Cilia and flagella play a crucial role in the development and function of most eukaryotic organisms. The structural core of the flagellum, the axoneme, is conserved in most eukaryotes and careful regulation of dynein motors within the axoneme is required for proper ciliary beating. The sperm flagellum from the American eel ( Anguilla rostrata ) has been shown to lack many of the canonical axonemal proteins, including the radial spokes, the central pair complex, and possibly even the outer row of dynein arms, presenting a “minimal” flagellar system. Here, we present cryo-electron tomography analysis of the eel sperm flagellum. We identified two states for the eel sperm flagellum within our tomograms, narrow and wide, and found that the flagellum started narrow near the sperm head and widened distally. Subtomogram averages revealed that the eel sperm flagellum has retained remnants of the missing regulatory complexes, including a short radial spoke 3 complex, basal components of radial spokes 1 and 2, and an outer dynein arm docking complex. We also describe unique structural features of the A. rostrata sperm flagellum, such as a unique pattern of holes at the inner junction and an accessory complex located at the “outer” junction. Finally, we discuss the consequences of losing key regulatory factors for the eel sperm flagellum and hypothesize several evolutionary factors that may have led to their loss. Together, our results shed light onto the structure and function of the eel sperm axoneme and provide insight into the minimum requirements for proper ciliary beating.

Zygotes of the fucoid brown algae are useful models for investigating the molecular and cellular mechanisms of cell polarization. These organisms are abundant in the marine intertidal zone, where they grow firmly anchored to rocks. In response to environmental cues like sunlight, zygotes generate asymmetries within the cell that ultimately establish an axis of growth. The transduction of these cues relies on Rac1-mediated signaling that remodels the actin cytoskeleton, alters patterns of endocytosis and secretion, and ultimately prepares the zygote for localized (tip) growth. This chapter presents protocols for obtaining synchronous populations of zygotes, and for detecting changes in filamentous actin arrays, endomembrane patterns, and secretion patterns that occur during light-induced polarization.

Intertidal macroalgae are resilient organisms, withstanding daily fluctuations in temperature, salinity and air exposure. These sessile seaweeds are exposed to anthropogenic pollution as an additional stressor in their natural habitat alongside coastal waterways, which are often adjacent to industrial centers. One such habitat in the New Bedford Harbor in Massachusetts has been recognized by government agencies as a site of National Priority after several decades of polychlorinated biphenyl and metal discharge. Research has focused on the effects of these contaminants among large-scale trophic cascades, with less emphasis on sessile primary productivity. Macroalgal members of the community were the focus of this study and were used to evaluate the presence and effect of environmental contaminants. Contaminants prominent in highly polluted regions of the harbor were elevated in tissue samples. Field surveys evaluating density and biomass were conducted in five areas of varying contamination and indicated a drastic inhibition of colonization with increasing contaminant load. No differences were detected between sites in terms of oocyte production in a single receptacle, but only individuals from less contaminated regions released those oocytes. Potential causes of decreased population size may therefore be reduction in individual biomass and a decreased ability to release eggs.

Brown algae are important residents of intertidal ecosystems like New Bedford Harbor of Massachusetts. Release of industrial waste has left this habitat high in metals and polychlorinated biphenyls, and the effects of exposure on coastal macroalgae are poorly understood. To determine whether chronic contaminant exposure affects offspring development, embryogenesis was evaluated in three populations of Fucus vesiculosus from sites of varying contamination. While the time to germination was unaffected, cytokinesis was delayed and growth was retarded in embryos from the more contaminated sites. A pulse exposure of polychlorinated biphenyls at fertilization did not affect early growth parameters.

This study investigates the pattern and performance of cellular structures during the early development of zygotes and embryos of Sargassum cymosum. The early development S. cymosum germlings has already been characterized and compared with the pattern of development established for all fucoid algae, in which the zygote remains attached to the receptacle by mucilage during the establishment of polarity and early cell division. As in the algae Fucus and Silvetia, the first division is transverse across the longer axis of the zygote of S. cymosum. However, the cell that will give rise to the rhizoids is not determined in the first division; rather, the formation of this cell occurs with the second division, forming a small cell in the embryo shaded site. Stabilizing polarity during the process of forming a multicellular embryo occurs rapidly. During development, significant cytoplasmic alterations take place. Initially, the cytoplasm shows large clusters of phenolic compounds located in specific parts, but later, in the course of development, these compounds are dispersed in the cytoplasm, although a significant amount remains confined to the nucleus. Moreover, to produce more zygotes and higher growth rates for the germlings, the best conditions found for the species S. cymosum were 22 and 26 °C, respectively.

