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The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the ‘Golgi-depleted’ karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.

The Golgi complex is a central organelle in the secretory pathway of all eukaryotes. It receives newly synthesized material from the endoplasmic reticulum (ER), which is processed and sorted at the trans-Golgi network for its final destination in the cell. Live-cell studies monitoring the dynamics of GFP-tagged Golgi proteins have demonstrated that this organelle is highly dynamic, raising the fundamental question of how it acquires and maintains its steady state architecture and function (Lippincott-Schwartz et al., 2000). Indeed, the molecular mechanisms of Golgi biogenesis and maintenance are presently only poorly understood (Lowe and Barr, 2007). In mammalian cells, Golgi growth occurs during interphase and relies on ER-to-Golgi transport (Guo et al., 2006), but whether Golgi biogenesis occurs from a pre-existing template or de novo remains to be resolved. Treatment of mammalian cells with the drug brefeldin A (BFA) disassembles the Golgi complex and most Golgi components redistribute to the ER. Interestingly, after drug washout, a fully intact Golgi complex can reassemble (Lippincott-Schwartz et al., 1989; Puri and Linstedt, 2003; Kasap et al., 2004), which is consistent with a de novo Golgi biogenesis scenario. However, to what extent this drug treatment represents a bona fide Golgi biogenesis model is not clear, because it has been shown that following BFA treatment, Golgi remnants remain throughout the cytoplasm (Orci et al., 1993; Seeman et al., 2000). It is therefore possible that these remnants act as a template for Golgi biogenesis after drug washout. More recent experiments, in which the Golgi complex was chemically inactivated in living cells, have shown that a new Golgi complex growing adjacent to the inactivated one lacks essential Golgi proteins and functionalities, including normal trafficking activity (Jollivet et al., 2007). This led to the conclusion that, although de novo biogenesis of Golgi-like structures can occur in mammalian cells, pre-existing Golgi templates are necessary for their organization into a morphologically and functionally intact organelle. Common to all of these approaches is the inability to remove the Golgi complex or its remnants from intact cells, and therefore, the fundamental question of whether a functional Golgi complex can be synthesized directly from ER membranes cannot be addressed. Alternative approaches to study Golgi biogenesis using cell microsurgery to generate Golgi- and nucleus-free cytoplasts were unable to synthesize a functional, transport-competent Golgi complex from the ER (Pelletier et al., 2000). Microsurgery has also been used to deplete the centrosome and Golgi from living cells (Maniotis and Schliwa, 1991) and subsequently study their biogenesis. The authors suggested that the Golgi complex, which was at least partially depleted from cells together with the centrosome, could be resynthesized to a normal extent within 20 hours of the surgery. However, as the focus of this study was the biogenesis of centrosomes, the extent of initial Golgi depletion by cell surgery was not addressed in detail. Therefore, despite the extensive amount of elegant work to characterize Golgi biogenesis, a clear description of how this occurs is currently lacking, largely because of technical limitations of the methods used.

Here, we describe a new method to study Golgi biogenesis by first displacing the Golgi complex from its juxtanuclear position, followed by its depletion from living mammalian cells using laser nanosurgery. Subsequently, we have followed the karyoplasts containing an intact nucleus using time-lapse microscopy, and investigated whether a functional Golgi complex can re-form under these conditions. In summary, our data not only demonstrate the feasibility of this approach, but also provide strong support for the hypothesis that the Golgi complex is capable of forming de novo in mammalian cells.

Fig. 1.

Immunofluorescence and ultrastructural analyses of karyoplasts after nanosurgery. (A) Brightfield and GalT–GFP fluorescence images of a BSC-1 cell before, during and after laser nanosurgery. The dashed line indicates the site of laser nanosurgery. Asterisks indicate the cell nucleus in the resulting karyoplast and the arrowheads point to the Golgiplasts. (B) GalT–GFP karyoplasts (asterisks), fixed 4 hours after Golgi removal, immunostained for GM130 or giantin. Control cells next to karyoplasts are indicated with arrowheads. (C) Electron microscopy analysis of a karyoplast and corresponding Golgiplast fixed 30 minutes after laser nanosurgery; n, nucleus; GC, Golgi complex; ERES, ER-exit site; c, centrioles. (D) Double immunostainings of a karyoplast (asterisk), Golgiplast (arrowhead) and neighboring control cell for the Golgi marker giantin and the centrosomal marker γ-tubulin 30 minutes after laser nanosurgery. The dashed squares mark areas that are shown enlarged threefold on the right. Arrows indicate centrioles.

Depleting the Golgi complex from living cells by laser nanosurgery

A BSC1 cell line, stably expressing GFP2-tagged galactosyltransferase (GalT–GFP) as a live-cell marker to visualize the Golgi complex, was used. Microdissection of cells was performed using a diffraction-limited laser nanosurgery device, which allows precise intracellular cutting of structures such as microtubules and actin stress fibers (Colombelli et al., 2004; Colombelli et al., 2005). Severing actin stress fibers leads to their retraction, and as a consequence, to the retraction of the plasma membrane and finally, dissection of the cell into two pieces (supplementary material Movie 1). To remove the Golgi complex in an efficient manner, we exploited our recent observations that BSC1 cells grown on 6-μm-wide fibronectin lines show a displacement of the Golgi complex and associated centrosome from the nucleus by distances of up to several micrometers (Pouthas et al., 2008) (Fig. 1A, supplementary material Figs S1 and S2). In experiments using cells growing on non-coated cover-glass surfaces, where the Golgi complex is often close to, or even overlapping, the cell nucleus, substantial depletion of the Golgi complex by laser nanosurgery was difficult and mostly incomplete, as judged by fluorescence-microscopy-based visual inspection using GalT–GFP as a Golgi marker (data not shown). By contrast, the procedure was significantly facilitated when cells were grown on fibronectin lines, resulting in a marked displacement of the Golgi complex from the cell nucleus. In this case, the entire GalT–GFP signal could be eliminated from the karyoplast by laser nanosurgery, as judged at the light microscopy level (Fig. 1A).

Electron microscopy analyses (Colombelli et al., 2008) of serial sections from karyoplasts fixed either immediately (n=3) or 4 hours after (n=4) laser nanosurgery confirmed the light microscopy observations. We could not detect any structures either morphologically similar to a stacked Golgi, Golgi mini-stacks or centrioles (Fig. 1C). Other membrane-bound organelles and structures of the early secretory pathway, including ER-exit sites, could however, be clearly identified in these karyoplasts (Fig. 1C). By contrast, Golgi stacks and associated centrosomes were only detected in the Golgiplasts (Fig. 1C). Consistent with these observations, presence of the Golgi markers GM130 (Golgin subfamily A member 2), giantin, TGN46 and the centrosomal marker γ-tubulin could not be detected above background staining in the karyoplasts up to 4 hours after laser nanosurgery (Fig. 1B,D; Table 1). From a total of 40 cells analyzed by immunofluorescence and by light microscopy at these early time-points after laser nanosurgery, 38 showed no Golgi-specific or centrosome-specific labeling, and only two karyoplasts showed a weak Golgi-like juxtanuclear presence of the Golgi markers (Table 1). By contrast, the Golgiplasts located next to the karyoplasts contained Golgi and centrosomal marker labeling that was similar to labeling in untreated neighboring control cells (Fig. 1D).

Table 1.

Immunofluorescence analysis of Golgi and organelle markers in karyoplasts at different time points after Golgi removal

Fig. 2.

Immunofluorescence analysis of endogenous Golgi and centrosomal markers in karyoplasts at later time-points. (A) Time-lapse analysis of GalT–GFP in a karyoplast. (B) A karyoplast (asterisk) with a control cell (arrowhead), which both express GalT–GFP. The karyoplast was followed until the first juxtanuclear punctate structures were visible, then fixed and double immunostained for the vesicular coat complexes COPI and COPII. The region within the dashed square is shown as an enlargement (threefold) adjacent to the main image. Arrows highlight three membrane structures positive for GalT–GFP, COPII and COPI. (C) GalT–GFP karyoplasts (asterisks) fixed 12 hours after Golgi removal, immunostained for GM130 or TGN46. Neighboring control cells are indicated with arrowheads. (D) Double immunostainings of a karyoplast (asterisks) and neighboring control cell for the Golgi marker giantin (left) and the centrosomal marker γ-tubulin (right) 24 hours after laser nanosurgery. The dashed squares mark areas that are shown enlarged threefold. Arrows indicate centrioles.

Fig. 3.

Golgi morphology and anterograde transport analyses. (A) Representative electron microscopy sections of karyoplasts showing Golgi ministacks (12 hours) and normal-sized Golgi stacks (16 and 24 hours). Control cells were fixed and processed similarly to the karyoplast. (B) Transport of GPI–CFP to the plasma membrane, visualized by anti-CFP staining in fixed cells 6 or 16 hours after nanosurgery. Arrows indicate the plasma membrane. The arrowhead points to an internal membrane structure in the karyoplast labeled with anti-CFP antibodies. Only half of the Golgi was removed from the ‘cut control’ cell. Pixel intensities for all images were adjusted according to the exposure times used during image acquisition to allow their direct comparison. (C) A karyoplast 6 hours after nanosurgery, showing anti-CFP-specific plasma membrane (arrow) and internal GPI–CFP labeling (arrowhead). The cell was permeabilized and stained for GM130. The cell nucleus (asterisk) and colocalizing GPI–CFP and GM130 structures (double arrows) are highlighted. Scale bars: 10 μm (B,C).

