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A key event in plant morphogenesis is the establishment of a division plane. A plant-specific microtubular preprophase band (PPB) accurately predicts the line of cell division, whereas the phragmoplast, another plant-specific array, executes cell division by maintaining this predicted line. Although establishment of these specific arrays apparently involves intracellular repolarization events that focus cellular resources to a division site, it still remains unclear how microtubules position the cell division planes. Here we study GFP-AtEB1 decorated microtubule plus-ends to dissect events at the division plane.
Early mitotic events included guided growth of endoplasmic microtubules (EMTs) towards the PPB site and their coincident localization with endocytic vesicles. Consequently, an endosomal belt lay in close proximity to the microtubular PPB at its maturation and was maintained during spindle formation. During cytokinesis, EMTs radiated from the former spindle poles in a geometrical conformation correlating with cell-plate navigation and tilt-correction. Naphthylphtalamic acid (NPA), an inhibitor of polar auxin efflux, caused abnormal PPBs and shifted division planes.
Our observations reveal a spatio-temporal link between microtubules and intracellular polarization essential for localized endocytosis and precise establishment of the division plane in plants. Additionally, they implicate the growth regulator, auxin, in this important cellular event.
In cell wall-encased, immobile plant cells, tight regulation of the cell division plane plays a crucial role in tissue and organ morphogenesis [1,2]. At the onset of mitosis a plant-specific cortical microtubule array, the PPB [3], emerges. Although the PPB disassembles as the cell enters mitosis, it precisely predicts where the new cell plate attaches to the parental cell walls at the end of cytokinesis [1,2]. Cell division planes are drastically affected by the absence of PPB [4], which may be achieved through experimental obliteration [5] or through genetic defects [6]. Since the discovery of the PPB four decades ago, the mechanism governing its creation and effect on delineating the future cell division plane has remained a mystery.
Though initially considered to be a cortical process, PPB formation involves two coincident cytoplasmic events too, namely the migration of the nucleus towards the cell center [7], and recruitment of numerous EMTs within motile cytoplasmic strands [8,9]. In centrifuged protonemata (consisting of a single elongated cell), nuclear displacement prior to pre-prophase induces the formation of a PPB around the new nuclear position, suggesting a role for the nucleus in PPB placement [10]. However, in plant cells under normal gravity conditions, the nucleus is positioned by microtubules, since its random migration is prevented by microtubular depolymerization [11]. These observations point towards a functional connection between the nucleus, microtubules and cell cortex for marking the PPB. In budding yeast, EMTs play a role in bringing the nucleus to the division plane by a cortical 'search and capture' mechanism [12-14]; and in fission yeast, EMTs position the nucleus by pushing against the cortex [15]. Under in vitro, simulated conditions, the microtubule seeds also probe the metallic boundaries of artificial chambers with cellular dimensions to bring nuclei to the center [16]. Together, these studies on diverse systems corroborate the role of microtubules in probing the cellular geometry to settle the nucleus at the center of the cell. In the absence of information on the polarity and dynamics of plant EMTs, their precise role in association with the nucleus and the PPB is unclear.
In plants, in addition to the PPB, the other two mitotic microtubular arrays, the spindle and the phragmoplast microtubules, are also of endoplasmic nature. During the spindle stage, the perpendicularity of the spindle to the cell division plane is often lost because of its rotation. In mammalian and yeast cells the spindle is kept at the center of the cell or at the bud site by the centrosome-originating astral microtubules [17,18]. In addition, any error in spindle orientation in these cell types is corrected by the outgoing astral microtubules, which probe the cortex to ensure that the segregated chromosomes are sufficiently distant and are not cleaved by the constricting actomyosin ring [19,20]. In acentrosomal plant cells, after PPB breakdown and disappearance of the nuclear surface-bound EMT, the factors maintaining the spindle at the cell centre still remain unresolved. Paradoxically, in plant cells possessing disoriented spindles, the cell plates are still able to anchor properly to the division sites marked perpendicularly to the cell axis. A mechanism obviously exists in plant cells to correct spindle disorientation and reinforce the line of cell division, but it remains obscure.
Here, we investigated the role of EMTs during PPB formation and their subsequent behavior following karyokinesis. Because of their uniform cell size, continuous cell division activity and absence of background fluorescence, Tobacco BY-2 cell suspensions remain a system of choice for plant cell cycle studies [21]. Therefore, we used tobacco BY-2 cells stably transformed with different microtubular markers, and performed a live cell time-lapse analysis to elucidate the role of EMTs in reinforcing the lines of the cell division planes in plants.
GFP-AtEB1 labeled plant microtubule plus ends exhibit guided growth to create bundles
Microtubule plus-end labeling has been achieved using a GFP-AtEB1 fusion protein and permits observations on microtubule growth directionality and dynamics [22]. In interphase cells, GFP-AtEB1 highlighted the bidirectional movement of comet-like structures, suggesting plus end growth of cortical microtubules of opposite growth polarities (Figure 1A, B; see also 1: Movie 1). In co-transformed cells, GFP-AtEB1 labeled the growing ends of YFP-MAP4 labeled microtubules (Figure 1C; see also 2: Movie 2) and co-localized with YFP-CLIP170 (Figure 1D; see also 3: Movie 3) on the growing microtubular plus ends, reconfirming the plus end-specific localization of GFP-AtEB1 in BY-2 cells.
Figure 1. Mechanisms for microtubule guidance and bundling. Green: GFP-AtEB1 (in A-H and N) and GFP-MAP4 (in I-M). Red: YFP-MAP4 (in C, E-G and N) and YFP-CLIP170 (in D). (A) 3-D maximum projection of interphase BY-2 cell, highlighting punctuate GFP-AtEB1 labeling in the cortex. (B) In interphase cells, GFP-AtEB1 comets at the cortex move in the same or opposite direction on the same track (arrowheads), and sometimes together (arrow) (See 1: Movie 1). (C) In co-transformed cells, GFP-EB1 labels the growing ends of microtubules labeled by YFP-MAP4 (See 2: Movie 2). (D) Co-localization of GFP-AtEB1 and YFP-CLIP170 on growing microtubule plus ends (See 3: Movie 3). (E) GFP-AtEB1 labeled growing microtubule changing from one microtubule track (labeled with YFP-MAP4) to another. [58] Growth of two separate unbundled microtubules (arrowheads) transiently meeting (arrow) and afterwards separating without inducing catastrophe. (G) Microtubules growing in opposite directions on the same track with similar speed (arrowheads) meet (arrow) and continue growing in opposite directions without inducing catastrophe. (H) Microtubule nucleation and growth (arrowheads) on an already existing microtubule (arrow). (I) Situation where attachment of a microtubule plus end (yellow arrowhead) to an existing microtubule induces a translocation of the minus end (red arrowhead) from one microtubule to another. (J) Microtubule plus end (yellow arrowhead) growing towards an existing microtubule, followed by guided growth in a new direction, causes bending at the point of previous attachment (red arrowhead). (K) Treadmilling microtubule (red arrowhead) with its plus end (red arrow) and minus end (yellow arrowhead) moving in the same direction (yellow arrowheads and red arrows) initiates bundling by bridging two other separate preexisting microtubules. (L) Long microtubule growing (arrowhead) and interacting with an existing microtubule induces reorientation of growth and bending, resulting in bundling with a shorter growing microtubule. (M) Depolymerization of one microtubule partially associated with a bundle (yellow arrowhead) causes bending of the remaining structure (red arrowhead). (N) Microtubule minus end detachment and subsequent movement (arrowheads) induces loss of GFP-AtEB1 from its plus end (yellow arrows); it recovers GFP-AtEB1 labeling and plus end growth once its minus end acquires new support on another polymer (arrowhead). Note that the same microtubule bends (red arrow) when its minus end is fixed and the plus end (yellow arrow) hits another microtubule. Time is indicated in seconds and bars represent 5 μm in A-D, 3 μm in K-M, 2 μm in E, I, J and 1 μm in F-H, N.
Additional File 1. GFP-AtEB1 labeled cortical microtubule plus-end dynamics exhibiting bidirectional growth during interphase. The movie is accelerated 80 times. The real time is 4 min.
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Additional File 2. GFP-AtEB1 (green) labels growing plus ends of YFP-MAP4 (red) decorated microtubules exhibiting microtubule guidance and bundling. The movie is accelerated 90 times. The real time is 3.75 min.
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Additional File 3. GFP-AtEB1 (green) colocalizes with YFP-CLIP170 (red) on growing microtubule plus ends. The movie is accelerated 60 times. The real time is 6 min.
