TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.     

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP₂) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP₂. However, the interaction with PIP₂ is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.Pollen tube growth provides a unique model system for studying the role of exocytosis in cell morphogenesis. Pollen tubes are characterized by a highly rapid polarized unidirectional tip growth. Given the relative simplicity of their structure, fast growth rates, haploid genome content, and ability to grow under in vitro culture conditions, pollen tubes provide an extremely attractive system for studying cell morphogenesis. Furthermore, the growth characteristics of pollen tubes resemble those of root hairs, moss protonema, and fungal hyphae and to some extent can be paralleled to neurite growth (Chebli and Geitmann, 2007; Cheung and Wu, 2008; Guan et al., 2013; Hepler and Winship, 2015).It is well established that oscillating polarized exocytosis is fundamental for pollen tube development and determines growth rate (Bove et al., 2008; McKenna et al., 2009; Chebli et al., 2013). Exocytosis is required for the delivery of membrane and cell wall components to the growing tip. Yet, the exact location where exocytosis takes place is under debate. Ultrastructural studies showing the accumulation of vesicles at the tip suggested that exocytosis takes place at the tip (Lancelle et al., 1987; Lancelle and Hepler, 1992; Derksen et al., 1995), which was further supported by studies on the dynamics of cell wall thickness (Rojas et al., 2011), secretion of pectin methyl esterase (PME) and PME inhibitor, and staining of pectin by propidium iodide (PI; Röckel et al., 2008; Rounds et al., 2014). Conversely, based on colabeling with FM1-43 and FM4-64, it was concluded that exocytosis takes place in a subapical collar located in the transition zone between the tip and the shank, as well as at the shank, but not at the tip (Bove et al., 2008; Zonia and Munnik, 2008). In agreement, the pollen tube-specific syntaxin GFP-SYP124 was observed in the inverted cone, 10 to 25 μm away from the tip (Silva et al., 2010), and fluorescence recovery after photobleaching experiments with FM dyes also have indicated that exocytosis takes place at the subapical region (Bove et al., 2008; Moscatelli et al., 2012; Idilli et al., 2013). Yet, based on pollen tube reorientation experiments in a microfluidics device, it was concluded that growth takes place at the tip rather than at a subapical collar located in the transition zone between the apex and the shank (Sanati Nezhad et al., 2014). The tip-based growth is in agreement with exocytosis taking place at the tip. Presumably, part of the disagreement regarding the site of exocytosis resulted from the lack of intracellular markers for exocytosis (Cheung and Wu, 2008; Hepler and Winship, 2015), and as a result, the relationship between the FM dye-labeled inverted cone and exocytotic events during pollen tube growth is not fully understood.In many cell types, the process of secretory vesicles tethering and docking prior to fusion with the plasma membrane is initially mediated by an evolutionarily conserved tethering complex known as the exocyst. The exocyst is a heterooligomeric protein complex composed of eight subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 (TerBush et al., 1996; Guo et al., 1999). Studies originally based on budding yeast (Saccharomyces cerevisiae) have shown that the exocyst functions as an effector of Rab and Rho small GTPases that specifies the sites of vesicle docking and fusion at the plasma membrane in both space and time (Guo et al., 2001; Zhang et al., 2001). Support for the function of the exocyst in vesicle tethering was demonstrated recently by ectopic Sec3p-dependent vesicle recruitment to the mitochondria (Luo et al., 2014).Land plants contain all subunits of the exocyst complex, which were shown to form the functional complex (Elias et al., 2003; Cole et al., 2005; Synek et al., 2006; Hála et al., 2008). Studies in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) have implicated the exocyst in the regulation of pollen tube and root hair growth, seed coat deposition, response to pathogens, cytokinesis, and meristem and stigma function (Cole et al., 2005; Synek et al., 2006; Hála et al., 2008; Fendrych et al., 2010; Kulich et al., 2010; Pecenková et al., 2011; Safavian and Goring, 2013; Wu et al., 2013; Safavian et al., 2015; Zhang et al., 2016). The growth arrest of pollen tubes in sec8, sec6, sec15a, and sec5a/sec5b single and double mutants (Cole et al., 2005; Hála et al., 2008) or following treatment with the EXO70 inhibitor ENDOSIDIN2 (Zhang et al., 2016), and of root hairs in maize root hairless1 (rth1) SEC3 mutant (Wen et al., 2005), the inhibition of seed coat deposition in the sec8 and exo70A1 