Fucoid zygotes use environmental vectors, including sunlight, to initiate a growth axis a few hours after fertilization. The first division is then transversely oriented by the growth axis, producing daughter cells of distinct fates. The tip growing rhizoid cell gives rise to the holdfast, anchoring the alga to the intertidal substratum, while the opposite thallus cell mainly generates the photosynthetic and reproductive stipe and fronds. Elaboration of this simple growth axis thus establishes the basic body plan of the adult; and elucidating the mechanisms responsible for formation of the growth axis is paramount to understanding fucoid morphogenesis. Recent studies have culminated in a model whereby sunlight, and perhaps other environmental cues, activate the signaling protein Rac1 at the rhizoid pole. Here it sets in motion nucleation of a patch of actin filaments that in turn, targets ions, proteins, and cellular processes to the future growth site. At germination, Rac1 initiates morphogenesis by inducing transformation of the patch of actin filaments to a structure that delivers vesicles to the growing tip, and a few hours later orients the spindle and cytokinetic plate.

Multicellular development has evolved independently on numerous occasions and there is great interest in the developmental mechanisms utilized by each of the divergent lineages. Fucoid algae, in the stramenopile lineage (distinct from metazoans, fungi and green plants) have long been used as a model for early development based on unique life cycle characteristics. The initially symmetric fucoid zygote generates a developmental axis that determines not only the site of growth, but also the orientation of the first cell division, whose products have distinct developmental fates. Establishment and maintenance of this growth axis is dependent on formation of a filamentous actin array that directs vesicular movement, depositing new membrane and wall material for development of the rhizoid. What is not well known, is how formation and placement of the actin array is regulated in fucoid algae. A candidate for this function is Rac1, a small GTPase of the highly conserved Rho family, which has been implicated in controlling the formation of actin arrays in diverse eukaryotes. We demonstrate that Rac1 is not only present during formation of the filamentous actin array, but that its localization overlaps with the array in polarizing zygotes. Pharmacologically inhibiting Rac1 activity was shown to impede formation and maintenance of the actin array, and ultimately polar growth. Evidence is provided that a requirement of Rac1 function is its ability to associate with membranes via a post-translationally added lipid tail. Taken together, the data indicate that Rac1 is a necessary participant in establishment of the growth pole, presumably by regulating the placement and formation of the actin array. A role for Rac1 and related proteins in regulating actin is shared by animals, plants, fungi and with this work, brown algae, thus a conserved mechanism for generating polarity is in operation in unique eukaryotic lineages.

Environmental contaminants, including poly‐chlorinated biphenyls ( PCB s), are enriched in coastal sediments, and despite a 1977 moratorium by the U nited S tates E nvironmental P rotection A gency on the production of PCB s, levels remain high, more so near former industrial plants. The effects of these contaminants on sessile species in the intertidal zone, particularly nonanimal species such as the ubiquitous fucoid brown algae, are not well known. We investigated the developmental effects of chronic PCB treatment beginning at fertilization on two species of marine rockweed, F ucus vesiculosus Linnaeus and S ilvetia compressa (J.Agardh) E.Serrão, T.O.Cho, S.M.Boo & Brawley. A mixture of the most widely used PCB congeners, Aroclors 1221, 1242, and 1254, was delivered at concentrations well below levels found in contaminated sediments, and resulted in severely delayed mitosis and cytokinesis in both species. In F. vesiculosus , this delay was accompanied by abnormal spindle morphology. PCB treatment also dramatically slowed or arrested rhizoid growth after 2–4 d, and by 7 d F . vesiculosus embryos were dead; in contrast, polar secretion of adhesive, germination, and photopolar germination were not affected. The dramatic delay in the first cell division and reduction in tip growth within the first week of development are likely to compromise S . compressa's ability to reproduce and establish new generations. Thus, the data presented here suggest that PCB s still present in coastal sediments may be inhibiting recruitment in these species. Moreover, as sediment dredging causes temporary spikes in PCB concentrations, these kinds of bioremediation steps may exacerbate the disruption of fucoid development.

The establishment of polarity is a fundamental property of most cells. In tip-growing plant and in fucoid algal cells, polarization specifies a growth pole, the center of localized secretion of new plasma membrane and cell wall material, generating a protrusion with a dome-shaped apex. Although much progress has been made concerning the cellular machinery required to execute tip growth, less is known regarding the signaling mechanisms involved in selecting the growth site and regulating vectorial cell division and expansion. Fucoid algal zygotes use extrinsic cues to orient their growth axes and are thus well-suited for studies of de novo selection of an axis. This process has been investigated largely by both pharmacological and immuno-localization studies. In tip growing plant cells, polarity is often predetermined, as in the formation of root hairs or moss protonema branches. More focus has been on genomic and genetic studies to reveal the molecules involved in expressing a growth axis. Here we review the common roles of the cytoskeleton and signal transduction pathways in the formation of a developmental axis in fucoid algal cells and the control of tip growth in higher plant cells.