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General observations on microtubule guidance and bundling formed the basis of our subsequent experiments. Tracking the GFP-AtEB1 comets, it was found that >80 % of freshly polymerizing microtubules exhibited guided growth on tracks established by existing microtubules, thereby creating bundles of microtubules (Figure 1C; see also 2: Movie 2). Preexisting microtubule-guided oriented bundling often involved i) two or more microtubules with apparently similar polarity moving one after the other, ii) microtubules with presumably opposite polarities moving in opposite directions to each other or iii) independent microtubules moving together as a pair with similar velocities (4.15 ± 0.41 μm/min, n = 29) (Figure 1B; see also 1: Movie 1). Although mammalian EB1 often induces microtubule bundling upon overexpression, AtEB1 did not cause the observed bundling in the plant cells since it lacks the microtubule bundling domain present in the mammalian ortholog [23]. Instead, intermicrotubular bridges [24] might be responsible for the observed bundle formation. In addition, when two microtubules grew together side-by-side on the same track, it was frequently observed that one of them shrank while the other continued growing (see 1: Movie 1). Further information on microtubular guidance and bundling mechanisms came from analysis of cells coexpressing GFP-AtEB1 and YFP-MAP4. A growing microtubule (green arrow, Figure 1E) could detach from an existing track and move on to another track where its growth became guided in another direction (Figure 1E). Interphase microtubules in mammalian cells show a similar guidance mechanism [25]. We also observed individual microtubule plus ends approaching each other from a similar (Figure 1F) or opposite (Figure 1G) direction and meeting without inducing catastrophe. Strikingly, microtubular nucleation was sometimes initiated on an existing microtubule (Figure 1H), an observation consistent with the plant-specific localization of gamma tubulin along the microtubule length [26] and with a study reporting microtubule nucleation from stable tubulin oligomers [27]. Microtubules also changed trajectories by reorienting their minus or plus ends when one of the ends was supported on the other polymer (Figure 1I, J). It was also observed that motile polymers exhibiting specialized treadmilling [28] initiated bundling by bridging the two preexisting and separate polymers (Figure 1K). In cases of guided growth-induced bundling, a shorter microtubule could adopt the growth direction of a preexisting longer microtubule and vice versa (Figure 1L). When one of the microtubules in a bundle retracted, it frequently caused the other to bend, implying the exertion of a pulling force (Figure 1M). We also observed that upon release of a minus end from a nucleation site, the opposite plus end depolymerized (with concomitant loss of GFP-AtEB1), whereas upon acquisition of a new support by its minus end the microtubule retained growth (and regained GFP-AtEB1) (Figure 1N). These observations indicate a provision for new nucleation on existing polymers, while suggesting that in certain cases the plus end somehow 'senses' the physical state of the opposite minus end. Together, these findings implicate intermicrotubular affinities and the capacity of the polymers to nucleate new or detached microtubules as a general mode of microtubule survival, reorientation and bundle creation.
Emergence, polarity and dynamics of EMTs at the onset of cell division
Equipped with information on the general polar behavior of microtubules in interphase cells we approached the questions of appearance, polarity and dynamics of the EMTs, specifically at the onset of cell division. During preprophase, more dynamic EMTs emerged, bridging the nucleus to the cortex and exhibiting considerable bidirectionality (Figure 2A–F; see also 4: Movie 4) with outgoing (from the nucleus towards the cortex) and incoming (from the cortex towards the nucleus) EMTs. More outgoing (80%) than incoming microtubules were observed though their growth rates were similar (5.86 ± 0.82 μm/min, n = 15 – outgoing; and 5.56 ± 0.47, n = 15 – incoming). Like cortical microtubules, EMTs also exhibited bundling and guidance characteristics indicating the existence of intermicrotubular affinities in the cytosol even in the absence of a cortical support. In contrast to yeast preprophase cells, where unidirectional microtubules (growing from the center towards the cell periphery) position the nucleus by pushing or pulling forces [12,15], our observations together with others [11] on the requirement of EMTs for nuclear displacement and their bidirectionality suggest that plant cells can utilize both outgoing and incoming EMTs for positioning the premitotic nucleus. Conversely, with bundling and track follow-up, the incoming microtubules might guide the outgoing ones to achieve selective cortical targeting. This may be an efficient mechanism for their navigation of intracellular space, since when many microtubules grow simultaneously in a bundle, the chance that microtubules will reach the cortical target(s) without becoming depolymerized in the process is expected to be substantially higher. Interestingly, EMTs maintained continuous contact with the cortical areas occupied by the developing PPB. At PPB maturation, the EMTs between the PPB and nuclear envelope (NE) remained bidirectional while those connecting to more distal cortical areas became unidirectional, displaying a radiating comet-like spectacular firework (Figure 2G–L; see also 5: Movie 5). At this stage, kymographs generated by tracking the GFP-AtEB1 comets clearly illustrate accelerated growth for both outgoing (8.33 ± 0.83 μm/min, n = 30) and incoming (8.02 ± 1.15 μm/min, n = 12) EMTs. Microtubule growth was maintained at ca. 6.78 ± 0.89 μm/min (n = 50) at the PPB [9] (Figure 2M–O). Consequently, the microtubule density on the NE gradually increased (see Figure 2G–K), confirming earlier observations on microtubular growth and stabilization on the NE in plant [9] and mammalian [29] cells. Our observations suggest that at the onset of mitosis, the outwardly-radiating EMTs position the nucleus in the center of the cell by pushing/pulling forces, while the bidirectional EMTs connecting the NE to the PPB position the nucleus at the centre of the PPB. Moreover, during PPB maturation, the change from bidirectional growth (from the distal cortex towards the NE and from the NE towards the distal cortex) to unidirectional growth (from the NE towards the distal cortex) of EMTs severely reduces their chance of survival and thereby causes their detachment and collapse.
Figure 2. Polarity and growth speed of EMTs bridging nucleus and cortex. Green: GFP-AtEB1 (in A-L). (A-F) EMTs exhibit bidirectional growth and microtubule bundling. Note that that the microtubule originating from the nuclear surface (outgoing) and the one coming from the cortex (incoming) cross each other (arrow) and, as in the cortical array, grow with similar speeds without interfering each other (arrowheads) (see 4: Movie 4). (G-L) EMT plus ends radiating mainly in an outward direction from the NE during PPB maturation (see 5: Movie 5). Kymograph projection of microtubule plus ends in the interphase cortex (M), PPB cortex (N) and preprophase cytoplasm (O) showing sustained polymerization. The horizontal axis, d, represents distance (18 μm in M, 13 μm in N and 20 μm in O), and the vertical axis, t, represents time (290 s in M, 140 s in N and 390 s in O). Note that for each of the 3 cases (M-O), the microtubules follow the tracks, exhibit bi-directionality and grow with the same speeds. By comparing the slopes between images M-O, it becomes evident that the microtubule growth speed increases from interphase to the PPB stage, as previously reported [9]. Note that the arrowhead in M shows the crossing of two EMTs growing on the same path at the same time but in opposite directions. Nucleus is marked by 'N', time is indicated in seconds and bars represent 8 μm.
Additional File 4. GFP-AtEB1 labeled endoplasmic microtubule plus ends display bidirectional (incoming and outgoing) growth polarity and bundling. The movie is accelerated 110 times. The real time is 6.5 min.
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Additional File 5. During PPB maturation, GFP-AtEB1 labeled endoplasmic microtubule plus ends radiate symmetrically, mainly in an outward direction from the NE. The movie is accelerated 100 times. The real time is 12 min.
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Role of EMTs in premitotic cytoplasmic organization
The implications of the EMT configuration for the organization of the premitotic cytoplasm were now investigated using GFP-MAP4 transformed BY-2 cells [9] together with various organelle markers. During G2-M transition, FM4-64-labeled endosomes (Figure 3A), Alexa 633-labeled pinocytic vesicles (Figure 3B), ST-YFP-labeled Golgi bodies (GA) (Figure 3C) and Mitotracker-labeled mitochondria (Figure 3D) all localized along the EMTs. In contrast to interphase, when the microtubules remain at the cortex and large vacuoles occupy the cell space (Figure 3E), the vacuoles appeared fragmented by intersecting EMTs during preprophase (Figure 3F). Previous studies have shown that the motility of cytoplasmic organelles in plants is mainly actin-based [30] and that the actin cytoskeleton co-exists with the mitotic microtubular arrays [31]. To further investigate the respective roles of microtubules and actin filaments in premitotic cytoplasmic organization, we treated the cells with latrunculin B (an actin polymerization inhibitor) and oryzalin (a microtubule depolymerizing herbicide). In latrunculin B-treated cells, the EMTs appeared stabilized and more intense with a normal cytoplasmic configuration (Figure 3G), whereas oryzalin destroyed the EMTs and caused cytoplasmic disorganization and nuclear displacement (Figure 3H). After combined oryzalin and latrunculin B treatment the nucleus completely lost its central position and the cytoplasm (stained with unbound GFP-MAP4) became completely disorganized (Figure 3I). After the oryzalin was washed out, the EMTs gradually reappeared and the cytoplasm regained its normal configuration with the nucleus replaced at the cell centre (data not shown). Together, these results suggest that EMTs have a major role in organizing the premitotic cytoplasm, but they do not discount the role of actin in mediating organelle motility.