Monitoring Golgi biogenesis by correlative light-electron microscopy

Time-lapse analyses of karyoplasts showed that approximately 1 hour after laser nanosurgery, the GalT–GFP-specific fluorescence started to increase steadily in the karyoplasts (Fig. 2A and supplementary material Movie 2) indicating a recovery of essential cell functions such as gene transcription and protein translation. At 6 hours and later, discrete GalT–GFP-specific structures appeared throughout the karyoplasts (Fig. 2A and supplementary material Movie 2). In karyoplasts 48±14% of these discrete GalT–GFP structures (n=267) colocalized with specific markers of the early secretory pathway vesicular coat, COPII and COPI (Fig. 2B). This was similar to the colocalization measured in unperturbed control cells (50±14% colocalization; n=112). Therefore, the discrete GalT–GFP-positive structures are probably transport carriers of the early biosynthetic transport route, as described in previous studies (Presley et al., 1997; Scales et al., 1997). At later time points (~8 hours or later after nanosurgery), these structures clustered in a juxtanuclear area where they colocalized with the endogenous Golgi markers GM130, giantin and TGN46 (Fig. 2C and Table 1). Interestingly, at all time points tested (up to 24 hours after laser nanosurgery) centrosomal γ-tubulin labeling was never observed in the karyoplasts (Fig. 2D). Electron microscopy analyses of serial sections from karyoplasts fixed 12 hours or later after nanosurgery revealed in 7 out of 11 karyoplasts analyzed, the presence of either several Golgi mini-stacks or normal-sized Golgi stacks, ranging from 500 nm to 2.2 μm in length (Fig. 3A), with the latter ones appearing highly similar to the single stacks observed in unperturbed control cells. Furthermore, the new Golgi stacks were always found adjacent to the nucleus. Consistent with the immunofluorescence analyses (Fig. 2D), we were unable to detect the presence of a centrosome or centrosome-like structures in any of these karyoplast serial sections.

Correlation of biosynthetic and retrograde transport activity with Golgi biogenesis

To investigate whether the newly synthesized Golgi complex was functionally intact, we tested the efficiency of biosynthetic transport in the karyoplasts. To this end, we expressed a GPI-anchored CFP (GPI–CFP) (Keller et al., 2001) in cells and followed its transport from the ER to the plasma membrane in karyoplasts at different time points after laser nanosurgery (Fig. 3B). In ‘Golgi-depleted’ karyoplasts, the anterograde transport of GPI-anchored CFP to the plasma membrane was not detectable within the first 6 hours after laser nanosurgery (Fig. 3B, n=5), although the karyoplasts expressed the marker (data not shown). In cells cut intentionally in such a manner that parts of the Golgi complex remained in the karyoplast (‘cut control’), transport of GPI–CFP to the plasma membrane occurred in a similar manner to that in unperturbed neighboring control cells (Fig. 3B, n=4). This suggests that in our assay, even parts of a functional Golgi complex are sufficient to allow GPI–CFP plasma membrane transport. In support of this, in one of the karyoplasts analyzed 6 hours after laser nanosurgery, and in which discrete juxtanuclear GM130-positive Golgi-like structures were observed, transport of GPI–CFP appeared to be restored to similar levels to those observed in control cells (Fig. 3C). This result is consistent with published data showing that a single mini-stack or Golgi fragment is sufficient to support transport from the ER to the plasma membrane (Pelletier et al., 2000). In karyoplasts analyzed 16 hours after laser nanosurgery, levels of GPI-anchored CFP at the plasma membrane were indistinguishable from those found in unperturbed control cells (Fig. 3B, n=4). Similar results demonstrating the transport competence of the newly synthesized Golgi complex were obtained in experiments following the Golgi-dependent transport of cholera toxin (CTX) (Fig. 4). Previous work has shown that the retrograde transport of this toxin from the plasma membrane to the ER requires an intact Golgi complex (Orlandi et al., 1993; Feng et al., 2004). In 70±5% of unperturbed control cells, fluorescently labeled CTX could be seen in internal membranes, including Golgi-like structures, the ER and the nuclear envelope (Fig. 4A). By contrast, we were unable to detect arrival of the toxin in the ER in any of the karyoplasts where the Golgi complex had been depleted by laser nanosurgery (n=6), 6 hours after laser nanosurgery (Fig. 4B). In these karyoplasts CTX-specific plasma membrane staining was predominantly observed, and very few internal membrane structures were labeled. In parallel experiments where cells were cut such that parts or the Golgi complex remained in the karyoplast, transport of the toxin to the ER occurred in a manner indistinguishable from that observed in unperturbed control cells (Fig. 4C, n=9). This result suggests that the laser nanosurgery procedure itself, or indeed removal of part of the Golgi complex, does not block trafficking of CTX through the Golgi complex to the ER. When karyoplasts were labeled with CTX 16 hours after laser nanosurgery, a time-point where Golgi mini-stacks were detectable in the karyoplasts (Fig. 3A), transport of CTX to the ER was restored to levels that were comparable with those seen in control cells (Fig. 4D, n=5).

Fig. 4.

Analysis of CTX retrograde transport after Golgi removal. Cells stably expressing GalT–GFP were labeled with Cy3-conjugated CTX 2 hours after nanosurgery and examined 4 hours after toxin binding and internalization from the plasma membrane. (A) Unperturbed control cells. Arrowheads in enlarged areas indicate CTX and GalT–GFP in the nuclear envelope. (B) Karyoplasts 6 hours after nanosurgery. The arrowhead and arrow in enlarged areas indicate the presence of GalT–GFP in the nuclear envelope and CTX at the plasma membrane, respectively. (C) A control cell cut such that a proportion of the Golgi complex remained in the karyoplast. Arrowheads in the areas enlarged twofold point to CTX and GalT–GFP in the nuclear envelope. (D) Karyoplasts labeled with CTX 24 hours after nanosurgery. Arrowheads in enlarged areas point to CTX and GalT–GFP in the nuclear envelope. Areas within dashed squares were enlarged threefold and are shown to the right. Scale bars: 10 μm.

Altogether, our data are consistent with the hypothesis that the Golgi complex can be substantially depleted from cells using laser nanosurgery and that this organelle is subsequently resynthesized within a period of 12 hours.

The appearance of endogenous Golgi matrix proteins and enzymes during Golgi biogenesis

To commence investigation of the mechanism by which the observed Golgi reassembly could occur, we quantified the appearance of endogenous Golgi matrix proteins (GM130, giantin, GRASP65) and enzymes (Mannosidase II, GalNacT1 and GalT; Fig. 5 and supplementary material Table S1) at different time points after nanosurgery. Consistently, all the matrix proteins tested began to show a juxtanuclear distribution coincident with a similar distribution of the ectopically expressed GFP-tagged Golgi marker (Fig. 5). Quantification of the Golgi-like signals for the matrix proteins showed that they appeared with similar efficiencies after nanosurgery (Fig. 5 and supplementary material Table S1). By contrast, endogenous Golgi enzymes only acquired a Golgi-like appearance at later time points (Fig. 5 and supplementary material Table S1). For example, at 12 hours after nanosurgery, when the newly formed Golgi complex was visible as a stacked structure (Fig. 3A) and was transport competent (Fig. 3B and Fig. 4), we could detect little Golgi-like labeling of endogenous Golgi enzymes in the majority of the cells analyzed (Fig. 5, supplementary material Table S1). These results indicate that a stacked Golgi complex can assemble and regain functionality despite lacking its full complement of resident enzymes.

Golgi biogenesis in GM130-downregulated cells

Based on these results, one possibility is that matrix proteins have a role in the early steps of Golgi assembly. To further test this hypothesis we used an RNA interference approach to specifically assess the role of GM130 in reassembly of the Golgi following nanosurgery. Although the small interfering RNA (siRNA) sequences used for the experiments was designed to target the human gene encoding GM130, a strong knockdown efficiency of up to 90% was also achieved in the monkey BSC1 cell line used in our experiments (see supplementary material Fig. S3A). At the immunofluorescence level, a significant reduction of GM130-specific staining, but not the Golgi marker giantin, was observed in most of the cells 72 hours after siRNA transfection (supplementary material Fig. S3B,D). Immunofluorescence and electron microscopy analyses showed a significant fragmentation of the Golgi complex in cells transfected with the siRNA targeting GM130 (supplementary material Fig. S3): a phenotype that is consistent with earlier work in which this protein was depleted from cells by RNA interference (Puthenveedu et al., 2006; Marra et al., 2007). No effect on GM130 levels or Golgi complex morphology was observed in cells transfected with non-silencing control siRNAs (supplementary material Fig. S3). Also consistent with earlier work (Puthenveedu et al., 2006; Marra et al., 2007), knockdown of GM130 did not interfere with the transport kinetics of a temperature-senitive variant of the vesicular stomatitis G-protein (VSV-G ts045) to the plasma membrane (supplementary material Fig. S4). The arrival of VSV-G at the Golgi complex following its export from the ER was also not significantly inhibited under the experimental conditions used here (supplementary material Fig. S4).