mutants (Kulich et al., 2010), and stigmatic papillae function in exo70A1 mutant plants (Safavian and Goring, 2013; Safavian et al., 2015) have implicated the exocyst in polarized exocytosis in plants. Given their function, it was likely that exocyst subunits could be used as markers for polarized exocytosis. Furthermore, it could also be hypothesized that, by studying the mechanisms that underlie the association of the exocyst complex with the plasma membrane, it should be possible to identify mechanisms underlying the regulation of polarized exocytosis (Guan et al., 2013). Moreover, since the interaction of exocytotic vesicles with the exocyst is transient and marks the site(s) of active exocytosis in the membrane, fluorescently labeled exocyst subunits could be used as markers for exocytosis while avoiding potential imaging artifacts stemming from pollen tube tips densely populated with vesicles.We have shown previously that the ROP effector ICR1 can interact with SEC3a and that ROPs can recruit SEC3a-ICR1 complexes to the plasma membrane (Lavy et al., 2007). However, ICR1 is not expressed in pollen tubes, suggesting that SEC3a membrane binding in these cells is likely dependent on other factors. In yeast, the interaction of Sec3p and Exo70p subunits with the plasma membrane is critical for exocyst function (He and Guo, 2009). It has been shown that the membrane binding of both Sec3p and Exo70p is facilitated by their interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂; He et al., 2007; Zhang et al., 2008). The yeast Exo70p interacts with PIP₂ via a number of positively charged residues distributed along the protein, with the highest number located at the C-terminal end (Pleskot et al., 2015). It has been suggested that yeast Sec3p interacts with PIP₂ through N-terminal basic residues (Zhang et al., 2008). These data were further corroborated by x-ray crystallography studies, which showed that the yeast Sec3p N-terminal region forms a Pleckstrin homology (PH) domain fold (Baek et al., 2010; Yamashita et al., 2010), a PIP₂ interaction motif (Lemmon, 2008).The localization of the exocyst subunits has been addressed in several studies. In Arabidopsis root hairs and root epidermis cells, SEC3a-GFP was observed in puncta distributed throughout the cell (Zhang et al., 2013). Studies on the Arabidopsis EXO70 subunits EXO70E2, EXO70A1, and EXO70B1 revealed them to be localized in distinct compartments that were termed exocyst-positive organelles (Wang et al., 2010). The exocyst-positive organelles, visualized mostly by ectopic expression, were shown to be cytoplasmic double membrane organelles that can fuse with the plasma membrane and secrete their contents to the apoplast in an exosome-like manner. It is not yet known whether other exocyst subunits also are localized to the same organelles and what might be the biological function of this putative compartment (Wang et al., 2010; Lin et al., 2015). In differentiating xylem cells, two coiled-coil proteins termed VESICLE TETHERING1 and VESICLE TETHERING2 recruit EXO70A1-positive puncta to microtubules via the GOLGI COMPLEX2 protein (Oda et al., 2015). Importantly, the functionality of the XFP fusion proteins used for the localization studies described above was not tested, and in most cases, the fusion proteins were overexpressed. Therefore, the functional localization of the exocyst is still unclear.Here, we studied the function and subcellular localization of the Arabidopsis exocyst SEC3a subunit using a combination of genetics, cell biology, biochemistry, and structural modeling approaches. Our results show that SEC3a is essential for the determination of pollen tube tip germination site and growth. Partial complementation of sec3a resulted in the formation of pollen with multiple pollen tube tips. In Arabidopsis growing pollen tubes, SEC3a localization is dynamic, and it accumulates in domains of polarized secretion, at or close to the tip plasma membrane (PM). Labeling of GFP-SEC3-expressing pollen with FM4-64 revealed the spatial correlation between polarized exocytosis and endocytic recycling. Furthermore, the association of SEC3a with PM at the tip marks the direction of tube elongation and positively correlates with the deposition of PI-labeled pectins and specific anti-esterified pectin antibodies in the cell wall. In tobacco (Nicotiana tabacum), the mechanisms underlying SEC3a interaction with the PM and its subcellular distribution depend on pollen tube growth mode and involve the interaction with PIP2 through the N-terminal PH domain. Collectively, our results highlight the function of SEC3a as a polarity determinant that links between polarized exocytosis and cell morphogenesis. The correlation between exocyst function and distribution in pollen tubes provides an explanation for some of the current discrepancies regarding the localization of exocytosis.     