Figure 3. EMTs configure the premitotic cytoplasm. Green: GFP-MAP4 (in A-I). Red: FM4-64 (in A, E and F), Alexa 633 (in B), ST-YFP (in C), Mitotracker (in D) FM4-64 labeled endosomes (A), Alexa 633 labeled pinocytic vesicles (B), ST-YFP labeled GA (C) and Mitotracker labeled mitochondria (D) all remain in the vicinity of GFP-MAP4 marked EMT tracks. (E) At interphase, GFP-MAP4 labeled microtubules remain in the cortex and FM4-64 labeled vacuoles occupy most of the endoplasmic space. [F] At preprophase, GFP-MAP4 labeled EMTs intersect the vacuoles labeled by FM4-64. Premitotic cells treated with latrunculin B (G), oryzalin (H) or both (I) show cytoplasmic disorganization in the presence of oryzalin (H-I). Bars represent 8 μm.
Guided growth of EMTs towards the PPB site and their coincident localization with endocytic vesicles
The implications of the observation, which differentiated between intracellular motility and intracellular compartmentalization, became apparent when we investigated the localization pattern for FM4-64 labeled endocytic vesicles in relation to the microtubules. During interphase, FM4-64 labeled endocytic vesicles were randomly localized in the cell (data not shown), but early in the G2-M transition they started coaligning with emerging EMTs (Figure 4A, B). In GFP-AtEB1 transformed cells, these endocytic vesicles displayed internalization paths along the EMT trajectories and their appearance coincided with the cortical sites approached by the EMT plus ends (Figure 4C–F; see also 6: Movie 6). Oryzalin-induced microtubule depolymerization immediately affected endocytic vesicular internalization, with complete disruption of their internalization routes (Figure 4G–I; see also 7: Movie 7). When the oryzalin was washed out, the reformed EMTs again approached the cortex and the internalization of the endocytic vesicle traffic resumed (Figure 4J). Most importantly, during PPB maturation the endocytic material aggregated at the cortical areas occupied by the PPB and approached by the radiating EMT plus ends (Figure 4K; see also 8: Movie 8). Consequently, the endocytic vesicles formed a cortical belt loosely co-localizing with the microtubular PPB (Figure 4L, M). Support for this observation comes from a recent electron microscope study analyzing the membrane architecture of the PPB, which revealed an accumulation of clathrin-coated and non-coated pits specifically in the PPB regions [32]. Moreover, the activity of an endosomal marker protein Ara7 (Rab5 homologue from Arabidopsis), is known to be up-regulated during mitosis [33], and a similar PPB belt was observed using GFP-Ara7 labeled endosomes (Figure 4N). Furthermore, the endosomal band we observed co-localized with a band comprising Golgi bodies (Figure 4O) [34]. It is noteworthy that during PPB maturation, the EMTs, which connect the nucleus to the cortical PPB, prohibit a continuous vacuolar structure and thereby create a cytoplasmic area proximal to the PPB (Figure 4P). This cytoplasmic area occupied by the EMTs at PPB maturation is still maintained at the spindle stage (Figure 5A). It has been proposed that the actin-depleted zone (ADZ), which appears during PPB breakdown and is also maintained throughout cytokinesis, participates in regulating the division plane, since actin disruption before ADZ formation affects the cell division planes [35]. We speculate that the lack of actin prohibits further transport of continuously endocytosed material, contributing to the formation of a coherent endosomal belt, for it has been shown that plant endosomal trafficking is mostly actin-based [36].
Figure 4. Endosomal belt co-localizes with microtubular PPB during preprophase. Green: GFP-MAP4 (in A, B, L, M and P), GFP-AtEB1 (in C-K), GFP-Ara7 (in N) and ST-YFP (in O). Red: FM4-64 (in A-M and O-P). Early in the G2-M transition, FM4-64 labeled endocytic vesicles follow the emerging EMTs labeled with GFP-MAP4, as shown in single median section (A) and 3D-projection (B). The marked rectangle in (C) is zoomed in for (D-I). FM4-64 labeled endocytic vesicles preferentially internalize from the cortical areas approached by the GFP-AtEB1 labeled EMT plus ends (D-F) (see 6: Movie 6), and oryzalin-induced microtubule depolymerization disrupts their internalization routes (G-I) (see 7: Movie 7) whereas the internalization paths are recovered after oryzalin removal (J). (K) Close-up of GFP-AtEB1 marked EMT plus ends bridging the NE and PPB. Note that during PPB narrowing, FM4-64 labeled endocytic vesicles preferentially internalize from the cortical areas approached by the GFP-AtEB1 (see 8: Movie 8). Formation of an FM4-64 labeled cortical belt at the PPB site (labeled with GFP-MAP4) is shown in a single median section (L) and in 3-D projection (M). (N) 3-D projection of GFP-Ara7 labeled endosomes exhibiting an endosomal belt at preprophase. (O) Both FM4-64 labeled endosomes and ST-YFP labeled GAs form a cortical belt at the PPB site. (P) GFP-MAP4 labeled EMTs connecting the nucleus to the PPB intersect FM4-64 labeled vacuoles. Time is indicated in minutes. Bars represent 7 μm in A, B, J, L, M, 10 μm in C, N-P and 5 μm in K.
Figure 5. PM targeted EMT plus ends probe the areas occupied by the preceding PPB and align the cell plates for proper docking at the parental walls. Green: GFP-MAP4 (in A, F-I), GFP-AtEB1 (in B-E, J-O and P). Red: FM4-64 (in A, F-O), YFP-MAP4 (in P). (A) Discontinuity of the vacuolar structures in the preceding PPB site (arrowheads) is maintained at the spindle stage, as visualized with FM4-64 labeled vacuoles and GFP-MAP4 labeled microtubules.(B-C) At the onset of the phragmoplast stage, GFP-AtEB1 labeled EMT plus ends (red arrowheads) originating from the former spindle poles grow towards the cortex (see 9: Movie 9). Occasionally, they grow towards the polar areas (yellow arrowhead). (D) GFP-AtEB1 labeled EMT plus ends (arrowheads) are attracted to the cortical areas marked by the preceding PPB. At late telophase, the distance through which GFP-AtEB1 labeled EMT plus ends reach towards the cortex is reduced. (E) 3-D projection showing GFP-AtEB1 labeled EMT plus end trajectories directed towards the cortex, which are different from the main phragmoplast structure. GFP-MAP4 labeled EMTs (F-I) or GFP-AtEB1 labeled EMT plus ends (J-M) continue to reach the cortex at the former PPB site and display close proximity to FM4-64 labeled endosomes (red arrow and arrowheads). These endosomes display movement towards the minus end of these EMTs. (N-O) GFP-AtEB1 labeled plus end growth of EMTs (arrowheads) towards opposite sides of the cortex is maintained during cell plate and phragmoplast tilting (see 10: Movie 10). (P) Enrichment of GFP-AtEB1 labeled microtubule plus ends (arrowhead) but not of YFP-MAP4 labeled microtubular parts at the phragmoplast midline. Time in F-I is given in seconds while that in J-O is indicated in minutes. Bars in A-O represent 8 μm while that in P represents 10 μm.
Additional File 6. FM4-64 labeled endocytic vesicles (red) preferentially internalize from the cortical areas approached by the GFP-AtEB1 labeled EMT plus ends (green). The movie is accelerated 80 times. The real time is 20 min.
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Additional File 7. Oryzalin-induced microtubule depolymerization disrupts internalization routes of FM4-64 labeled endosomes. The movie is accelerated at 80 times. The real time is 7 min.
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Additional File 8. GFP-AtEB1 marked EMT plus ends bridging NE and PPB. Note that during PPB narrowing, FM4-64 labeled endocytic vesicles preferentially internalize from the cortical areas approached by the GFP-AtEB1. The movie is accelerated at 120 times. The real is 18 min.
