Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes.Pollen tubes play a crucial role in flowering plant reproduction. A pollen tube is the vegetative cell of the male gametophyte. It undergoes rapid polarized growth in order to transport the two nonmotile sperm cells to an ovule. This rapid growth is supported by the constant delivery of secretory vesicles to the pollen tube tip, where they fuse with the plasma membrane to enlarge the cell (Bove et al., 2008; Bou Daher and Geitmann, 2011; Chebli et al., 2013). This vesicle delivery is assumed to be driven by the rapid movement of organelles and cytosol throughout the cell, a process that is commonly referred to as cytoplasmic streaming (Shimmen, 2007). Cytoplasmic streaming in angiosperm pollen tubes forms a reverse fountain: organelles moving toward the tip travel along the cell membrane, while organelles moving away from the tip travel through the center of the tube (Heslop-Harrison and Heslop-Harrison, 1990; Derksen et al., 2002). Drug treatments revealed that pollen tube cytoplasmic streaming and tip growth depend on actin filaments (Franke et al., 1972; Mascarenhas and Lafountain, 1972; Heslop-Harrison and Heslop-Harrison, 1989; Parton et al., 2001; Vidali et al., 2001). Curiously, very low concentrations of actin polymerization inhibitors can prevent growth without completely stopping cytoplasmic streaming, indicating that cytoplasmic streaming is not sufficient for pollen tube growth (Vidali et al., 2001). At the same time, however, drug treatments have not been able to specifically inhibit cytoplasmic streaming; thus, it is unknown whether cytoplasmic streaming is necessary for pollen tube growth.Myosins are actin-based motor proteins that actively transport organelles throughout the cell and are responsible for cytoplasmic streaming in plants (Shimmen, 2007; Sparkes, 2011; Madison and Nebenführ, 2013). Myosins can be grouped into at least 30 different classes based on amino acid sequence similarity of the motor domain, of which only class VIII and class XI myosins are found in plants (Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Class VIII and class XI myosins have similar domain architecture. The N-terminal motor domain binds actin and hydrolyzes ATP (Tominaga et al., 2003) and is often preceded by an SH3-like (for sarcoma homology3) domain of unknown function. The neck domain, containing IQ (Ile-Gln) motifs, acts as a lever arm and is bound by calmodulin-like proteins that mediate calcium regulation of motor activity (Kinkema and Schiefelbein, 1994; Yokota et al., 1999; Tominaga et al., 2012). The coiled-coil domain facilitates dimerization (Li and Nebenführ, 2008), and the globular tail functions as the cargo-binding domain (Li and Nebenführ, 2007). Class VIII myosins also contain an N-terminal extension, MyTH8 (for myosin tail homology8; Mühlhausen and Kollmar, 2013), and class XI myosins contain a dilute domain in the C-terminal globular tail (Kinkema and Schiefelbein, 1994; Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Recently, Mühlhausen and Kollmar (2013) proposed a new nomenclature for plant myosins based on a comprehensive phylogenetic analysis of all known plant myosins that clearly identifies paralogs and makes interspecies comparisons easier (Madison and Nebenführ, 2013).The localization of class VIII myosins, as determined by immunolocalization and the expression of fluorescently labeled full-length or tail constructs, has implicated these myosins in cell-to-cell communication, cell division, and endocytosis in angiosperms and moss (Reichelt et al., 1999; Van Damme et al., 2004; Avisar et al., 2008; Golomb et al., 2008; Sattarzadeh et al., 2008; Yuan et al., 2011; Haraguchi et al., 2014; Wu and Bezanilla, 2014). On the other hand, class XI myosin mutants have been studied extensively in Arabidopsis (Arabidopsis thaliana), which revealed roles for class XI myosins in cell expansion and organelle motility (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Very few studies have examined the reproductive tissues of class XI myosin mutants. In rice (Oryza sativa), one myosin XI was shown to be required for normal pollen development under short-day conditions (Jiang et al., 2007). In Arabidopsis, class XI myosins are required for stigmatic papillae elongation, which is necessary for normal fertility (Ojangu et al., 2012). Even though pollen tubes of myosin XI mutants have not been examined, the tip growth of another tip-growing plant cell has been thoroughly examined in myosin mutants. Root hairs are tubular outgrowths of root epidermal cells that function to increase the surface area of the root for water and nutrient uptake. Two myosin XI mutants have shorter root hairs, of which the myo11e1 (xik; myosin XI K) mutation has been shown to be associated with a slower root hair growth rate and reduced actin dynamics compared with the wild type (Ojangu et al., 2007; Peremyslov et al., 2008; Park and Nebenführ, 2013). Higher order mutants have a further reduction in root hair growth and have altered actin organization (Prokhnevsky et al., 2008; Peremyslov et al., 2010). Disruption of actin organization was also observed in myosin XI mutants of the moss Physcomitrella patens (Vidali et al., 2010), where these motors appear to coordinate the formation of actin filaments in the apical dome of the tip-growing protonemal cells (Furt et al., 2013). Interestingly, organelle movements in P. patens are much slower than in angiosperms and do not seem to depend on myosin motors (Furt et al., 2012).The function of myosins in pollen tubes is currently not known, although it is generally assumed that they are responsible for the prominent cytoplasmic streaming observed in these cells by associating with organelle surfaces (Kohno and Shimmen, 1988; Shimmen, 2007). Myosin from lily (Lilium longiflorum) pollen tubes was isolated biochemically and shown to move actin filaments with a speed of about 8 µm s⁻¹ (Yokota and Shimmen, 1994) in a calcium-dependent manner (Yokota et al., 1999). Antibodies against this myosin labeled small structures in both the tip region and along the shank (Yokota et al., 1995), consistent with the proposed role of this motor in moving secretory vesicles to the apex.In Arabidopsis, six of 13 myosin XI genes are highly expressed in pollen: Myo11A1 (XIA), Myo11A2 (XID), Myo11B1 (XIB), Myo11C1 (XIC), Myo11C2 (XIE), and Myo11D (XIJ; Peremyslov et al., 2011; Sparkes, 2011). The original gene names (Reddy and Day, 2001) are given in parentheses. Myo11D is the only short-tailed myosin XI in Arabidopsis (Mühlhausen and Kollmar, 2013) and lacks the typical myosin XI globular tail involved in cargo binding (Li and Nebenführ, 2007). The remaining genes have the same domain architecture as the conventional class XI myosins that have been shown to be involved in the elongation of trichomes, stigmatic papillae, and root hairs (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Therefore, we predicted that these five pollen-expressed, conventional class XI myosins are required for the rapid elongation of pollen tubes. In this study, we examined transfer DNA (T-DNA) insertion mutants of Myo11A1, Myo11A2, Myo11B1, Myo11C1, and Myo11C2 for defects in fertility and pollen tube growth. Organelle motility and actin organization were also examined in myo11c1 myo11c2 pollen tubes.     