When we analyzed Golgi biogenesis in GM130-knockdown cells 12 hours after nanosurgery, giantin labeling was largely absent and GalT–GFP distribution was confined to the ER and discrete structures throughout the cytoplasm (Fig. 6A and not shown). By contrast, in cells transfected with control siRNAs, a substantial amount of giantin staining and GalT–GFP fluorescence could be observed in juxtanuclear Golgi-like structures (Fig. 6). At later time points, giantin staining could be detected in discrete structures in GM130-knockdown cells and 24 hours after laser nanosurgery, its cellular distribution was similar to that in neighboring control cells not subjected to nanosurgery (Fig. 6). Ultrastructural analyses by electron microscopy confirmed these observations. In GM130-knockdown cells 12 hours after nanosurgery, we could not identify any stacked Golgi-like structures, but rather only carrier-like membranes that were occasionally associated with microtubules or vesicular clusters in the juxtanuclear area (Fig. 7A). A stacked Golgi structure, although largely fragmented, was only observed in GM130-knockdown cells 24 hours after nanosurgery (Fig. 7B).

Fig. 5.

Monitoring Golgi enzymes and matrix proteins during Golgi biogenesis. Karyoplasts generated from BSC1 GalT–GFP or from YT2 cells were fixed in paraformaldehyde 12 hours after nanosurgery and then immunostained for two different Golgi markers. Confocal z-stacks of images covering the entire cell were acquired and the maximum intensity projection of these is shown. The lower panels show enlargements (fourfold) of the boxed Golgi area in the upper panel. The matrix proteins GM130, GRASP65 (A) and Giantin (B) were visualized. The enzymes mannosidase II [MannII (C)] and galactosyltransferase [GalT (D)] remain absent 12 hours after nanosurgery, although the same cells show a Golgi-like labeling for the matrix proteins GM130 and giantin, and the ectopically expressed GFP-tagged Golgi markers GalT–GFP and GalNAcT2–YFP.

Fig. 6.

Golgi biogenesis in GM130-knockdown cells. (A) Cells transfected with either control siRNAs, or siRNAs targeting GM130 were dissected with laser nanosurgery, fixed at the indicated time points and double-immunostained for GM130 and giantin. The arrow indicates the karyoplast and insets represent 2.5-fold enlargements of the respective juxtanuclear area. (B) Quantification of the juxtanuclear giantin signal in GM130-knockdown or control karyoplasts at 12 and 24 hours after nanosurgery. Cells were treated as described in A and z-stacks of stained cells were acquired by confocal microscopy. Using maximum projection images of the z-stacks, the ratio between the giantin-specific Golgi fluorescence in karyoplasts and the mean giantin-specific Golgi intensity in neighboring control cells was calculated. The graph shows data from between five and seven independent experiments for each condition, with the horizontal lines representing the median values of the quantifications.

Here, we describe and apply a new approach to deplete the Golgi complex from living cells and subsequently follow its biogenesis using correlative time-lapse and electron microscopy. Our results show that ‘Golgi-depleted’ karyoplasts are capable of synthesizing a stacked and transport-competent Golgi complex within 12 hours, which is consistent with models for de novo Golgi biogenesis. Surprisingly, Golgi biogenesis apparently occurs in the absence of a centrosome, although it requires the Golgi matrix protein GM130. This result is also consistent with our observation that during Golgi biogenesis, matrix proteins display a juxtanuclear Golgi-like appearance when Golgi enzymes are still largely absent from this organelle.

Fig. 7.

Ultrastructural characterization of GM130-knockdown karyoplasts. (A) GM130-knockdown karyoplasts were fixed 12 hours after nanosurgery and processed for electron microscopy. No stacked Golgi-like structures could be detected. Two examples of putative Golgi intermediates are shown. In the left image, a putative peripheral carrier (arrow) in close proximity to a microtubule (indicated by arrowheads) is highlighted. In the right image, vesicles (asterisks), carriers and larger structures can be seen clustered in the juxtanuclear area (N). (B) GM130-knockdown and control karyoplasts fixed 24 hours after nanosurgery. Golgi ministacks or more organized Golgi complex (GC) structures were found in both GM130-knockdown and control cells.

The phenomenon of Golgi biogenesis has been extensively studied by a number of laboratories using various approaches, in turn resulting in a variety of results and interpretations (for a review, see Lowe and Barr, 2007). Our results appear to be in contrast to earlier work in mammalian cells (Pelletier et al., 2000; Jollivet et al., 2007), which suggested that de novo biogenesis of a stacked and transport-competent Golgi complex is not possible, and that Golgi biogenesis more likely involves templated growth. One reason why Jollivet and colleagues (Jollivet et al., 2007) might not have observed de novo biogenesis of a stacked and transport-competent Golgi complex could be that their time of observation after chemical inactivation of the existing Golgi complex (max. 4 hours) was shorter than the time actually required for Golgi biogenesis in karyoplasts as we observed here (12 hours). Pelletier and colleagues (Pelletier et al., 2000) clearly showed that cytoplasts generated by microsurgery fail to synthesize a Golgi or Golgi-like structures from the ER and are not capable of supporting biosynthetic transport to the plasma membrane. One limitation of this earlier study, compared with the approach that we have used, is that the cytoplasts did not contain a nucleus, and therefore transcription to ultimately drive new protein synthesis could most likely not occur. These cellular events might be essential for de novo Golgi biogenesis. However, our data are consistent with earlier studies that have used BFA in combination with the kinase inhibitor H89, which inhibits COPII recruitment and cargo exit from the ER (Puri and Linstedt, 2003), to induce Golgi complex disassembly. Because Golgi components are only redistributed rather than actually depleted in these approaches, a Golgi complex could therefore rapidly reassemble within 30 minutes after washout of the drugs. The time required for the biogenesis of a morphologically and functionally intact Golgi complex, as we have observed here, strongly argues that a number of factors first need to be synthesized by the cells before Golgi assembly can occur. Our data are supported by analyses using light microscopy and electron microscopy that show a substantial, if not complete, depletion of components of the Golgi complex immediately after nanosurgery. Our observation that RNA-interference-mediated knockdown of GM130 can delay Golgi reformation in karyoplasts for several hours further strengthens the evidence that matrix proteins such as GM130 had been substantially depleted in the karyoplast by the nanosurgery. Although a definitive demonstration of the complete absence of any remaining ‘Golgi seed(s)’ after laser nanosurgery is formally impossible, our results strongly suggest the existence of de novo Golgi biogenesis in mammalian cells.

A close association and functional interplay between the Golgi complex, the centrosome and associated microtubules has been established in recent years (for a review, see Sütterlin and Colanzi, 2010). Surprisingly, our data here suggest that the biogenesis of a stacked and transport-competent Golgi complex can occur in the absence of a centrosome. This raises the immediate question of how juxtanuclear positioning of the newly synthesized Golgi complex, as we have observed here, is achieved. One possible explanation might be through the use of a self-organization process in which microtubules nucleating from the membranes of the nuclear envelope and/or ER-derived Golgi precursors is sufficient to facilitate clustering of the precursors into larger pre-Golgi structures that are then able to mature into a complete Golgi complex. In addition to such a self-organizing mechanism, a number of factors associated with Golgi precursors that would need to be resynthesized after Golgi depletion, and thereby help to form a functionally and structurally normal Golgi complex, should also exist. Likely candidates are proteins proposed to be part of a Golgi matrix, such as GM130 (Shorter and Warren, 2002; Lowe and Barr, 2007). The approach described here should help to further characterize the role of these proteins in relation to Golgi biogenesis. In line with the hypothesis for a crucial role of Golgi matrix proteins in maintaining and organizing Golgi structure and function, the knockdown of GM130 in our experiments delayed Golgi biogenesis in karyoplasts for several hours. Moreover, observations that knockdown of GM130 under our experimental conditions had no apparent effect on secretory transport, excludes the possibility that a lack of GM130 causes a general inhibition of ER export and therefore a subsequent delay in the arrival at the Golgi of material such as lipids and proteins that are necessary to build a new Golgi complex. Therefore, these data are consistent with a structural role of GM130 in Golgi biogenesis at the level of vesicle tethering or lateral cisternal fusion, for example, as suggested previously (Lowe and Barr, 2007; Puthenveedu et al., 2006; Marra et al., 2007).

Altogether, our data strongly support the hypothesis that Golgi biogenesis can occur de novo in mammalian cells, even in the absence of a centrosome. The Golgi matrix protein GM130 appears to have a crucial role in the efficiency with which this biogenesis occurs. With the approach described here, it will now be possible to investigate the role of other factors in Golgi biogenesis more systematically.