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EMTs radiating from the former spindle poles attain a geometrical conformation correlating with cell-plate navigation and tilt-correction
Following observations on microtubule and organelle behavior during the early stages of mitosis, we analyzed events at later stages. Immediately after chromosomal separation at anaphase, the EMTs emanating from the region occupied by spindle poles 'probed' the cell cortex (Figure 5B, C; see also 9: Movie 9) while exhibiting unidirectional growth at speeds of 8.52 ± 1.23 μm/min (n = 45). During late telophase, these EMTs mainly probed the cortical areas previously occupied by the PPB (Figure 5D). In addition, the EMTs originating from the non-facing surfaces of the daughter nuclei appeared fewer than the phragmoplast microtubules and occasionally exhibited growth trajectories towards the cell poles (Figure 5E). Because of the remarkable co-incidence between the EMTs and endocytosed material during preprophase, we investigated whether these EMTs approaching the cortex co-localized with FM4-64-labeled endocytic material during telophase. Such co-localization was observed both for GFP-MAP4 (Figure 5F–I) and GFP-AtEB1 (Figure 5J–M) labeled EMTs which were approaching the former PPB sites. During cell plate expansion, the EMTs appeared to navigate the cell plate and to align it to establish a clear line of division (Figure 5N, O; see also 10: Movie 10). EMT plus ends within the phragmoplast midline (where the cell plate subsequently formed) were labeled strongly with GFP-AtEB1 but less strongly with YFP-MAP4, indicating the polarity of the phragmoplast microtubules, oriented with their plus ends towards the developing cell plate (Figure 5P).
Additional File 9. At the onset of the phragmoplast stage, GFP-AtEB1 labeled EMT plus ends originating from the former spindle poles grow towards the cortex. The movie is accelerated 60 times. The real time is 3 min.
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Additional File 10. GFP-AtEB1 labeled plus end growth of EMTs towards opposite sides of the cortex is maintained during cell plate and phragmoplast tilting. The movie is accelerated 60 times. The real time is 2 min.
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In budding yeast, cytokinesis is delayed until the spindle is properly positioned, but in a mutant for Bim1 (EB1 homologue) this delay is abolished, resulting in abnormal cell division [37]. This suggests that Bim1 has an important role in sensing and positioning the spindle. Cytokinesis in plants often begins with tilted spindles, indicating the absence of a spindle alignment checkpoint in plant cells. However, this spindle tilting is corrected later during the progression of cytokinesis, which may indicate the involvement of a positioning sensor and correction mechanism in plant cells. We propose that the EMTs initiating from the spindle poles and approaching the cortex (Figure 5B–O) are involved in this sensor and tilting mechanism. Though different scenarios can be evoked for the correction mechanism, in each case a specialized cortical reference site would be required. We therefore investigated the effect of an auxin efflux inhibitor, NPA, which has been shown to block both vesicular trafficking and internalization of plasma membrane-localized proteins [36] without directly affecting microtubules or actin filaments [38].
Polarity inhibitor induces abnormal PPBs and shifts cell division planes
Prolonged NPA treatment in BY-2 cells caused inclined and periclinal cell divisions (Figure 6C) instead of normal anticlinal cell divisions (Figure 6A), as in tobacco VBI-0 cells [39]. Cells with aberrant division planes also exhibited major alterations in interphase cortical microtubule alignments in the daughter cells (Compare Figure 6A, B with Figure 6C, D). NPA treatment caused formation of abnormal PPBs (Figure 6E) that resulted in inclined spindles (Figure 6E) and phragmoplasts (Figure 6F), resulting in shifted division planes. In some NPA-treated cells, two separate PPBs were observed and the inclined cell plate was attached to the parental cell walls, with either end docking at one of the places marked by these two PPBs (Figure 6G–L; see also 11: Movie 11). Closer observation of this two-PPB situation revealed a preferential attachment of the cell plate at the PPB sites connecting with the largest number of EMTs (Figure 6H). These results, together with observations from other laboratories, implicate a link between the intracellular establishment of polarity, endocytosis, the placement of initial PPBs and the final cell division planes.
Figure 6. NPA induces abnormal PPBs and altered cell divisions. (A-B) Normal anticlinal cell divisions (arrowheads in A) and transverse organization of cortical microtubules in NPA untreated cells. (C-D) Inclined and periclinal cell divisions (red arrowheads in C) with altered organization of GFP-MAP4 labeled cortical microtubules (red arrowheads in D) in NPA treated cells. A, C show single median sections and B, D show 3-D projections. Note that the first round of cell division (yellow arrowheads) is normal and a shift in the cell division planes occurs in the second round. (E-F) Formation of periclinal PPBs and spindles (arrowheads in E) and periclinal phragmoplasts (arrowhead in F). Bidirectional arrows in A, C, E and F show the long axes of the cells. (G-L) NPA treatment sometimes causes formation of two separate PPBs (arrowheads in G) equidistant from the nucleus, which results in tilted spindle formation (I) and phragmoplast initiation (J), phragmoplast growth (K) and cell plate docking (L) at sites marked by either of the PPBs (arrowheads) (see 11: Movie 11). G shows 3-D projection and H-L show single median sections. Bars represent 10 μm and time is indicated in minutes.
Additional File 11. NPA treatment sometimes causes formation of two separate PPBs equidistant from the nucleus, resulting in tilted spindle formation and phragmoplast initiation, growth and cell plate docking at sites marked by either of the PPBs. The movie is accelerated 550 times. The real time is 120 min.
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In mammalian and yeast cells, a microtubule plus end-mediated 'search and capture' mechanism has been credited with positioning and aligning the spindles [17,19] and determining the plane of cell division [40]. Our observations suggest that EMTs in plant cells may behave similarly to establish and regulate the cell division plane. Interestingly, it has been shown previously that injuries caused by inserting microneedles at these specific cortical sites, probed by EMTs during cytokinesis, affect cell plate alignment [41].
In mammalian and yeast cells, EB1 binds to adenomatous polyposis coli (APC). In epithelial cells, APC is mainly found at specialized cortical sites [42], providing a planar cue [43]. In mammalian cells, microtubule-mediated APC delivery to specialized cortical sites has been demonstrated [44,45]. A mechanism for attaining polarity cues is proposed for mammalian cells, according to which the EB1-labeled microtubule plus ends target to the specialized APC-marked cortical sites [46]. A similar role in attracting EB1-labeled microtubular plus ends towards the cortex has been attributed to Kar9p in budding yeast [17] and to Moe1 in fission yeast [47]. Moreover, it has been suggested that LIS1 is a regulated adapter between CLIP170 and cytoplasmic dynein at sites involved in cargo-microtubule loading and/or the control of microtubule dynamics [48].
Interestingly, plants seem to possess homologues for APC, Kar9p and Moe1, and the plant cytoskeletal-related Tonneau2 [49] protein contains a LisH domain present in LIS1 (our unpublished results based on the NCBI search engine). In maize, the Tangled1 (which is distantly related to the APC) mutant displays altered PPBs, spindles and phragmoplasts and shifted cell division planes. Furthermore, Tangled1 expression and its microtubule localization correlate with the cell division stage [50]. In mammalian cells, EB1 is required for microtubule tip-specific localization of APC but not vice versa. In the absence of EB1, APC localizes all along the microtubule lengths [50,51]. In addition, APC assembly in the cortical clusters is EB1-independent but depends on the existence of the armadillo domain [52]. In plants, it remains to be determined how EB1 localizes in the Tangled1 mutant and vice versa. From parallels in the mammalian cell literature one might hypothesize that Tangled1-mediated EB1 targeting to specialized cortical sites regulates the cell division planes in plants. However, there are several problems with this hypothesis. For instance, Tangled1 contains only the microtubule binding domain and lacks both the EB1 binding and the armadillo domains of APC [52]. Inversely, AtEB1 possesses an APC interaction domain [23] and a unique C-terminal acidic tail [22], and armadillo domain-containing proteins exist in Arabidopsis [53]. This leaves open the possibility that EB1 interacts with other PM-localized basic protein(s). In addition, plants lacking Tonneau2 fail to assemble PPB [4] and Tonneau2 possesses a LisH domain. From the parallel mammalian cell literature one might hypothesize that the LisH domain in Tonneau2 may mediate EMT plus end cortical interactions. Hence it will be interesting to determine how EB1 localizes in Tangled1 and Tonneau2 mutants and vice versa.