Morphogenesis in fucoid algae begins with adhesive secretion and rhizoid germination, developmental events that secure the alga within the intertidal zone. The importance of the actin cytoskeleton during these processes has been well established; but in general, little is known about actin regulation within the stramenopile lineage. Based on conserved strategies for regulation of actin in other lineages, co-localization of the Arp2/3 complex with actin structures that are essential for rhizoid formation may implicate members of the Rho family of small GTPases in the signaling pathway(s) regulating actin polymerization during fucoid development. Our lab recently demonstrated Rac1 dependent regulation of endomembrane polarization, polarization of adhesive secretion, germination and tip growth in the fucoid brown alga Silvetia compressa. We also present new evidence revealing Rac1 localization during germination in S. compressa, and show that membrane localization is essential for proper Rac1 function.

Proper cell morphogenesis is dependent on the establishment and expression of cellular polarity. In the fucoid zygote, cell shape is critical for establishing the developmental pattern of the adult, and is achieved by guiding insertion of new membrane and wall to the rhizoid tip. Selection and growth of the appropriate tip site are accompanied by formation of dynamic actin arrays associated with the actin-nucleating Arp2/3 complex. In eukaryotes, a major pathway for activation of the Arp2/3 complex is via the Rho family GTPase, Rac1, which stimulates the Scar/WAVE complex. To determine whether Rac1 controls actin nucleation in Silvetia compressa (J. Agardh) E. Serrao, T. O. Cho, S. M. Boo et Brawley, we tested the effects of the Rac1-specific inhibitory compound, NSC23766, on actin dependent processes and on actin arrays. We found that NSC23766 disrupted polar secretion of adhesive, polarization of endomembranes, and tip-focused growth in the rhizoid. Similarly, NSC23766 altered actin and Arp2 localization in the growing rhizoid. In contrast, NSC23766 had no effect on selection of the growth site or on cytokinesis. These data suggest that Rac1 participates in nucleation of specific actin arrays in the developing zygote.

Research Article| November 01 2008 Tissue Regeneration in the Classroom! Lama Hassoun, Lama Hassoun Search for other works by this author on: This Site PubMed Google Scholar Whitney Hable, Whitney Hable Search for other works by this author on: This Site PubMed Google Scholar Tracie Payne-Ferreira Tracie Payne-Ferreira Search for other works by this author on: This Site PubMed Google Scholar The American Biology Teacher (2008) 70 (9): 546–549. https://doi.org/10.2307/27669340 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Lama Hassoun, Whitney Hable, Tracie Payne-Ferreira; Tissue Regeneration in the Classroom!. The American Biology Teacher 1 November 2008; 70 (9): 546–549. doi: https://doi.org/10.2307/27669340 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentThe American Biology Teacher Search This content is only available via PDF. Copyright National Association of Biology Teachers Article PDF first page preview Close Modal You do not currently have access to this content.

Research Article| November 01 2008 Tissue Regeneration in the Classroom! Lama Hassoun, Lama Hassoun Search for other works by this author on: This Site PubMed Google Scholar Whitney Hable, Whitney Hable Search for other works by this author on: This Site PubMed Google Scholar Tracie Payne-Ferreira Tracie Payne-Ferreira Search for other works by this author on: This Site PubMed Google Scholar The American Biology Teacher (2008) 70 (9): 546–549. https://doi.org/10.2307/27669340 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Lama Hassoun, Whitney Hable, Tracie Payne-Ferreira; Tissue Regeneration in the Classroom!. The American Biology Teacher 1 November 2008; 70 (9): 546–549. doi: https://doi.org/10.2307/27669340 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentThe American Biology Teacher Search This content is only available via PDF. Copyright National Association of Biology Teachers Article PDF first page preview Close Modal You do not currently have access to this content.

BACKGROUND:Fucoid zygotes are excellent experimental organisms for investigating mechanisms that establish cell polarity and determine the site of tip growth. A common feature of polarity establishment is targeting endocytosis and exocytosis (secretion) to localized cortical domains. We have investigated the spatiotemporal development of endomembrane asymmetry in photopolarizing zygotes, and examined the underlying cellular physiology.RESULTS:The vital dye FM4-64 was used to visualize endomembranes. The endomembrane system preferentially accumulated at the rhizoid (growth) pole within 4 h of fertilization. The polarized endomembrane array was initially labile and reoriented when the developmental axis changed direction in response to changing light cues. Pharmacological studies indicated that vesicle trafficking, actin and microtubules were needed to maintain endomembrane polarity. In addition, endocytosis required a functional cortical actin cytoskeleton.CONCLUSION:Endomembrane polarization is an early event in polarity establishment, beginning very soon after photolocalization of cortical actin to the presumptive rhizoid site. Targeting of endocytosis and secretion to the rhizoid cortex contributes to membrane asymmetry. We suggest that microtubule-actin interactions, possibly involving microtubule capture and stabilization at actin-rich sites in the rhizoid, may organize the endomembrane array.