Materials and Methods

Reagents were purchased as follows: fibronectin (from bovine plasma, Sigma), Alexa-Fluor-647-conjugated fibrinogen (Molecular Probes), Sylgard 184 (Dow Corning). Suberic acid bis (3-sulfo-N-hydroxysuccinimide ester) sodium salt (BS3, Sigma), 3-aminopropyltriethoxysilane (APTS, Sigma), Poly(L)-lysine hydrobromide (Sigma), mPEG-SPA-2000 or mPEG-SPA-5000 (Shearwater), silicon-free grease (Glisseal N, Borer Chemie), Enhanced GFP-Golgi mammalian expression vector (Clontech), Monosialoganglioside GM1 from bovine brain (Sigma), Cholera toxin (CTX, from Vibrio cholerae, Sigma), Neomycin (G418, Calbiochem) and GFP2 expression vector (Perkin Elmer). Anti-GM130 (Silencer pre-designed siRNA ID 145093) and Scramble (Silencer Negative Control siRNA 1) siRNA were purchased from Ambion. All the cell culture reagents were purchased from Gibco, unless otherwise noted.

Cell lines, cell culture and transfection

BSC1 (ATCC CCL-26) and BSC1-GalT–GFP2 cells were cultured in modified Eagle's medium (MEM, Life Technologies) supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively) at 37°C in 5% CO2. Cells were seeded on patterned surfaces or on live-cell dishes (MatTek) at a density of 15,000 cells/ml. Experiments were performed 24 hours or 48 hours after seeding of the cells. One hour before nanosurgery, the medium was exchanged to CO2-independent medium.

Stable cell lines were generated by transfecting BSC1 cells using Fugene6 (Invitrogen) according to the manufacturer's protocol with a modified version of the EGFP–Golgi vector containing a neomycin-resistance cassette (Clontech). It encodes a fusion protein consisting of modified green fluorescent protein (GFP2) and a sequence encoding the N-terminal 81 amino acids of human β1,4-galactosyltransferase (Gal-T). This region contains the membrane-anchoring signal peptide that targets the fusion protein to the trans-medial region of the Golgi complex. Addition of neomycin or G418 (a neomycin analog) selected for cells that had incorporated the vector. Resistant clones were screened for GFP2–Golgi expression levels, growth rates and Golgi morphology, and subject to a second round of selection by cell sorting.

YT2 cells [BSC1 stably transfected with the GalNAcT2-YFP (Axelsson and Warren, 2004)] were a gift from Graham Warren (Max Perutz Laboratories, Vienna, Austria) and were cultured in D-MEM (Dulbecco's modified Eagle medium, GIBCO) supplemented with 10% FCS and Geneticin (800 μg/ml) at 37°C in 5% CO2.

For transient transfection of siRNAs, Oligofectamine (Invitrogen) was used in accordance to the manufacturer's instructions. For the assessment of the effects of GM130 knockdown (supplementary material Fig. S3B–E) cells were analyzed 72 hours after transfection, whereas laser nanosurgery was performed 12–24 hours after transfection, because the removal of the Golgi-complex-associated GM130 in the presence of the siRNA (and thus of new protein translation) allowed us to have a very efficient knockdown efficiency immediately after transfection, without waiting for degradation of the pre-existing protein.

Antibodies and immunofluorescence

Antibodies were used as follows: monoclonal antibodies GM130 (BD Biosciences), Golgin97 (Molecular Probes), GalNAcT1 and GalT (CellMAb), α-tubulin (clone GTU-88, Sigma); polyclonal antibodies Giantin (Abcam), Mannosidase II (AbD Serotec) and Sec31 (BD Biosciences). Rabbit antibodies against recombinant GFP and beta-prime COPI were raised by standard protocols. Sheep antibodies against TGN46 (Biozol) and GRASP65 (a gift from Martin Lowe, University of Manchester, UK) were used. All fluorescently conjugated secondary antibodies were purchased from either Molecular Probes or Amersham.

Cells were fixed with either −20°C methanol for 4 minutes or 4% paraformaldehyde (PFA) for 15 minutes. Cells were subsequently treated with 0.1 M glycine in 1× PBS for 5 minutes and then in 0.1% Triton X-100 in 1× PBS for a further 5 minutes. Fixed and permeabilized cells were incubated with primary antibodies in 1× PBS for 30 minutes. Samples were washed three times for 5 minutes in 1× PBS, and incubated for 30 minutes in fluorescently conjugated secondary antibodies, before being washed three times for 5 minutes in 1× PBS. Where required, cells were permeabilized with 0.1% saponin. Coverslips were finally mounted on glass slides with Mowiol (Calbiochem).

Patterning techniques

The patterning was performed as previously described (Pouthas et al., 2008). Briefly, the layout design of the stamp was done using CleWin Software (WieWeb) and translated into 5 inch chromium photolithography mask by Delta Mask V.O.F. (Enschede) then used to produce a structured silicon master (3 μm depth) using a positive tone resist process. Poly(dimethylsiloxane) stamps (PDMS stamps) were fabricated by curing Sylgard 184 (Dow Corning) at 60°C for 24 hours. After curing, the elastomer was peeled off from the master. The PDMS stamps were incubated with 100 μl aliquot of 1:2 mixture of Alexa-Fluor-647-conjugated fibrinogen and fibronectin solution, or only fibronectin solution, at a final protein concentration of 50 μg/ml in 1× PBS for 30 minutes. The stamps were then washed in deionized water and blow-dried with a stream of nitrogen (FN-stamp). Silanization of the slides was performed by immersion in 5% 3-aminopropyltriethoxysilane (APTS), acetone solution for 30 seconds followed by several washing steps in 1× PBS. The slides were then incubated in bis(sulfosuccinimidyl)suberate (BS3; approx. 0.5 mg/ml in 1× PBS) for 10 minutes. Blow-dried FN stamps were then placed in contact with silanized glass surfaces for 5 minutes. After removal of the stamp, the slides were incubated in a 1 mg/ml poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) for 30 minutes for back-fill. All these processes were done at room temperature. Finally, the patterned glass slides were attached to drilled 10 mm holes at the bottom of 35 mm plastic Petri dish using silicon-free grease (Glisseal N, Borer Chemie).

Generation of karyoplasts devoid of Golgi by laser nanosurgery

Cell nanosurgery by low intensity and shockwave-free plasma ablation was achieved with a frequency tripled Nd:YAG-pulsed laser (JDSUniphase), at a wavelength of 355 nm and theoretical pulse duration of less than 500 pseconds on a Zeiss Axiovert 200M using a Zeiss heating stage insert. All irradiations were performed along a manually oriented laser line-target, defined interactively on the image with a graphical mouse interface. Pulse density along the line was set such that the optical intensity was approximately constant with a linear density of about 5 pulses per μm at a speed of 100–1000 pulses per second. In this study, actin severing was successful with typical energy of 200 nJ per pulse. Cell dissection was also successful with lower energies down to about 100 nJ and with repeated irradiations in several axial positions to insure fast and efficient separation of karyoplast and Golgiplast. Alternatively, an Olympus Fluoview 1000 system coupled with a second scanning unit (355 nm pulsed laser) was used for the nanosurgery.

After cell dissection, the positions of successfully cut cells were inscribed inside the glass coverslip about 10 μm deep with the same laser at higher power (Colombelli et al., 2008). By doing so, a square box could be placed around the recently cut cell and a number could be written to clearly identify it during the following steps.

Live-cell imaging

Cells and karyoplasts were imaged using either a multi-position time-lapse microscope: CellR, or CellM (Olympus) with the following objectives: 20× (UPlanSApo, NA 0.75), 40× (UPlanSApo, NA 0.9). Alternatively, cells were imaged using a DeltaVision RT system (Applied Precision) with a 20× (NA 0.4) objective. After immunofluorescence, images were captured using a Zeiss Axiovert 200 microscope, Zeiss LSM 710, or Leica SP2 AOBS confocal microscope (Leica Microsystems).

Transport assays

Cholera toxin (CTX) was labeled with Cy3 dye (Amersham) according to the manufacturer's instructions. Karyoplasts were followed by time-lapse microscopy for 2 hours or 12 hours after laser nanosurgery, incubated with GM1 receptor (final solution 1 μM in MEM without FCS) on ice for 20 minutes, then with CTX (1:750) for an additional 20 minutes followed by 5 minutes at 37°C (toxin release). Unbound toxin was removed and CTX uptake was followed for 4 hours by time-lapse microscopy, before fixation with 4% PFA.

GPI–CFP adenovirus was a gift from Kai Simons (Max Planck Institute of Cell Biology and Genetics, Dresden, Germany) and prepared as described previously (Keller et al., 2001), diluted 1:20 in medium (MEM with 10% FCS) and incubated on cells (100 μl/dish, 1 hour, 37°C). Cells were washed once and left for 10 minutes at 37°C. The laser nanosurgery was performed within 1 hour to avoid any transport of the GPI to the plasma membrane before karyoplast generation. The karyoplasts were followed for 6 hours before fixation with 4% PFA. Alternatively, the karyoplasts were followed for 12 hours or longer. In all cases, the GPI–CFP plasma membrane signal was enhanced using a polyclonal antibody against GFP, without membrane permeabilization.