In mammalian cells, many proteins have been shown to associate in a microtubule plus end complex, which has been described as a plus end raft [54]. As with lipid rafts, protein concentration at the distal ends may allow a cascade of interactions in the restricted area of a microtubule plus end. This may, in turn, control the dynamic behavior of this cytoskeletal network and its anchoring to other structures [54]. An alternative to this would be that EMTs, by interpreting cell geometry and polarity cues, deposit protein(s) at the PPB, which subsequently attracts the phragmoplast microtubules. Conversely, the connection between EMT plus ends and endocytosis may indicate a role for localized endocytosis in modifying the PM architecture, which may transmit the memory for re-attracting EMT plus ends during cytokinesis. We consider that the polarity inhibitor NPA affects PPB formation by modulating endocytosis, as it does not affect microtubules but interferes with endocytosis and with polarity-based EMT plus end targeting to specialized areas of the PM. Thus, feedback loops, comprising polarity establishment-endocytosis-microtubule plus end guidance-further endocytosis, appear to be essential for defining and creating planes of cell division in plant cells.
In conclusion, our results suggest that in mitotic plant cells, EMT plus ends may act as cell shape/polarity sensing and orienting machines by their sustained cortical targeting, as shown for yeast [15,55]. EMTs in premitotic plant cells are bundled and bidirectional, as reported very recently in fission yeast by analysis of the EB1 homologue mal3p [56], indicating evolutionary conservation of the processes involved in defining cell division planes. Importantly, we show that at preprophase the targeting of EMT plus ends coincides with endocytosis events to establish a plant-specific cortical endocytic belt. During cytokinesis, this same belt again interacts with the EMT plus ends of the expanding phragmoplast to ensure proper cell plate navigation and docking. Our results reveal a link between the position of EMT plus ends, the establishment of intracellular polarity and the localization of endocytosis that is essential for the regulation of cell division planes in plants.
Plant material and growth conditions
Tobacco BY-2 cells were cultured and transformed as reported previously [9].
Construction of reporter genes
The construction of GFP-MAP4, YFP-MAP4, YFP-CLIP170 and GFP-AtEB1 was described previously [9,22]. GFP-Ara7 in vector pBSIIKS+ [33] was excised with HindIII-XbaI and sub-cloned into the binary vector pBINPLUS. STtmd-YFP in vector pMON [57] was digested with PstI -SmaI and cloned into the binary vector pCAMBIA 1390.
Fluorescent dyes and drugs
FM4-64 (Molecular Probes) dissolved in water was applied to the BY-2 cells at 2 μM final concentration for 5 min. The cells were washed with BY-2 medium to remove excess dye and were observed immediately. Alexa 633 (Molecular probes) and Mitotracker (Molecular probes) dissolved in water were also applied at 2 μM final concentration and the cells were observed immediately. Stock solutions of taxol (Sigma-Aldrich), latrunculin B (Sigma-Aldrich) and NPA (Sigma-Aldrich) in DMSO were applied to the cells to give final concentrations of 10 μM, 10 μM and 50 μM respectively. Stock solutions of oryzalin (Greyhound Chromatography and Allied Chemicals, Merseyside, UK) were prepared in ethanol and used at 10 μM final concentration.
Live cell analysis
For live cell analysis, the Zeiss CLSM510 system implemented on an inverted (Axiovert 100) microscope was used. The microscopy system, sample preparation, single wavelength scanning, image processing and movie generation were as previously described [9]. Dual color imaging was performed using dual excitation/emission scanning in multitracking mode. For GFP /YFP dual scanning, we used excitation/emission combinations of 458 nm/ BP 475–525 for GFP and 514 nm/ BP 530–600 for YFP, in combination with the HFT 458/514 primary and NFT515 secondary dichroic splitters. For GFP/ FM4-64 dual scanning, we used excitation/emission combinations of 488 nm/ BP 505–550 for GFP and 543 nm/ LP585 for FM4-64, in combination with the HFT 488/543 primary and NFT545 secondary dichroic splitters. For GFP/ Alexa dual scanning, we used excitation/emission combinations of 488 nm/ BP 505–550 for GFP and 633 nm/ LP650 for Alexa, in combination with the HFT UV/488/543/633 primary and NFT545 secondary dichroic splitters. All filters were from Zeiss. For time-lapse analysis, images were obtained at 1–10 s time intervals. All experiments were repeated 3–5 times. Acquired images were processed using LSM510 Image Browser version 3.0 (Zeiss Corp.). Maximum projections were obtained from 0.5 μm spaced serial optical sections and were exported as TIFF files. For time-series scans, all the images were exported as time-series TIFF files. The exported images were processed with Adobe Photoshop version 5.0 (Adobe Systems Inc.). For individual plant microtubule growth measurements, all time scans were analyzed in the animation mode of LSM510 Image Browser 3.2 (Zeiss Corp.) by marking the single ends of individual microtubules in each image by a zoom function and tracking them for several minutes. The shortest displacement of the plus ends resolvable in this analysis was 0.1 μm. The time values were obtained from the respective frame times in the time-lapse. Thereafter, the data were manually transferred into Excel and processed. The microtubule growth histories (kymographs) were obtained by processing the raw data in LSM510 Image Browser 3.2 (Zeiss Corp.).
Authors' contributions
PD designed the experiments, acquired the data, analyzed and interpreted them and drafted the manuscript. TWJG supervised the research. PD, JM, MH and TWJG participated in manuscript designing, coordination and editing. All authors read and approved the final manuscript.
We acknowledge F. Baluska for stimulating discussions. We are grateful to A. Nakano (RIKEN, Saitama, Japan) for the GFP-Ara7 construct and J. Carette (Wageningen Univ., the Netherlands) for the ST-YFP construct. P.D. and T.W.J.G. were supported by NWO FOM-ALW 805.47.012 and by NWO van der Leeuw 835.25.004. J. M. was supported by a Volkswagon Stiftung grant to M.H.
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Controlling uric acid eases gout
Published: Monday, November 4, 2013 at 5:30 a.m.
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Gout is a condition caused by the deposit of uric crystals in the joints, resulting in severe swelling and pain in various joints of the body. This disease mostly affects men ages 30 to 45, and women ages 55 to 70. It occurs more often in people who are obese and already suffer from hypertension, diabetes or high cholesterol.
Uric acid is a substance that results from the breakdown of purine, a substance found in food that also exists naturally in our bodies. Purine usually dissolves in our blood, passes through the kidneys, and is excreted through urine. However, if the body produces more uric acid than the kidneys can excrete, then uric acid can accumulate in the joints as well as the kidneys. This build-up of uric acid in our joints creates urate crystals and it is these crystals that can trigger intense swelling and pain. This process can occur in almost any joint, including toes, ankles, elbows, wrists, or fingers.
High uric is not only a true risk factor for gout, but also plays a significant role in developing hypertension, cardiovascular or renal disease. Two factors can contribute to high uric acid in our blood: genetic factors, which cause an overproduction or underexcretion of urate; or lifestyle factors.
The best way to help reduce your risk of subsequent gout attacks is to reduce uric acid in the blood to less than 6 milligrams per milliliter. Medications such as Allopurinol or Fubuxostat can decrease uric acid production while another drug, Probenecid, can increase the amount of uric acid passed into your urine. Lifestyle habits are also a crucial factor in reducing uric acid. Since there is a link between high uric acid, gout, and obesity, maintaining both a healthy weight and eating habits are essential. Consumption of foods to that contain large amounts of purine, such as all meats, seafood, yeast, peas, beans, lentils, asparagus and mushrooms, should be reduced while alcohol consumption should be limited. Large servings of meat and heavy beers should be avoided. Proper dieting and exercise not only leads to a fall in serum urate concentration due to an increased excretion of urate through kidneys, but a reduction in the amount of urate produced within the body as well.
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Create Your Own Economy: The Path to Prosperity in a Disordered World
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How will we live well in a super-networked, information-soaked, yet predictably irrational world? The only way to know is to understand how the way we think is changing. As economist Tyler Cowen boldly shows in "Create Your Own Economy," the way we think now is changing more rapidly than it has in a very long time. Not since the Industrial Revolution has a man-made creation--in this case, the World Wide Web--so greatly influenced the way our minds work and our human potential. Cowen argues brilliantly that we are breaking down cultural information into ever-smaller tidbits, ordering and reordering them in our minds (and our computers) to meet our own specific needs. "Create Your Own Economy" explains why the coming world of Web 3.0 is good for us; why social networking sites such as Facebook are so necessary; what's so great about "Tweeting" and texting; how education will get better; and why politics, literature, and philosophy will become richer. This is a revolutionary guide to life in the new world.
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Tyler Cowen is a professor of economics at George Mason University. An "AudioFile" Earphones Award winner and Audie Award finalist, Patrick Lawlor is also an accomplished stage actor.