Comparing two methods for estimating floral resource availability for insect pollinators in semi-natural habitats

Pollinator and flowering plant interactions play a critical role in maintaining most terrestrial ecosystems, including agroecosystems. Although estimates of floral resource availability are essential to understand plant–pollinator relationships, no generally accepted methodology exists to date. We compared two methods for sampling floral resources in a single meadow. About every three days, we recorded species lists of insect-pollinated plants with abundance categories assigned to each species (hereafter referred to as scanning) and we counted the flowering shoots in 36 2 × 2 m quadrats (hereafter quadrat sampling). These methods were compared with respect to (i) the number of species detected, (ii) estimated floral resource abundance, and (iii) temporal changes in flowering. With scanning, we found more potential nectar-plant species and species were found earlier than with quadrat sampling. With the latter, abundant species were found with higher probability than the scarce. Flower abundances were correlated between the two methods. We predicted that a cover of 6.3 ± 3.6% should be used for an appropriate estimate of flower abundance in our study site, although the optimal cover probably varies across different habitats. Furthermore, flower abundance changed 6% per day compared to the flowering peak. Overall, scanning seems to be more appropriate for detecting presence and the timing of species, while quadrats may provide higher resolution for abundance estimates. Increased sampling coverage and frequency may enhance research accuracy and using scanning and quadrat sampling simultaneously may help to optimize research effort. We encourage further development of sampling protocols.     

Relationships between wild bees,hoverflies and pollination success in apple orchards with different landscape contexts

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Dynamic evaluation of blood flow microcirculation by combined use of the laser Doppler flowmetry and high‐speed videocapillaroscopy methods

The dynamic light scattering methods are widely used in biomedical diagnostics involving evaluation of blood flow. However, there exist some difficulties in quantitative interpretation of backscattered light signals from the viewpoint of diagnostic information. This study considers the application of the high‐speed videocapillaroscopy (VCS) method that provides the direct measurement of the red blood cells (RBCs) velocity into a capillary. The VCS signal presents true oscillation nature of backscattered light caused by moving RBCs. Thus, the VCS signal can be assigned as a reference one with respect to more complicated signals like in laser Doppler flowmetry (LDF). An essential correlation between blood flow velocity oscillations in a separate human capillary and the integral perfusion estimate obtained by the LDF method has been found. The observation of blood flow by the VCS method during upper arm occlusion has shown emergence of the reverse blood flow effect in capillaries that corresponds to the biological zero signal in the LDF. The reverse blood flow effect has to be taken into account in interpretation of LDF signals.      

Roadside verges and cemeteries: Comparative analysis of anthropogenic orchid habitats in the Eastern Mediterranean

Several important habitats have become threatened in the last few centuries in the Mediterranean Basin due to major changes adopted in land‐use practices. The consequent loss of natural and seminatural orchid habitats leads to the appreciation of small anthropogenic habitats, such as cemeteries and roadside verges. Colonization of cemeteries and roadside verges by orchids has long been known, but no study to date compared the suitability of these two anthropogenic habitats for orchids. Therefore, in this paper our aim was to survey cemeteries and roadside verges and to compare these two habitats regarding their role in conserving Mediterranean terrestrial orchids. We conducted field surveys in three Mediterranean islands, Cyprus, Crete, and Lesbos, where both cemeteries and roadside verges were sampled on a geographically representative scale. We found a total of almost 7,000 orchid individuals, belonging to 77 species in the two anthropogenic habitat types. Roadside verges hosted significantly more individuals than cemeteries in Crete and Lesbos, and significantly more species across all three islands. Our results suggest that although cemeteries have a great potential conservation value in other parts of the world, intensive maintenance practices that characterized cemeteries in these three islands renders them unable to sustain valuable plant communities. On the other hand, roadside verges play a prominent role in the conservation of Mediterranean orchids in Cyprus and Greece. The pioneer status of roadside verges facilitates their fast colonization, while roads serve as ecological corridors in fragmented landscapes.     

A Cross-Species Analysis Reveals a General Role for Piezo2 in Mechanosensory Specialization of Trigeminal Ganglia from Tactile Specialist Birds

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Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFβ, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.