For the VSV-G ts045 transport assay, cells were transfected with GM130 or scramble siRNA and 55 hours thereafter they were infected with an adenovirus vector for the expression of ts045–ECFP (Keller et al., 2001). After incubation at 40°C for 17 hours (which results in the accumulation of the protein in the ER), cells were either fixed immediately in PFA, or the temperature was shifted to 32°C to release the ER-exit block and then cells were fixed at different time points after the temperature shift.

Cells were then immunolabeled for VSV-G without permeabilization to identify only the surface fraction and the nuclei were stained with Hoechst 33342. 16 images were randomly acquired for each time point with an automated Olympus widefield microscope (operated with the software ScanR) and the analysis was done as described above.

Correlative light and electron microscopy

Correlative light and electron microscopy was performed as previously described (Colombelli et al., 2008). Briefly, cell locations were marked with the laser inside the glass coverslip and further fixed for flat embedding with 2.5% glutaraldehyde in Cacodylade buffer. Samples were washed with CaCo buffer, and kept cold on ice during incubation with 2% OsO4 in CaCo buffer for 40 minutes. The sample was rinsed with water, before uranyl acetate (0.5% in water, 30 minutes) was added to increase the contrast. After several washes with water, the samples were moved back to the laser nanosurgery microscope for laser surface etching. Two lines perpendicular to the fibronectin pattern, one on each side of the cell of interest, were etched at the surface of the coverslip with a depth of a few microns. Additional washing with PBS was done to remove glass debris from the top of the cells, which were then dehydrated with ethanol. The coverslips were embedded in Epon and left to polymerize at 60°C (24 hours). After polymerization and removal of the coverslip, the etched marks were clearly visible on the Epon surface, so that the samples could be trimmed only around the cell of interest. Ultra thin (50–60 nm) sections were made [on average 58±21 (s.d.), n=18] serial sections covered the complete cytoplasm of the cell) and transferred to copper-palladium slot grids (2×1 mm, Plano, Wetzlar) freshly coated with Formvar. The sections were post stained with uranyl acetate and lead citrate. The pictures were taken using a BioTwin CM120 electron microscope (FEI Company).

Quantification protocols

Golgi complex displacement

Analysis of a complete Golgi complex displacement was done by measuring the immunofluorescence intensity of different Golgi markers in the Golgi area compared with areas adjacent to the nucleus. Fixed control cells grown on fibronectin lines were selected for a displaced Golgi complex in the GalT–GFP channel and confocal images were acquired of the complete cell volume. From the maximum intensity projections in ImageJ, overlay pictures were made from the different channels. RGB pictures were used to create plot profiles, by placing a 70-μm-long line along the cell, through the centre of the nucleus and the Golgi complex.

Colocalization analysis

Colocalization analysis between GalT–GFP and COPI structures was performed as follows. Images were acquired with a Zeiss Axiovert 200 microscope, 63× (NA 1.4) or a Leica SP2 AOBS confocal microscope, 63× (NA 1.4) and loaded in ImageJ (version 1.39p, NIH). Each channel image (GalT–GFP, COPI and COPII) was separately analyzed and fluorescently positive structures were marked, using the ‘point selection’ function. The three channel pictures were thereafter overlaid, and the colocalization in each category was measured (GalT–GFP, GalT–GFP/COPI, GalT–GFP/COPII and GalT–GFP/COPI/COPII), and used for calculation of the mean ± s.d.

Appearance of endogenous proteins in the new Golgi complex

The sequential order of appearance of the different Golgi proteins in the de novo Golgi complex was analyzed as follows. Immunofluorescence images of GM130 and an additional marker of interest were acquired on a Zeiss LSM 710 confocal microscope as a maximum projection of Z stacks across the whole cells. Images were low-pass filtered to remove image noise and background signals were subtracted. Corrected images were then thresholded such that the entire Golgi-marker-specific signal in non-treated control cells was detected. The total fluorescence in each thresholded Golgi structure was then determined and the ratio between the karyoplast under view and the average of the neighboring control cells was calculated, to assess the recovery of the Golgi complex in the karyoplast. GM130 endogenous levels were used as a reference in all cases, except for the GalT measurement shown in Fig. 5, where Giantin was used. The quantification of these experiments is shown in supplementary material Table S1.

The analysis of the Giantin signal in GM130-knockdown or control cells 12 or 24 hours after nanosurgery was carried out as described above, and GM130 signal was in this case used to check the efficiency of the protein knockdown. The ratio of total Giantin signal in the karyoplast and the average in control cells was used here as a measure of the Golgi reformation process and is shown in Fig. 6.

Automated analysis of VSV-G transport assay

Automated image analysis procedures were implemented as modules in a MATLAB-based version of the open-source software CellProfiler (Lamprecht et al., 2007). The quantification of VSV-G surface transport assay was performed as described previously (Simpson et al., 2007). Briefly, from the identified cell nuclei, the cytoplasmic extension of a cell (cell mask) was approximated by step-wise dilation of the nuclear area until neighboring areas touch or the dilation exceeds a user defined maximal radius, which was chosen to match the typical size of a cell. After subtracting background intensities from each image, the integrated optical density within each cell mask was computed for both VSVG-CFP (Total ts045) and anti-VSVG–Alexa-Fluor-647 (Surface ts045). The ts045 surface transport efficiency of a cell was assessed by dividing Surface ts045 by Total ts045. The histogram in supplementary material Fig. S4D shows the mean transport efficiency calculated on more than 100 cells for each experimental condition.

For automated detection of Golgi structures in fluorescence images (GalNAcT2–YFP), we developed a MATLAB-based image-processing module for the segmentation of locally bright objects of arbitrary shape. In brief, we separated locally bright Golgi foreground signals from background signals by a morphological top-hat filter with a circular structural element, whose radius was chosen to match the typical size of Golgi objects. The resulting foreground image was then subjected to a local adaptive thresholding algorithm, neighboring thresholded pixels were combined, and connected components with a minimal number of 4 pixels were assigned as Golgi objects. CFP–ts045 images were subjected to a morphological top-hat filter with a circular structural element, whose radius was chosen to keep locally bright CFP–ts045 signals such as that due to accumulation within the Golgi, but to subtract ‘background’ intensities such as CFP-ts045 residing in the ER. Next, for each cell we computed the integrated optical density of the background-corrected CFP–ts045 images within Golgi masks and this value was divided by the Total-ts045 value to obtain the ts045 Golgi fraction.

SDS-PAGE and western blotting

BSC1 cells were lysed in sodium dodecyl sulphate (SDS) sample buffer, boiled for 10 minutes and resolved in NuPage 4–12% Bis-Tris polyacrylamide gradient gel (Sigma). Proteins were transferred onto PVDF membranes (BioRad) using a Mini Trans-Blot cell (BioRad) at 100 V for 80 minutes. Membranes were blocked in Tris-buffered saline, 0.1% Tween 20, 2% milk powder, probed using antibodies against GM130 (BD Biosciences) and α-tubulin (NeoMarkers) and specific binding was detected via an Alexa-Fluor-680-conjugated secondary antibody (Molecular Probes) with an Odyssey Infrared Imaging System (LI-Cor Biosystems).

We are grateful to F. Pouthas and P. Girard for providing expertise in micropatterning and T. Wolf for providing wafers. We thank C. Tischer for help in the automated image analysis, the ALMF team at EMBL Heidelberg for their continuous support, and C. Schuberth and E. San Pietro in the Pepperkok team for critical reading of the manuscript. P.R. is a recipient of a von Humboldt exchange fellowship.

  • * These authors contributed equally to this work

  • Present address: Institute for Research in Biomedicine (IRB) Parc Científic de Barcelona, C/ Baldiri Reixac 10, 08028 Barcelona, Spain

  • § Present address: School of Biology and Environmental Science, University College Dublin (UCD), Belfield, Dublin 4, Ireland

  • Supplementary material available online at

  • Accepted November 17, 2010.

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Apple Tree Granny Smith Variety Grafted

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Apple Tree Granny Smith Variety Grafted in a 3 Gallon Container. Granny Smith Apples are crisp, juicy, and tart which makes them perfect for either baking in pies, stewed in sauces or eating out of hand. They're also great in salads because once cut, they keep their color longer than other apples. Low chill requirement good for Southern climates.

Origin of Name
Botaniocal Information
Apple Breeding
Maturation And Harvest
Food Value
Medicinal Use

Apple tree (Malus domestica) Scientific classification Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Rosales Family: Rosaceae Subfamily: Maloideae Genus: Malus Species: M. domestica Binomial name Malus domestica Borkh. The apple is the pomaceous fruit of the apple tree, species Malus domestica in the rose family Rosaceae. It is one of the most widely cultivated tree fruits. The tree is small and deciduous, reaching 5-12 m tall, with a broad, often densely twiggy crown. The leaves are alternately arranged simple ovals 5-12 cm long and 3-6 cm broad on a 2-5 cm petiole with an acute tip, serrated margin and a slightly downy underside. Flowers are produced in spring simultaneous with the budding of the leaves. The flowers are white, five petaled, 2.5-3.5 cm in diameter, white with a pink tinge that gradually fades. The fruit matures in autumn, and is typically 5-9 cm diameter (rarely up to 15 cm). The centre of the fruit contains five carpels arranged in a five-point star, each carpel containing one to three seeds.