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""Patrick Lawlor tackles this study in behavioral economics with ease."" ---AudioFile


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Young Voters Could Again Play Key Role in US Election Cindy Saine October 05, 2012 President Barack Obama has said young voters were crucial to his victory over Republican John McCain in 2008. In 2008, they turned out in record numbers. Four years later, enthusiasm on college campuses for the contest between Obama and Republican Mitt Romney seems less visible. Both campaigns are reaching out to voters between the ages of 18 and 29. At the University of Maryland near Washington D.C., young Democrats meet to phone voters in nearby "battleground states" - states that tend to swing between Democratic and Republican candidates. Maryland itself tends to vote Democratic in presidential races. Engaging college voters Surveys show that young people are less engaged this year than in 2008. But at Maryland, College Democrat President Tyler Grote said there is a lot of enthusiasm for Obama. "People are very passionate about what they believe in, especially when there are people with two very different approaches for how they want to run this country," said Grote. A few hundred Maryland students turned out to watch the first presidential debate between Obama and Romney. College Republican Caroline Carlson said the economic situation has dampened the president's appeal. "In 2008 it was all about hope and change. They thought they were voting for a celebrity, whereas now, they realize when they are graduating from college, I think the unemployment rate for people who are 20-24 is about 14 percent, which is pretty high," said Carlson. Courting youth The Romney campaign is trying to win over young voters who turned out for Obama in 2008 by highlighting the economic situation.
Republican Vice Presidential Candidate Paul Ryan appealed to young voters in his convention speech in Tampa. "College graduates should not have to live out their 20s in their childhood bedrooms, staring up at fading Obama posters and wondering when they can move out and get on with their lives," said Ryan. The president has visited a number of college campuses and has spoken about keeping student loans affordable. He asked Ohio students for their votes. "And if you're with me and if you work with me, we will win Wood County again. We'll win Ohio again. We will finish what we started," said Obama. Taking notice of campaigns Young voters traditionally favor Democrats. Heather Smith is president of the non-partisan group "Rock the Vote," which organizes rock concerts and voter registration drives on college campuses. She said young people who turned 18 since the last election are different from those four years ago. "They have to be engaged by the candidates to show up and repeat the record-breaking decision-making impact that their older brothers and sisters had," she said. Smith said young people have not been following the 2012 race as closely as they did in 2008. But they're now starting to pay attention and are lining up to register.
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Replies are closed for this discussion.
@ Blaine L. - Just call me Forrest. :)
I learned long ago to keep my eyes open and move away from trouble, I credit that policy with saving my bacon on more then one occasion.
Let me leave I'll leave, back me into a corner different story.
My position:
former NRA member who left the organization due to it's total intransigence to any kind of gun control; military vet; former prison corrections officer trained w/ multi round clip .223 (used in towers); active hunter.
As a citizen of this country I am comfortable permitting the restriction of certain classifications of military style weaponry. In particular the banning of semi-automatic assault style firearms. These currently account for approximately 1.7% of total US gun sales. That still leaves over 98% of all current firearms available for continued purchase. The previous ban on these weapons did not result in a further erosion of my constitutional rights to keep and operate a plethora of firearms.
If the 'no restrictions whatsoever' gun owners still feel it is unnecessary to make these specific weapons unavailable then I believe they should be willing to compromise in another area. The production or importation of any ammunition used in these weapons should be halted and a hefty fine imposed if you're caught in the possession of said ammo.
I would be willing to grant access to these style weapons in the context of an indoor gun range where the weaponry is maintained under lock and key and strict supervision is observed during use.
Restrictions on specific weapons availability is not a cure all. But in combination with other measures it is a start to trying to reduce these instances of mass slaughter. It is selfish to consider the importance of having legal access to a weapon designed for the military/swat environment more valid than the need to concretely address our country's ongoing problem of mass shootings.
I am a staunch defender of my right to possess and use firearms as a citizen. I also recognize that in a sane society we understand that certain restrictions are necessary to attempt to minimize the number of senseless deaths we endure each year.
while i respect your position and could probably agree with far too many caveats to list....questions
1) would the banning of handguns be a far more effective option to stopping senseless deaths according to your position? looking at the FBI crime data, i ask. this would of course effect concealed carry, and the ability to "defend" yourself outside of your owned private property. still an effective option, possibly.
2) Since neither of us are very young men, and served...the ar 15 has been available to U.S. civilians for nearly 50 years, with no major changes to its design or operation....have we "evolved" past the trusted ownership of them as civilians?
I dont believe 0 gun control is the answer, but i take exception to the insane majority of views on it...they are flawed before they take flight. i do agree it is a far more complex problem then guns..
And now a word from Sam Harris:
"The fantasies of many martial artists aside, to go unarmed against a person with a knife is to put oneself in very real peril, regardless of one's training."
This statement only proves that Sam Harris knows inferior martial artists.
That is one of the best videos for getting the point (please excuse the pun) across about the limitations of firearms in a hand to hand combat situation. I run into too many people who think a handgun solves all problems under all circumstances, it doesn't.
I met a Special Forces veteran once and we got to talking about weapons. One thing he said stuck with me. "If I'm in nose-to-nose combat, I'll take a knife over a gun any day of the week."
A big 10-4, a guy who knows how to handle a knife scares the crap outa me.
I agree. But a guy who doesn't know how to handle a gun scares me just as much.
Hahaha, I would have to agree with that.
You've probably been to one of "those ranges" then.
Yea, we have a public range that has no range masters and vague rules, I don't go to that one. :)
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Phenflu CDX use while Breastfeeding
Important: Phenflu CDX is a drug containing multiple ingredients. Please check each of the links below, where breastfeeding (lactation) information is available. This information is not intended as a substitute for professional judgment. Always consult your physician.
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More about acetaminophen/codeine/guaifenesin/phenylephrine
Consumer resources
- Phenylephrine/guaifenesin/codeine/acetaminophen
- Acetaminophen, codeine, guaifenesin, and phenylephrine
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Biographies of Performers: Main Page | A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | Explanation | Acronyms
Edith Selig (Soprano)
Born: Germany (?)
Édith Selig-Papée is a German-born French classical soprano in concert, opera and Lied, known for singing music of J.S. Bach and French composers. She is also a music pedagogue. In 1958 Édith Selig recorded Gustav Mahler's Symphony No. 2 with the mezzo-soprano Eugenia Zareska, the Choeurs et Orchestre national de la Radiodiffusion française conducted by Carl Schuricht. She recorded Bach Cantatas with Fritz Werner, Heinrich-Schütz-Chor Heilbronn and Pforzheim Chamber Orchestra, including Ich hatte viel Bekümmernis, BWV 21, an early ambitious work, performed in Weimar in 1714 on the 3rd Sunday after Trinity. She also recorded with the same ensemble J.S. Bach's Actus tragicus (BWV 106) and Easter Oratorio (BWV 249). In the 1960's she recorded J.S. Bach's Magnificat (BWV 243) and Missa in F Major, BWV 233, with Karl Ristenpart and Saar Radio Chamber Orchestra, alto Claudia Hellmann, tenor Georg Jelden, bass Jakob Stämpfli and Maurice André (trumpet). Edith Selig has specialized in singing works of French composers. In 1960 she recorded in Paris Gluck's Orfeo ed Euridice, conducted by Charles Bruck, with Rita Gorr and Nadine Sautereau. In 1963 she recorded Rameau's Acte de ballet Pigmalion, conducted by Marcel Couraud. She recorded the Te Deum of Esprit Joseph Antoine Blanchard, conducted by Louis Frémaux. In 1996 she recorded the Te Deum of Marc-Antoine Charpentier with the Paul Kuentz Chamber Orchestra, conducted by Paul Kuentz. Edith Selig has been teaching at the École Normale de Musique de Paris. Among her pupils there were sopranos Qiu Shuwei, Lillian Sit, Satoko Yaba and Chenxing Yuan and pianist Kanae Endo. Selig also taught at the Conservatoire Régional de Rouen (Normandie, France). Among her pupils there were Rocamora Dominique (between 1984 and 1988) and bass-baritone Josselin Michalon. Along with voice teachers such as Régine Crespin and Jakob Stämpfli, Edith Selig-Papée was a founding member of the European Voice Teachers Association (EVTA) in 1988. She has lectured internationally, such as on La Mélodie Francaise in Detmold and Philadelphia. She has served on the jury of international competitions such as the 23" Concours international de chant de Paris in 2001.