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions,Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.     

Prohibitins Are Required for Cancer Cell Proliferation and Adhesion

Prohibitin 1 (PHB1) is a highly conserved protein that together with its homologue prohibitin 2 (PHB2) mainly localizes to the inner mitochondrial membrane. Although it was originally identified by its ability to inhibit G1/S progression in human fibroblasts, its role as tumor suppressor is debated. To determine the function of prohibitins in maintaining cell homeostasis, we generated cancer cell lines expressing prohibitin-directed shRNAs. We show that prohibitin proteins are necessary for the proliferation of cancer cells. Down-regulation of prohibitin expression drastically reduced the rate of cell division. Furthermore, mitochondrial morphology was not affected, but loss of prohibitins did lead to the degradation of the fusion protein OPA1 and, in certain cancer cell lines, to a reduced capability to exhibit anchorage-independent growth. These cancer cells also exhibited reduced adhesion to the extracellular matrix. Taken together, these observations suggest prohibitins play a crucial role in adhesion processes in the cell and thereby sustaining cancer cell propagation and survival.     

Arabidopsis RETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.     

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 Are Polygalacturonases Required for Cell Separation during Reproductive Development in Arabidopsis

Cell separation is thought to involve degradation of pectin by several hydrolytic enzymes, particularly polygalacturonase (PG). Here, we characterize an activation tagging line with reduced growth and male sterility caused by increased expression of a PG encoded by QUARTET2 (QRT2). QRT2 is essential for pollen grain separation and is part of a small family of three closely related endo-PGs in the Arabidopsis thaliana proteome, including ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2. Functional assays and complementation experiments confirm that ADPG1, ADPG2, and QRT2 are PGs. Genetic analysis demonstrates that ADPG1 and ADPG2 are essential for silique dehiscence. In addition, ADPG2 and QRT2 contribute to floral organ abscission, while all three genes contribute to anther dehiscence. Expression analysis is consistent with the observed mutant phenotypes. INDEHISCENT (IND) encodes a putative basic helix-loop-helix required for silique dehiscence, and we demonstrate that the closely related HECATE3 (HEC3) gene is required for normal seed abscission and show that IND and HEC3 are required for normal expression of ADPG1 in the silique dehiscence zone and seed abscission zone, respectively. We also show that jasmonic acid and ethylene act together with abscisic acid to regulate floral organ abscission, in part by promoting QRT2 expression. These results demonstrate that multiple cell separation events, including both abscission and dehiscence, require closely related PG genes.     