Origin of Name

The word "apple" comes from the Old English word æppel, which in turn has recognisable cognates in a number of the northern branches of the Indo-European language family. The prevailing theory is that "apple" may be one of the most ancient Indo-European words (*abl-) to come down to English in a recognisable form. The scientific name malus, on the other hand, comes from the Latin word for apple, and ultimately from the archaic Greek malon (melon in later dialects). The legendary placename Avalon is thought to come from a Celtic evolution of the same root as the English "apple"; the name of the town of Avellino, near Naples in Italy is likewise thought to come from the same root via the Italic languages. Linnaeus assigned the apple to the genus Pyrus, along with pears and quinces. Philip Miller subsequently separated the apple into its own genus, a division repeatedly ratified over many years.

Wild Malus sieversii apple in Kazakhstan Apple cut horizontally, showing seedsThe wild ancestor of Malus domestica is Malus sieversii. It has no common name in English, but is known in Kazakhstan, where it is native, as 'alma'; in fact, the region where it is thought to originate is called Alma-Ata, or 'father of the apples'. This tree is still found wild in the mountains of Central Asia in southern Kazakhstan, Kyrgyzstan, Tajikistan, and Xinjiang, China. For many years, there was a debate about whether M. domestica evolved from chance hybridisation among various wild species. Recent DNA analysis by Barrie Juniper, Emeritus Fellow in the Department of Plant Sciences at Oxford University and others, has indicated, however, that the hybridisation theory is probably false. Instead, it appears that a single species still growing in the Ili Valley on the northern slopes of the Tien Shan mountains at the border of northwest China and the former Soviet Republic of Kazakhstan is the progenitor of the apples we eat today. Leaves taken from trees in this area were analyzed for DNA composition, which showed them all to belong to the species M. sieversii, with some genetic sequences common to M. domestica.  Some individual M. sieversii, recently planted by the US government at a research facility, resist many diseases and pests that affect domestic apples, and are the subject of continuing research to develop new disease-resistant apples.  Other species that were previously thought to have made contributions to the genome of the domestic apples are Malus baccata and Malus sylvestris, but there is no hard evidence for this in older apple cultivars. These and other Malus species have been used in some recent breeding programmes to develop apples suitable for growing in climates unsuitable for M. domestica, mainly for increased cold tolerance. The apple tree was perhaps the earliest tree to be cultivated, and apples have remained an important food in all cooler climates. To a greater degree than other tree fruit, except possibly citrus, apples store for months while still retaining much of their nutritive value. Winter apples, picked in late autumn and stored just above freezing, have been an important food in Asia and Europe for millennia, as well as in Argentina and in the United States since the arrival of Europeans.

Apple Breeding

Like most perennial fruits, apples are ordinarily propagated asexually by grafting. Seedling apples are different from their parents, sometimes radically. Most new apple cultivars originate as seedlings, which either arise by chance or are bred by deliberately crossing cultivars with promising characteristics. The words 'seedling', 'pippin', and 'kernel' in the name of an apple cultivar suggest that it originated as a seedling. Apples can also form bud sports (mutations on a single branch). Some bud sports turn out to be improved strains of the parent cultivar. Some differ sufficiently from the parent tree to be considered new cultivars.. Some breeders have crossed ordinary apples with crabapples or unusually hardy apples in order to produce hardier cultivars. For example, the Excelsior Experiment Station of the University of Minnesota has, since the 1930s, introduced a steady progression of important hardy apples that are widely grown, both commercially and by backyard orchardists, throughout Minnesota and Wisconsin. Its most important introductions have included 'Haralson' (which is the most widely cultivated apple in Minnesota), 'Wealthy', 'Honeygold', and 'Honeycrisp'.

Apples are self-incompatible and must be cross-pollinated to develop fruit. Pollination management is an important component of apple culture. Before planting, it is important to arrange for pollenizers, cultivars of apple or crab apple that provide plentiful, viable and compatible pollen. Orchard blocks may alternate rows of compatible cultivars, or may have periodic crab apple trees, or grafted-on limbs of crab apple. Some cultivars produce very little pollen, or the pollen is sterile, so these are not good pollenizers. Quality nurseries have pollenizer compatibility lists. Growers with old orchard blocks of single cultivars sometimes provide bouquets of crab apple blossoms in drums or pails in the orchard for pollenizers. Home growers with a single tree, and no other cultivars in the neighbourhood can do the same on a smaller scale. During the flowering each season, apple growers usually provide pollinators to carry the pollen. Honeybee hives are most commonly used, and arrangements may be made with a commercial beekeeper who supplies hives for a fee. Orchard mason bees are also used as supplemental pollinators in commercial orchards. Home growers may find these more acceptable in suburban locations because they do not sting. Some wild bees such as carpenter bees and other solitary bees may help. Bumble bee queens are sometimes present in orchards, but not usually in enough quantity to be significant pollinators. Symptoms of inadequate pollination are excessive fruit drop (when marble sized), small and misshapen apples, slowness to ripen, and low seed count. Well pollinated apples are the best quality, and will have 7 to 10 seeds. Apples having fewer than 3 seeds will usually not mature and will drop from the trees in the early summer. Inadequate pollination can result from either a lack of pollinators or pollenizers, or from poor pollinating weather at flowering time. It generally requires multiple bee visits to deliver sufficient grains of pollen to accomplish complete pollination. Apple tree in flower a common problem is a late frost that destroys the delicate outer structures of the flower. It is best to plant apples on a slope for air drainage, but not on a south facing slope (in the northern hemisphere) as this will encourage early flowering and increase susceptibility to frost. If the frost is not too severe, the tree can be wetted with water spray before the morning sun hits the flowers, and it may save them. Frost damage can be evaluated 24 hours after the frost. If the pistil has turned black, the flower is ruined and will not produce fruit. Growing apples near a body of water can give an advantage by slowing spring warm up, which retards flowering until frost is less likely. In some areas of the USA, such as the eastern shore of Lake Michigan, the southern shore of Lake Ontario, and around some smaller lakes, this cooling effect of water, combined with good, well-drained soils, has made apple growing concentrations possible. However, the cool, humid spring weather in such locations can also increase problems with fungal diseases, notably apple scab; many of the most important apple-growing regions (e.g. northern China, central Turkey, and eastern Washington in the USA) have climates more like the species' native region well away from the sea or any lakes, with cold winters leading to a short, but warm spring with low risk of frost. Home growers may not have a body of water to help, but can utilise north slopes or other geographical features to retard spring flowering. Apples (or any fruit) planted on a south facing slope in the northern hemisphere (or north facing in the southern hemisphere), will flower early and be particularly vulnerable to spring frost.

Maturation And Harvest

Cultivars vary in their yield and the ultimate size of the tree, even when grown on the same rootstock. Some cultivars, if left unpruned, will grow very large, which allows them to bear a great deal more fruit, but makes harvest very difficult. Mature trees typically bear 40-200 kg of apples each year, though productivity can be close to zero in poor years. Apples are harvested using three-point ladders that are designed to fit amongst the branches. Dwarf trees will bear about 10-80 kg of fruit per year.

Food Value

Apples, with skin (edible parts) Nutritional value per 100 g (3.5 oz)
Energy 50 kcal 220 kJ
Carbohydrates 13.81 g
Sugars 10.39 g
Dietary fiber 2.4 g
Fat 0.17 g
Protein 0.26 g
Thiamin (Vit. B1) 0.017 mg 1%
Riboflavin (Vit. B2) 0.026 mg 2%
Niacin (Vit. B3) 0.091 mg 1%
Pantothenic acid (B5) 0.061 mg 1%
Vitamin B6 0.041 mg 3%
Folate (Vit. B9) 3 µg 1%
Vitamin C 4.6 mg 8%
Calcium 6 mg 1%
Iron 0.12 mg 1%
Magnesium 5 mg 1%
Phosphorus 11 mg 2%
Potassium 107 mg 2%
Zinc 0.04 mg 0%
Percentages are relative to US recommendations for adults.
 Source: USDA Nutrient database

Medicinal Use

An old proverb attests to the health benefits of the fruit: "An apple a day keeps the doctor away." Research suggests that apples may reduce the risk of colon cancer, prostate cancer and lung cancer.  Like many fruits, apples contain Vitamin C as well as a host of other antioxidant compounds, which may reduce the risk of cancer by preventing DNA damage. The fibre content, while less than in most other fruits, helps regulate bowel movements and may thus reduce the risk of colon cancer. They may also help with heart disease, weight loss and controlling cholesterol, as they do not have any cholesterol, have fibre (which reduces cholesterol by preventing reabsorption), and are bulky for their caloric content like most fruits and vegetables. A group of chemicals in apples could protect the brain from the type of damage that triggers such neurodegenerative diseases as Alzheimer's and Parkinsonism. Chang Y. 'Cy' Lee of the Cornell University found that the apple phenolics, which are naturally occurring antioxidants found in fresh apples, can protect nerve cells from neurotoxicity induced by oxidative stress. The researchers used red delicious apples from New York State to provide the extracts to study the effects of phytochemicals. Lee said that all apples are high in the critical phytonutrients and that the amount of phenolic compounds in the apple flesh and in the skin vary from year to year, season to season and from growing region to growing region (November/December 2004 issue of the Journal of Food Science). The predominant phenolic phytochemicals in apples are quercetin, epicatechin, and procyanidin B2 (PMID 14558772). The seeds are mildly poisonous, containing a small amount of amygdalin, a cyanogenic glycoside, but a large amount would need to be chewed to have any toxic effect.  Pesticide contamination is linked to an increasing number of diseases, and they are mostly found on the outside of fruits and vegetables. Washing or peeling before eating may reduce pesticide intake but peeling will also reduce the intake of the beneficial nutrients. Apple consumption can help remove trapped food and clean between the teeth, but the malic acid contained within the fruit is also capable of eroding tooth enamel over time, and through excess consumption.