Previous work has demonstrated that dynamic actin arrays are important for axis establishment and polar growth in the fucoid zygote, Silvetia compressa. Transitions between these arrays are mediated by depolymerization of an existing array and polymerization of a new array. To begin to understand how polymerization of new arrays might be regulated, we investigated the role of the highly conserved, actin-nucleating, Actin-related protein 2/3 (Arp2/3) complex. Arp2, a subunit of the complex, was cloned and peptide antibodies were raised to the C-terminal domain. In immunolocalization studies of polarizing zygotes, actin and Arp2 colocalized around the nucleus and in a patch at the rhizoid pole. In germinated zygotes, a cone of Arp2 and actin extended from the nucleus to the subapex. Within the rhizoid tip, three structural zones were observed in the majority of zygotes: the extreme apex was devoid of label, the subapex was enriched for Arp2, and further back both actin and Arp2 were present. This zonation suggests that actin nucleation occurs at the leading edge of the cone, in the Arp2-enriched region. In two sets of experiments, we showed that tip zonation is important for growth. First, pharmacological treatments that disrupted Arp2/actin zonation arrested tip growth. Second, changes in the direction of tip growth during negative phototropism were preceded by a reorientation of the zonation in accordance with the new growth direction. This work represents the first investigation of Arp2/3 complex localization in tip-growing algal cells.

Although much studied, the role of Ca2+ in early fucoid development remains unclear. One technique to investigate Ca2+ function that has not been fully exploited is the use of ionophores to drastically increase cytosolic Ca2+ activity. We have therefore conducted an analysis of the effects of uniform application of the Ca2+ ionophores, A23187 and ionomycin, on early development of fucoid algae. Both ionophores had substantially the same effects. Cell adhesive secretion, rhizoid growth and negative phototropism were reduced but not abolished by ionophore treatment, and germination was delayed. One plausible interpretation of these data is that secretion is partially compromised in the treated zygotes. Surprisingly, photopolarization and cytokinesis were unaffected, indicating that Ca2+ homeostasis may not be required for these processes.

There are two quite different modes of polar cell expansion in plant cells, namely, diffuse growth and tip growth. The direction of diffuse growth is determined by the orientation of cellulose microfibrils in the cell wall, which in turn are aligned by microtubules in the cell cortex. The orientation of the cortical microtubule array changes in response to developmental and environmental signals, and recent evidence indicates that microtubule disassembly/reassembly and microtubule translocation participate in reorientation of the array. Tip growth, in contrast, is governed mainly by F-actin, which has several putative forms and functions in elongating cells. Longitudinal cables are involved in vesicle transport to the expanding apical dome and, in some tip growers, a subapical ring of F-actin may participate in wall-membrane adhesions. The structure and function of F-actin within the apical dome may be variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in elongating tips suggests extensive regulation of this cytoskeletal array.

During the establishment of polarity, fucoid algal zygotes adhere to the substratum and select a growth axis according to environmental cues. Since little is known about the early events leading to axis selection, we investigated the chronology of cell adhesion, adhesive deposition, and axis selection induced by light (photopolarization). The requirements for secretion and the cytoskeleton in these processes and in the process of changing the orientation of an axis in response to new environmental cues (axis realignment) were also tested. Adhesive deposition occurred in two distinct stages: it was deposited uniformally on young zygotes (uniform primary adhesive) and later was deposited asymmetrically (polar secondary adhesive). Uniform primary adhesive deposition, cell adhesion, and photopolarization occurred simultaneously, and shortly thereafter, polar secondary adhesive deposition occurred at the future growth site. Uniform primary adhesive deposition and cell adhesion required secretion, but were independent of filamentous-actin (F-actin) and microtubule function. Photopolarization of young zygotes and polar secondary adhesive deposition required secretion but not microtubules. F-actin served to localize secondary adhesive deposition at the rhizoid pole; its function in polarization was more complex. F-actin was required for axis selection; however, its role in realignment of an axis depended on the light regime. The differing requirements for F-actin during development indicates that the axis is not static, but changes with time. These findings indicate that previous and future work on "axis formation" must be interpreted in the context of the developmental stage of the zygote.