Source: Wikipedia Website (December 2012); Rocamora Dominique (May 2013); Bits & pieces from barious websites Contributed by
Recordings of Bach Cantatas & Other Vocal Works
Karl Ristenpart
Fritz Werner
Links to other Sites
Edith Selig (Wikipedia)
Introduction | Cantatas | Other Vocal | Instrumental | Performers | General Topics | Articles | Books | Movies | New Biographies | Texts & Translations | Scores | References | Commentaries | Music | Concerts | Festivals | Tour | Art & Memorabilia Chorale Texts | Chorale Melodies | Lutheran Church Year | Readings | Poets & Composers | Arrangements & Transcriptions Search Website | Search Works/Movements | Terms & Abbreviations | Copyright | How to contribute | Sitemap | Links
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Avon Manor B & B & 3 bedroom Cottage, Avon by the Sea
Lowest price$217 CAD
Nice owner and easy to get along with. Rooms very clean and 1 block from the ocean.…
The innkeepers have thought of everything to make you feel the mood of the shore. Best…
TripAdvisor Traveller Rating: 3.5
From 49 reviews
Enter dates to check prices
Hotel Info: 866-266-9798
from $217 CAD- Cottage, 3 Bedrooms
- Suite, 1 King Bed with Sofabed
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- Standard Room, 1 Queen Bed with Sofabed
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A play on geometric shapes, three brass rectangles are linked up for a modern look. Each brass rectangle in this necklace is connected with a small ball chain and hangs from a delicate brass chain.
- Raw brass
- Brass chain
- Total necklace length is 16"
Shipping: This product ships from the maker within 5 days, via USPS
Returns: Full refund issued for items returned within 30 days of receipt


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OXFORD TOWN – The public is invited to venture out onto the Square and enjoy the Mississippi blues with your family and friends July 16-18 at Oxford's first Blues Festival. Page 8 of this week's Oxford Town has a schedule, photos and other details about the festival that will be held at several great venues around town, concluding on Sunday at Foxfire Ranch in Marshall County. (July 15, 2010, Page 8)
Monday, May 4, 2015
Copyright © 2015 The Oxford Eagle. All rights reserved Visit the old Oxford EAGLE site


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Molecular specificity in photoacoustic microscopy by time-resolved transient absorption |
Optics Letters, Vol. 39, Issue 11, pp. 3102-3105 (2014)
Enhanced HTML Acrobat PDF (339 KB)
We have recently harnessed transient absorption, a resonant two-photon process, for ultrahigh resolution photoacoustic microscopy, achieving nearly an order of magnitude improvement in axial resolution. The axial resolution is optically constrained due to the two-photon process unlike traditional photoacoustic microscopy where the axial resolution is inversely proportional to the frequency bandwidth of the detector. As a resonant process, the arrival time of the two photons need not be instantaneous. Systematically recording the signal as a function of the delay between two pulses will result in the measurement of an exponential decay whose time constant is related to the molecular dynamics. This time constant, analogous to the fluorescence lifetime, but encompassing nonradiative decay as well, can be used to differentiate between molecular systems with overlapping absorption spectra. This is frequently the situation for closely related yet distinct molecules such as redox pairs. In order to enable the measure of the exponential decay, we have reconfigured our transient absorption ultrasonic microscopy (TAUM) system to incorporate two laser sources with precisely controlled pulse trains. The system was tested by measuring Rhodamine 6G, an efficient laser dye where the molecular dynamics are dominated by the fluorescence pathway. As expected, the measured exponential time constant or ground state recovery time,
© 2014 Optical Society of America
OCIS Codes
(110.5120) Imaging systems : Photoacoustic imaging
(170.3650) Medical optics and biotechnology : Lifetime-based sensing
(170.6920) Medical optics and biotechnology : Time-resolved imaging
(180.4315) Microscopy : Nonlinear microscopy
ToC Category:
Medical Optics and Biotechnology
Original Manuscript: January 22, 2014
Revised Manuscript: April 21, 2014
Manuscript Accepted: April 21, 2014
Published: May 19, 2014
Virtual Issues
Vol. 9, Iss. 8 Virtual Journal for Biomedical Optics
Ryan L. Shelton, Scott P. Mattison, and Brian E. Applegate, "Molecular specificity in photoacoustic microscopy by time-resolved transient absorption," Opt. Lett. 39, 3102-3105 (2014)
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- K. Maslov, H. F. Zhang, S. Hu, and L. V. Wang, Opt. Lett. 33, 929 (2008). [CrossRef]
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- R. L. Shelton, S. P. Mattison, and B. E. Applegate, "Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy," J. Biophoton., doi:10.1002/jbio.201300059 (2013). [CrossRef]
- S. W. Huang, J. F. Eary, C. X. Jia, L. Y. Huang, S. Ashkenazi, and M. O'Donnell, Opt. Lett. 34, 2393 (2009). [CrossRef]
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- K. Dalziel and J. R. P. O'brien, Biochem. J. 78, 236 (1961).
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Birthday Gift Ideas On 12-12-12
GIFT IDEAS On 12-12-12
And what famous people share my birthday in which December 12th? They are Ai Kato, Bill Nighy, Bob Barker, Bruce Kulick, Connie Francis, Daniel Magder, Emerson Fittipaldi and 107 more great men and women.
IF you were born on December 12, it means your birthday is oddy unique and significant for the year 2012. The day would be on Wednesday, 12-12-2012 or Monday for 12-12-2011. Have you been planning what are you up to for this special day? We think you should celebrate your birthday differently than usual celebrations since it's a lifetime moment.
Some people are wondering and are wanting to know what will happen in 12/12/12 and will the world really end on Dec 12, 2012? Let's just forget the forecast of Mayan Calendar and leave it that way.
According to astrological signs and dates system, people who were born on December 12th are known by their signs: SAGITTARIUS – The Archer. It is the 10th sign of the zodiac. Based on typical Sagittarius people, they are mostly freedom lovers, independence, masculine, overly optimistic, creative and very honest type of people. But the Sagittarius men sometimes are emotionally uncontrol with lack of reliability and also unresponsible for their duties.
Are you wondering what to buy your Sagittarius Man for his birthday on December 12? Buying a gift for a man can be a daunting task for some. But, the list bellow might give you a few inspiration of birthday gifts on 12/12/12.
Unique 12/12/12 Birthday Gift Ideas
- For a pet lover: buy him a Zoo Med Naturalistic Terrarium- 12x12x12, which is an awesome and ideal reptile or amphibian housing designed of water tight base for his home or office. Offering lockable door for safety and security.
- For a rock music lover: "VooDoo Shoppe 12X12X12," an album created by New Orleans indie rock group Cowboy Mouth.
- For men who love to play with kitchen utensils: an Acrylic Desiccator Cabinet 12X12X12 made of Bronze. It's a sturdy cabinets which is designed to provide low humidity and particulate-free storage, use in refrigerators or at room temperature.
- For boyfriend who love watching a movie or his pavourite sports team on TV: get him 12 boxes of 12x12x12 size box contains 4 JUMBO size bags of popcorn. Offered delicious flavor combos like caramel, bacon cheddar, sweet cajun cheddar, sweet caramel peanut and more plavors. Then, set 12 days romantic scenes and watching load of programs together during the night.
- For him video games lover: celebrates his birthday with NBA 2K12 – a basketball video game features the great 15 legendary NBA players like Michael Jordan, Larry Bird, Magic Johnson, Kareem Abdul-Jabbar, Dr. J and more. It provides excellent animation make him feel like actually playing in a NBA game. Or, FIFA Soccer 12, If he like soccer games, this is the best one out there on the market which can build his ultimate team from the best football stars in the massive FIFA Ultimate Team online community. Good for one to make new friends online.
- For someone who loves arts: a 12×12 size Ferris Wheel Poster created by Santiago Poveda. It is made with high quality poster paper and is designed for years of enjoyment. Or a wooden human mannequin, 12 inches tall produced by Alvin. It's a useful tool for learning to draw people.
- For the "retro" style boyfriend: get him an Original Penguin Men's Jingle Sneakers. It has cool color will help him stand out from the crowd. The padded insole is extra comfortable for long hours of wear. Original Penguin is known today for its unique modern, fashionable takes on heritage American looks.
- For him who is Sci-Fi fan and computer nut and is always running out of USB slots: its time for you to buy him a Star Wars R2D2 USB Hub – an ideal birthday gift for boyfriend this year. It looks great on his desk too.
- For guys who have everything: the choice of double Swarovski crystal cufflinks, or England flavor black cufflinks
12 Original Present Ideas:
- Put your Sagittarius Man into unique personalized picture and give him a treat from
- Turning 18: a book entitled "LoveScopes: What Astrology Knows about You and the Ones You Love"
- Turn 20: Personalized Birthday Cheers Photo Frame
- For boyfriend who is turning 21: Personalized Make Your Own Stainless Steel Flask
- Turn 25: Your Faces on Adam and Eve Artwork
- Turning 30: Vintage Personalized Birthday T-Shirt for husband who loves great looking
- Turning 40: Golfer's Flask for the one who is into playing golf
- Turning 50: Birthday Party Hat or Birthday Memories Personalized Wood Frame
- Turning 60: Old Geezer Car Decorating Kit or the 60th birthday vintage wine glass
- Turning 70: Reluctant Patient Funny Birthday Card
- Elvis Presley 75th birthday collection – for men who turn 75
- Buy your close man designer clothes as Sagittarius Man loves to be masculine and being the centre of people attention
No matter what kind of gift you buy for your Sagittarius Man, a thoughtful one, should be something memorable which can always be treasured by him.