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Apple Tree Granny Smith Variety Grafted

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Mark was always athletic growing up but never had a filter when it came to food. So when college arrived, the workouts stopped, but the overeating did not. Tipping the scales at almost 450 lbs, he decided to do something about it. Enter: The Biggest Loser. Mark tore through season 10 losing the most weight of any contestant by far that year (213 lbs.) and captured the 100K prize! Since then he has dedicated his life to health and fitness by spreading the word to as many people as possible through public speaking, brand marketing, and social media. He has also started his own line of athletic apparel (Bare Fit Wear) that carries clothing to fit anyone, no matter what point of their fitness journey they are on.

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Critically Evaluate Freuds Theory Psychology Essay

Sigmund Freud (1856-1939) he was Jewish and educated in Vienna, where he trained in medicine. Freud was the founder of psychoanalysis. He was the first psychologist to recognise the importance of the conscious and unconscious mind. His theory was psychoanalysis. The value and validity of his theory has been greatly questioned, since its inception in the early 1900s.

His critics and devotees, see the answer lying in opposite extremes. Having him all the magician or all the messiah, was he either? Was Sigmund Freud a great medical scientist who uncovered important truths about human psychology? Or was he something different? His theories offer a science, but his critics of psychoanalysis question whether or not it is indeed a science; the value of Freud's data, the methods that he used and the effectiveness of the treatments. Throughout this essay I intend to show the positive and negative criticisms of Freud's theory psychoanalysis. This work from

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Freud spent many years hypothesizing about the role of dreams and their interpretation. He argued that dreams allowed a person to discharge otherwise unacceptable and unconscious wishes and urges. He defines the state of sleep to be a period of uproar and chaos during which the unconscious thoughts of the (Id) attempt to force their way into consciousness. More specifically, a dream was the disguised fulfilment of a repressed desire. It had to be disguised because the repressed desires could be sexual or aggressive urges unacceptable to the dreamer when awake.

However, the major problem with Freud's theory of dream function is that interpretation of a dream function is not something that can be objectively achieved.

Webb and Cartwright 1978 see dreams as a way of dealing with problems relating to work, sex and relationships that occur during working hours. Cartwright 1984 argued that whatever is symbolised in a dream are the dreams 'true meaning' and unlike Freud, he saw no reason to distinguish between dreams content and meaning. This dissertation from

Jung and Freud had worked together at first interpret ting dreams, and then had a major falling out over their different ideas of dreaming. Jung did not agree with the distinction between content and meaning. To him dreams had no disguised meaning, but directly reflected the minds current state. Their content included thoughts, memories and emotions from the day's conscious events and images reflecting our unconscious world. A major difference between Jung and Freud was Freud emphasised the dark and destructive nature of the unconscious influences on dream imagery, Jung emphasised the positive and constructive nature of these influences.

On a positive note one similarity between Freud and Jung on dreaming was they both used imagery and symbolism for dream interpretation. However, Jung's framework was totally different to Freud's.

Moving on to one of the more controversial aspects of Freud's theory, and one that probably caused the greatest problem for his critics, was his ideas of sexual instinct and its importance in the development of the individual. Freud's account on development is closely related to other aspects of his theory, in particular the structure of personality and the stages of psychosexual development. Freud believed that the personality comprises of three parts the id, the ego and the superego. The id contains everything inherited at birth. The ego is the part of the id which has been modified by the direct influence of the external world. The superego represents morals and values. Freud's psychosexual development stages; oral stage 0-1 yrs, pleasure associated with the mouth. Anal stage 1-3 yrs pleasure focuses o n the bowel/bladder, phallic stage 3-5yrs p pleasure zone genitals. Latency period 5-6 yrs associated with puberty and finally genital stage, associated with puberty and maturity. This dissertation from

According to Freud how well we deal with each of these stages, and whether or not we receive the optimal amount of gratification, determine psychologically healthy we are as an adult. The major conflict in the Freudian view in the development is the oedipal conflict, this in brief is where Oedipus unwittingly kills his father, and marries his mother. In the female version the Electra complex girls between the ages of 4-6yrs envy their father for possessing a penis that they have been denied.

This particular part of Freud's theory has many supporters and critics. One of the most important implications of adopting this view of human behaviour is its reliance on the concept of an unconscious mind, which influences our thinking and behaviour in ways we are not aware. If we are unaware of the forces that guide us, then it follows that we are not capable of intervening to change or go against them. (Slife&Williams 1995).

Jung and Adler disagreed with Freud about the idea of such a pervasive sexual instinct, and proposed their own ideas of what motivated human behaviour. Grunbaun(1986) believes that the reasoning on which Freud based his entire psychoanalytic theory was "fundamentally flawed, even if the validity of his clinical evidence were not in question" but that "the clinical data are themselves suspect; more often than not, they may be patients responses to the suggestions and expectations of the analyst".cobg bgr sebgbgw orbg bgk inbg fobg bg.

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Additional critics contend that Freud's clinical data are flawed or invalid. Greenberg (1986) believes that Freud's case studies do not place enough stress on revealing the outcome of the treatment and that Freud's aim was more to illustrate his theoretical points. In addition Freud fully presented only 12 cases, but mentioned over 100 minor cases. Greenberg asserts that many of the presented cases would not even be considered acceptable examples of psychoanalysis, and that all of Freud's cases had basic shortcomings. Finally Greenberg finds it "both skirting and curious that Freud chose to illustrate the usefulness of psychoanalysis through the display of unsuccessful cases"

Other powerful critics, (Colby 1960) claim Freud's evidence flawed due to lack of an experiment, the lack of a control group and lack of observation that went unrecorded. In addition, (Holt 1986) found fault with the demographically restricted sample of individuals on which Freud based the majority of his data and theory. However in favour of Freud (shrevin 1986) insists that "Freud's admirable heuristic hypotheses did not come out of thin air or out of his imagination".coba bar sebabaw orba bak inba foba ba:

Freud used a technique called free association to evaluate his patients. This involved the patients lying on a couch in Freud's consultation room, and he would sit behind their heads, so they could not see him. Patients would be encouraged to relax and to talk about their life and feelings. They were encouraged by Freud to remember events from their childhood and feelings, associated with the event. Freud often became personally involved in the therapeutic conversation and would explain his thinking to the patient. Freud would then interpret what had been said through transference and relay his thoughts on to the patient.

Critics of this technique including (Storr 1986) claim "Free association" is a method employed in psychoanalysis where the patients speak about any subject matter whatsoever and the analyst draws conclusions based on what is said. "Grunbaun forcefully argues that free association is neither free nor validating evidence for psychoanalytic theory." "For my own part, however", Grunbaun concludes "I find it unwarranted to use free association to validate casual inferences". Grunbaun contends that free association is not a valid method of accessing the patients repressed memories, because there is no way of ensuring that the analyst is capable of distinguishing between the patients actual memories and imagined memories, constructed due to the influence of the analyst leading questionscocc ccr seccccw orcc cck incc focc cc.

According to Thomas (1990) transference has become so central to the theory and practice of psychoanalysis that many analysts believe that making interpretations about transference is what distinguishes psychoanalysis from other forms of psychotherapy. Thomas maintains that "in Freud's time, counter- transference feelings were considered to be failing on the part of the analyst. These feelings were to be controlled absolutely now, counter- transference is considered an unavoidable outcome of the analytic process, irrespective of how well prepared the analyst is by analytic training and its years of required personal analysisââ‚¬Â¦most modern analysts are trained to observe their own counter- transference feelings and to use these to increase their understanding of the patients transference and defences".

In order to evaluate the strength of Freud's theory of psychoanalysis one must consider a few of the qualities that make a theory of personality or behaviour "great". Among the many qualities that people consider important are that the theory addresses its problem, and can be applied in a practical ways, fits with other theories and withstands the test of time. In addition according to many philosophers of science, is falsifiable, able to be generalised, leads to new theories and ideas, and is recognised by others in the field. Clearly psychoanalysis meets many of these criteria. Freud coined the term "psychoanalysis" in 1856, even today as we are approaching the twenty first -century, psychoanalysis remains as a valid option for patients suffering from mental illness. The theory psychoanalysis was innovative and revolutionary, and clearly has withstood the test of time.

