 motors are required for organization of spindle microtubules in Silvetia compressa zygotes

BackgroundMonastrol, a chemical inhibitor specific to the Kinesin-5 family of motor proteins, was used to examine the functional roles of Kinesin-5 proteins during the first, asymmetric cell division cycle in the brown alga Silvetia compressa.ResultsMonastrol treatment had no effect on developing zygotes prior to entry into mitosis. After mitosis entry, monastrol treatment led to formation of monasters and cell cycle arrest in a dose dependent fashion. These findings indicate that Kinesin-5 motors maintain spindle bipolarity, and are consistent with reports in animal cells. At low drug concentrations that permitted cell division, spindle position was highly displaced from normal, resulting in abnormal division planes. Strikingly, application of monastrol also led to formation of numerous cytasters throughout the cytoplasm and multipolar spindles, uncovering a novel effect of monastrol treatment not observed in animal cells.ConclusionWe postulate that monastrol treatment causes spindle poles to break apart forming cytasters, some of which capture chromosomes and become supernumerary spindle poles. Thus, in addition to maintaining spindle bipolarity, Kinesin-5 members in S. compressa likely organize microtubules at spindle poles. To our knowledge, this is the first functional characterization of the Kinesin-5 family in stramenopiles.

Asymmetric microtubule arrays organize the endoplasmic reticulum during polarity establishment in the brown alga Silvetia compressa.

Polarity is a fundamental characteristic of most cell types, and is crucial to early development of the brown alga Silvetia compressa. In eukaryotes the cytoskeleton plays an important role in generating cellular asymmetries. While it is known that F‐actin is required for polarization and growth in most tip‐growing cells, the roles of microtubules are less clear. We examined the distribution and function of microtubules in S. compressa zygotes as they polarized and initiated tip growth. Microtubules formed asymmetric arrays oriented toward the rhizoid hemisphere early in the polarization process. These arrays were spatially coupled with polar adhesive deposition, a marker of the rhizoid pole. Reorientation of the light vector during polarization led to sequential redistribution of polar axis components, with the microtubules and the polar axis reorienting nearly simultaneously, followed by cell wall loosening and then deposition of new polar adhesive. These findings suggested that microtubules may organize and target endomembrane arrays. We therefore examined the distribution of the endoplasmic reticulum during polarization and found it colocalized with microtubules and became targeted toward the rhizoid pole as microtubule asymmetry was generated. Endoplasmic reticulum association with microtubules remained fully intact following pharmacological disruption of F‐actin, whereas microtubule disruption led to aggregation of the endoplasmic reticulum around the nucleus. We propose that brown algae utilize microtubules for organization of the endoplasmic reticulum and migration of exocytotic components to the rhizoid cortex, and present a model for polarity establishment to account for these new findings.

Genetic Crosses between Ectocarpus Strains

This article describes a procedure for conducting crosses between different strains of Ectocarpus. Crossing gametophytes to obtain the sporophyte generation is the most technically challenging stage of this process because diploid sporophytes have to be distinguished from the haploid partheno-sporophytes that result from the parthenogenetic germination of unfused gametes. This requires careful monitoring of the progeny of the genetic cross until they have developed sufficiently to be transferred to a separate Petri dish. Genetic crosses allow several classical genetic methodologies to be applied in Ectocarpus, including allelic complementation tests, backcrosses, combination of different genetic mutations, and outcrosses to create mapping populations.

Non‐cell autonomous regulation of life cycle transitions in the model brown alga Ectocarpus

The model brown alga Ectocarpus has a haploid-diploid life cycle, involving alternation between two independent multicellular generations, the gametophyte and the sporophyte. Recent work has shown that alternation of generations is not determined by ploidy but is rather under genetic control, involving at least one master regulatory locus, OUROBOROS (ORO). Using cell biology approaches combined with measurements of generation-specific transcript abundance we provide evidence that alternation of generations can also be regulated by non-cell autonomous mechanisms. The Ectocarpus sporophyte produces a diffusible factor that causes major developmental reprogramming in gametophyte cells. Cells become resistant to reprogramming when the cell wall is synthetized, suggesting that the cell wall may play a role in locking an individual into the developmental program that has been engaged. A functional ORO gene is necessary for the induction of the developmental switch. Our results highlight the role of the cell wall in maintaining the differentiated generation stage once the appropriate developmental program has been engaged and also indicate that ORO is a key member of the developmental pathway triggered by the sporophyte factor. Alternation between gametophyte and sporophyte generations in Ectocarpus is surprisingly labile, perhaps reflecting an adaptation to the variable seashore environment inhabited by this alga.

Phospholipid signaling during stramenopile development

Development of sessile organisms requires adaptation to an ever-changing environment. In order to respond quickly to these challenges, complex signaling mechanisms have evolved to facilitate cellular modifications. The importance of phospholipid-based signaling pathways in plants, as well as animals, has recently been gaining attention. Both the PLD and PLC pathways produce the signaling molecule PA, which modulates MTs, F-actin, and endomembrane trafficking. We have examined the roles of the PLD signaling pathway during development of the marine brown alga Silvetia compressa. Zygotes were treated with 1- and 2-butanol, both of which activate the PLD enzyme. However, only 1-butanol competes with water as a transphosphatidylation substrate, at the expense of PA production. Interestingly, we found that 1- and 2-butanol both disrupted MT organization and thereby cell division, with 1-butanol being more potent. These findings question whether the effects of butyl alcohol treatment are due to lowered PA levels or activation of the PLD enzyme. Additionally, preliminary results show that inhibition of DAGK results in loss of centrosomal MTs and formation of cortical MT cages that are strikingly similar to those formed following 1-butanol treatment. These data suggest that perturbation of the PLD or PLC pathway leads to cortical stabilization and/or nucleation of MT arrays.

Isolation and Regeneration of Protoplasts from Ectocarpus

This article describes how to obtain isolated cells with no surrounding cell wall by enzymatic digestion of Ectocarpus filaments. The resultant protoplasts are totipotent and regenerate to produce individual algae under appropriate culture conditions. The yield of protoplasts and their capacity to regenerate are highly dependent on the Ectocarpus strain used, the stage of the life cycle, and the culture conditions. The highest yields are obtained with young gametophyte filaments cultivated at low density. The naked, wall-less cells produced by this protocol can be used for several applications, including studies of cell wall regeneration, investigation of the role of the cell wall in determining cell fate, and as a source of naked cells for the development of methods for introducing diverse molecules into the cell.

Immunostaining of Ectocarpus Cells

This article describes an immunostaining protocol for Ectocarpus that was optimized for the detection of tubulin but could be used with any suitable antibody. Ectocarpus has small but relatively transparent cells and the uniseriate filaments can be grown directly attached to the surface of microscope slides. These features make Ectocarpus particularly suitable for high resolution imaging approaches, both in vivo or after fixation. All incubations described below are carried out on a platform shaker at room temperature. Use high-quality microscope slides to avoid imperfections in the glass that can be a problem for confocal laserscan microscopy analysis.