Famous People With Odd Birthday:
Here are the inspiring 107 famous, rich or wealthy celebrities, actors, actresses, leaders, sport figures or historic people who come from various different places such as United States, United Kingdom, Canada, Australia, Japan, Russia and Brazil – Page 1/3:
1982-12-12 | AI KATO | Japanese actress |
1963-12-12 | AI ORIKASA | Japanese voice actress and singer |
1949-12-12 | BILL NIGHY | English actor |
1950-12-12 | BILLY SMITH | Canadian ice hockey player |
1923-12-12 | BOB BARKER | American television game show host |
1923-12-12 | BOB DOROUGH | American jazz pianist/vocalist |
1932-12-12 | BOB PETTIT | American basketball player |
1952-12-12 | Brenton Broadstock – Australian composer and academic | |
1977-12-12 | BRIDGET HALL | American supermodel |
1953-12-12 | BRUCE KULICK | American guitarist (Kiss) |
1969-12-12 | CARRIE WESTCOTT | American model and actress |
1952-12-12 | CATHY RIGBY | American gymnast and actress |
1985-12-12 | CHRIS JENNINGS | American football player |
1977-12-12 | COLIN WHITE | Canadian ice hockey player |
1938-12-12 | CONNIE FRANCIS | American singer |
Famous People Who Share My Birthday in December | ||
1975-12-12 | CRAIG MOORE | Australian footballer |
1958-12-12 | DAG INGEBRIGTSEN | Norwegian musician |
1976-12-12 | DAN HAWKINS | English guitarist (The Darkness) |
1984-12-12 | DANIEL AGGER | Danish footballer |
1991-12-12 | DANIEL MAGDER | Canadian actor |
1984-12-12 | DANIEL MERRETT | Australian Rules Football |
1953-12-12 | DAVE MENIKETTI | American guitarist |
1985-12-12 | DAVID VEIKUNE | American football player |
1967-12-12 | DEKE SHARON | American musician |
1943-12-12 | DICKEY BETTS | American musician |
1940-12-12 | DIONNE WARWICK | American singer |
1982-12-12 | DMITRY TURSUNOV | Russian tennis player |
1924-12-12 | ED KOCH | American politician |
1955-12-12 | EDDY SCHEPERS | Belgian cyclist |
1946-12-12 | EMERSON FITTIPALDI | Brazilian race car driver |
1940-12-12 | FRANCES WILLARD | American magician |
Page 1 of 3 – Gift Ideas on 12/12/12 and what famous people share my birthday – December 12th:


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So when will Hansen Road get a good repave from the foundation up?
Why don't we just shut it down if nobody wants to claim responsibility? It's clearly a dangerous road.
Let's see what happens to Dairy Road if Hansen Road gets shut down. Hansen Road is obviously not a important road, is it?
Burt Sato


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Home > Cyclone summary > Work updates >Cyclone update 9 |
Cyclone updateJanuary 24th, 2012 |
General report on cyclone Thane in Auroville |
Tar road between Edayanchavadi and Kuilappalayam. (Photo by Manohar) |
A Finance and Accounts team has been busy disbursing money from Auroville's reserves to cover the immediate and urgent needs of the community, and is keeping careful account of these transactions and the generous donations that have been sent in by individuals, institutions and businesses around the world. (Please see attached financial report.)
Auroville Universal Township firstname.lastname@example.com |


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Media: Australia Gives $14 Million to Support Language Resources
Submitter: |
Submitter Email: | firstname.lastname@example.com |
Linguistic Field(s): |
Language Documentation |
Media Body: |
The "Creative Australia" act adds nearly $14 million to support language resources and documentation: "Provide new funding of $13.983 million over four years to develop new community-driven language resources and activities, an extension of the Indigenous Languages Support program…". Columnist Greg Dickson wonders: while it may sound like a lot of money, does it adequately recognize the dire situation of the many endangered Indigenous languages in Australia? Read more: http://blogs.crikey.com.au/fullysic/2013/03/13/creative-australia-lends-extra-support-to-indigenous-languages-but-is-it-enough/ |
Issue Number: | 24.1264 |
Date Posted: | March 13, 2013 |


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Ah yes, finally found it. Should have told me she passed through here that long ago. Let's see.Velinde. Booked passage to Booty Bay on the Black Osprey. I don't have anything here saying otherwise, so I'd assume it arrived in port safely.
Not much more help I can be to you, but she asked about overland travel over on that side of the world, and I mentioned Ruzzgot, a caravan driver based out of Booty Bay. Might be that this Velinde traveled with him.
Move along, now. I haven't all day for you.
Upon completion of this quest you will gain:- 610 experience
- 25 reputation with Booty Bay


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Forum migration mostly complete. Please see this post for more info and post any issues.
This is the problem. Trying to activate the Vector Control Center I get:user/bin/basmCC not found.Sorry, should this discussion be entered elsewhere?
Using terminal I got to 'vasm' and activated CUPS. Using Control Center, I got into Printer setup and found my HP Laserjet IIP. When I went to test print a page, I could not get my user name authenticated (also tried to print as root). No luck.
The community involved in Vector is great, but clearly my needs would try the members.
The free download version is available here http://vectorlinux.osuosl.org/veclinux-5.9/iso-release/VL5.9.1-SOHO.iso
QuoteThe free download version is available here http://vectorlinux.osuosl.org/veclinux-5.9/iso-release/VL5.9.1-SOHO.isoVery popular download. Takes some 7 hours so I gave up for today. Need to shut down computer in a few hours. Late at night here.
QuoteIs this version a live CD version then? Can I use it in a Frugal install. Will it save to the NTFS hdd?
Is this version a live CD version then? Can I use it in a Frugal install. Will it save to the NTFS hdd?


text:
Title: | Trinity Cathedral, Port of Spain. |
Abstract: | Image depicts the Trinity Cathedral in Port of Spain Trinidad, in the Republic of Trinidad and Tobago. This is a view of the cathedral from the western corner of Hart Street, just inside the intersection with Abercromby Street. Almost all of the western end of the building is obscured by trees. |
Description: | Colour: Sepia; Style: Landscape; Other: Unbordered, Divided. |
URI: | http://hdl.handle.net/2139/7252 |
Date: | 2010-05-18 |
Files | Size | Format | View |
GoldF6_107C.pdf | 124.6Kb |


text:
- Event URN:
- 249553
- Start date:
- Sun, 7th Apr 2013
- Event Type:
- Cycle Sportive (Non-Competitive)
- Venue:
- Hildenborough Village Hall, 10 Riding Lane Hildenborough, Tonbridge, Kent, TN11 9HY ( View map )
- Online Entry FAQs
- Event website:
- http://www.riderhq.com/events/thefoolserrandii-morefoolyou
- Event organiser:
- Mr Alex Brooks-Johnson
email@example.com - Cheques payable to:
- enter online at event website
- Download Entry Form
- Info for Riders:
- The Fool's Errand Sportive starts and finishes at Hildenborough Village Hall and is mostly on quiet country lanes and roads through the beautiful countryside and picturesque villages of Kent. With the first shoots of spring there couldn't be a better time to see the glorious sweeping valley views, green hills and distant horizons. The route itself is a challenging 120k with 1500 metres of climbing. Heading west on a fast undulating stretch across the Kent Weald you will cycle past Bough Beech Reservoir and up the notorious Toys Hill. Sweeping down through the Westerham Valley the route soon starts to head east towards Otford and the tough climb up Rowdow. After a gentle descent into the Darenth Valley you will take some short, sharp climbs up onto the top of the North Downs and then heads south-east into the rolling hills along the Medway Valley, finishing at the start point in Hildenborough. One feed/toilet stop in Wrotham at Widehorizons Margaret McMillan House.
- Preview Rides:
Date Start Ride name Grade BCG Duration 7 Apr 13 08:00 The Fool's Errand II - More Fool You --- --- 120km / 75m
View All
Event Location:
Ride: The Fool's Errand II - More Fool You
Date | Start | Distance | BCG | Est. Field Size |
7 Apr 13 | 08:00 | 120km / 75m | --- | --- |