Notch Signaling is Required for Dendritic Cell Maturation and T Cell Expansion in Paracoccidioidomycosis

The Notch signaling pathway participates in several cellular functional aspects. This signaling has an important role in targeting both DC maturation and DC-mediated T cell responses. Thus, it is essential to investigate the influence of this signaling pathway in the role played by DCs in the pathogenesis of experimental paracoccidioidomycosis. This disease is a granulomatous and systemic mycosis that mainly affects lung tissue and can spread to any other organ and system. In this study, we demonstrated that bone marrow-derived DCs infected with yeasts from Paracoccidioides brasiliensis strain 18 performed efficiently their maturation after the activation of Notch signaling, with an increase in CD80, CD86, CCR7, and CD40 expression and the release of cytokines such as IL-6 and TNF-α. We observed that the inhibition of the γ-secretase DAPT impaired the proliferation of T cells induced by DC stimulation. In conclusion, our data suggest that Notch signaling contributes effectively to the maturation of DCs and the DC-mediated activation of the T cell response in P. brasiliensis infections.     

Ultraconserved Enhancers Are Required for Normal Development

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ABCG Transporters Are Required for Suberin and Pollen Wall Extracellular Barriers in Arabidopsis

Effective regulation of water balance in plants requires localized extracellular barriers that control water and solute movement. We describe a clade of five Arabidopsis thaliana ABCG half-transporters that are required for synthesis of an effective suberin barrier in roots and seed coats (ABCG2, ABCG6, and ABCG20) and for synthesis of an intact pollen wall (ABCG1 and ABCG16). Seed coats of abcg2 abcg6 abcg20 triple mutant plants had increased permeability to tetrazolium red and decreased suberin content. The root system of triple mutant plants was more permeable to water and salts in a zone complementary to that affected by the Casparian strip. Suberin of mutant roots and seed coats had distorted lamellar structure and reduced proportions of aliphatic components. Root wax from the mutant was deficient in alkylhydroxycinnamate esters. These mutant plants also had few lateral roots and precocious secondary growth in primary roots. abcg1 abcg16 double mutants defective in the other two members of the clade had pollen with defects in the nexine layer of the tapetum-derived exine pollen wall and in the pollen-derived intine layer. Mutant pollen collapsed at the time of anther desiccation. These mutants reveal transport requirements for barrier synthesis as well as physiological and developmental consequences of barrier deficiency.     

ROS Are Required for Mouse Spermatogonial Stem Cell Self-Renewal

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Arabidopsis ABCG Transporters,Which Are Required for Export of Diverse Cuticular Lipids,Dimerize in Different Combinations

ATP binding cassette (ABC) transporters play diverse roles, including lipid transport, in all kingdoms. ABCG subfamily transporters that are encoded as half-transporters require dimerization to form a functional ABC transporter. Different dimer combinations that may transport diverse substrates have been predicted from mutant phenotypes. In Arabidopsis thaliana, mutant analyses have shown that ABCG11/WBC11 and ABCG12/CER5 are required for lipid export from the epidermis to the protective cuticle. The objective of this study was to determine whether ABCG11 and ABCG12 interact with themselves or each other using bimolecular fluorescence complementation (BiFC) and protein traffic assays in vivo. With BiFC, ABCG11/ABCG12 heterodimers and ABCG11 homodimers were detected, while ABCG12 homodimers were not. Fluorescently tagged ABCG11 or ABCG12 was localized in the stem epidermal cells of abcg11 abcg12 double mutants. ABCG11 could traffic to the plasma membrane in the absence of ABCG12, suggesting that ABCG11 is capable of forming flexible dimer partnerships. By contrast, ABCG12 was retained in the endoplasmic reticulum in the absence of ABCG11, indicating that ABCG12 is only capable of forming a dimer with ABCG11 in epidermal cells. Emerging themes in ABCG transporter biology are that some ABCG proteins are promiscuous, having multiple partnerships, while other ABCG transporters form obligate heterodimers for specialized functions.     

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

Class I Myosins Have Overlapping and Specialized Functions in Left-Right Asymmetric Development in Drosophila

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left–right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.