Finally to evaluate the critics Grunbaun (1986) asserts that "while psychoanalysis may thus be said to be scientifically alive, it is currently hardly well". The criticisms of Freud's theory can be grouped into three general categories. First, critics contend that Freud's theory was lacking in empirical evidence and relies too heavily on therapeutic achievements, whereas others assert that even Freud's clinical data are flawed, inaccurate and selective at best. Second, the actual method or technique involved in psychoanalysis, such as Freud's ideas on the interpretation of dreams and the role of free association, have been criticised. Finally, some critics assert that psychoanalysis is simply not a science and many of the principles upon which it is based are inaccurate. Eysenck and Wilson (1973) there is not one study which one could point to with confidence and say "Here is definitive support of this or that Freudian notion."

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In conclusion many of the critics and devotees of Sigmund Freud all have valuable opinions, to which I have shown throughout this essay. However, despite the weaknesses of psychoanalysis I believe that the many strengths of the theory are extremely significant and have withstood the test of time and are widely used today.

Therefore in my own opinion psychoanalysis should not be disregarded. Although many criticisms exist, there are as many arguments, if not more, in support of the rich and diverse contributions that Freud's theory gave to psychoanalysis. His legacy lives on indisputable form.

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Julian Lennon Releases Video For New Single 'Lookin 4 Luv'

The first solo recording from Julian Lennon since 1998, his brand new studio album entitled ‘Everything Changes’ is released on October 3rd and features twelve original tracks, the album is preceded by lead single ‘Lookin’ 4 Luv’, which is currently play-listed at BBC Radio 2.

‘Everything Changes’ came about over a period of time, as Julian gathered together the basic formations for 30+ songs by picking up a guitar, or sitting at the piano every now and again. After inviting some friends over to build upon these ideas, the result was ‘Everything Changes’.

'Lookin' 4 Luv' was released on Monday 12th September.


Five Considerations for Borrowers Completing a Disposition

Posted in Contractual Matters
Ana Badour

In my prior post, Seven Considerations for Borrowers Completing an Acquisition, I described seven key issues that borrowers should consider when completing an acquisition.   Similarly, if your company is considering a disposition (either of assets or of shares) and is a borrower under a credit facility, it is important to consider whether the proposed disposition will result in a breach of any of the provisions of your credit documentation.  Here are five key questions to ask when undertaking a disposition:

  1. Is the disposition permitted under the credit agreement? Many credit agreements contain negative covenants that prohibit dispositions without consent of the lenders or impose restrictions on dispositions. For example, some credit agreements will impose limits on the dollar amount of permitted dispositions (either in the aggregate over the term of the credit facility, in the aggregate over a fiscal year or individually) or a requirement that the disposition meet certain requirements, such as being on reasonable commercial terms with a third party. If the disposition is not permitted, you will need to seek consent of the lenders.
  2. Is there a requirement to use the proceeds of the disposition in a specific manner?  Some credit agreements require either that the borrower re-invest the proceeds of a disposition in the business within a certain time, or that all, or a portion of, the proceeds of the disposition be applied as a repayment of the credit facilities.
  3. Are there several steps to the disposition? If so, each step should be reviewed to determine whether it is permitted under the credit agreement or whether consents or notices are required under the terms of the agreement in order to implement any step or series of steps.
  4. Will the disposition affect the calculation of financial covenants?  The completion of a disposition may affect the calculation of various financial covenants under the credit agreement.
  5. Is there a need for a release of security?  If the credit facilities are secured, the purchaser may require the delivery of a specific release of security from the lenders whereby the lenders agree to release their security interest against the assets or shares being sold.  There may be timing concerns since lenders generally do not wish to provide a release of security prior to the disposition having been successfully completed. These timing issues are typically addressed through escrow arrangements.

As the structure of the disposition transaction evolves, you should regularly review and consider your existing credit documentation to ensure that such changes do not result in non-compliance with the credit documentation.


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Yesterday, the European Commission took a tentative step towards solving this problem by changing its previous policy of increasing biofuel use for cars and trucks by proposing to cap it at 5%.

So why the change of heart?

In a recent report, Oxfam concluded that the demand for biofuels has pushed millions of people to poverty through rising food prices. In 2008, when biofuels accounted for 3.5% of transport fuel in the EU, a study for the Commission estimated that 70,000 km² of land was needed to grow biofuel crops and that close to half of that area would be located outside of Europe. If this amount of land had been used to produce wheat and maize instead, it could have fed 127 million people for an entire year.

Increasing the price of food as well as reducing its availability should be enough reasons to suspend biofuel mandates. However, expanding biofuel production also has severe negative climate impacts as it often displaces crop production farther into threatened forests, savannahs and peatland or increases the use of oil-based fertilisers and pesticides. The destruction of ecosystems for biofuel crop production leads to massive greenhouse gas emissions as intact ecosystems are natural carbon sinks.

Nevertheless, the Commission proposal does not go far enough. It does not require governments or fuel suppliers to account for indirect emissions from biofuels. EU countries can still continue to use biofuels that have higher emissions than crude oil.

In the coming months, the European Parliament and the Council will negotiate towards a final adoption of EU biofuels policy. They should amend the Commission proposal to properly account for the full climate change impact of biofuels.

Used wisely, biofuels can be part of the solutions needed for a fossil fuel-free future. But if current biofuels policy increases food prices, while making climate change worse through additional deforestation and unsustainable farming practices, then the system isn’t working and has to change.

Sini Harkki is a campaigner with Greenpeace Nordic


Gretta Vosper Sermons

I’ve just discovered that West Hill United Church, where Gretta Vosper is the minister, has a videoblog (or ‘vlog’) with clips of her sermons. I’ve just discovered the site, and so may have more to say about specific sermons at some point. Anyone exploring what “progressive Christianity” means will presumably find this a useful resource.

Fear of Refugees: A Timely Reminder
Christians in Name Only
Making the Canon Game
God in the Hands of Angry Sinners


Milo and Olive

American | Santa Monica

"Close your eyes and pick a dish – it'll be delicious" at this Santa Monica "favorite" (sib of Huckleberry and Rustic Canyon) offering a range of American "staples" all day, including "exceptional" wood-fired pizzas, "must-have" salads and pastas, plus "artful" espresso drinks and pastries "made with expertise and love"; decor is minimal-"chic" but be warned: despite an expanded dining room, they "still have long waits" and seating is first come, first served.

2723 Wilshire Blvd | Map
Santa Monica, California 90403

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7:00am - 11:00pm
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Milo and Olive

2723 Wilshire Blvd
Santa Monica, California 90403

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Gigantic explosions buried in dust: ALMA probes environment around dark gamma-ray bursts

June 11, 2014 in Astronomy & Space / Astronomy
This is an artist's conception of the environment around GRB 020819B based on ALMA observations. Credit: NAOJ

Observations from the Atacama Large Millimeter/submillimeter Array (ALMA) have for the first time directly mapped out the molecular gas and dust in the host galaxies of gamma-ray bursts (GRBs)—the biggest explosions in the Universe. In a complete surprise, less gas was observed than expected, and correspondingly much more dust, making some GRBs appear as "dark GRBs". This work will appear in the journal Nature on 12 June 2014 and is the first ALMA science result on GRBs to appear. It shows ALMA's potential to help us to better understand these objects.

Gamma-ray bursts (GRBs) are intense bursts of extremely high energy observed in distant galaxies—the brightest explosive phenomenon in the Universe. Bursts that last more than a couple of seconds are known as long-duration gamma-ray bursts (LGRBs) and are associated with supernova explosions—powerful detonations at the ends of the lives of .

In just a matter of seconds, a typical burst releases as much energy as the Sun will in its entire ten-billion-year lifetime. The explosion itself is often followed by a slowly fading emission, known as an afterglow, which is thought to be created by collisions between the ejected material and the surrounding gas.

However, some mysteriously seem to have no afterglow—they are referred to as dark bursts. One possible explanation is that clouds of dust absorb the afterglow radiation.

In recent years, scientists have been working to better understand how GRBs form by probing their host galaxies. Astronomers expected that the massive stars that were GRB progenitors would be found in active star-forming regions in these galaxies, which would be surrounded by a large amount of molecular gas—the fuel for star formation. However, there had been no observational result to back up this theory, leaving a long-standing mystery.

For the first time, a Japanese team of astronomers led by Bunyo Hatsukade from the National Astronomical Observatory of Japan, have used ALMA to detect the radio emission from molecular gas in two dark LGRB hosts—GRB 020819B and GRB 051022—at about 4.3 billion and 6.9 billion light-years, respectively. Although such radio emission had never been detected in the GRB host galaxies, ALMA made it possible with its unprecedentedly high sensitivity.

Kotaro Kohno, a professor at the University of Tokyo and a member of the research team, said, "We have been searching for molecular gas in GRB host galaxies for over ten years using various telescopes around the world. As a result of our hard work, we finally achieved a remarkable breakthrough using the power of ALMA. We are very excited with what we have achieved."

Another remarkable achievement made possible by the high resolution of ALMA was uncovering the distribution of molecular gas and dust in GRB host galaxies. Observations of the GRB 020819B revealed a remarkably dust-rich environment in the outskirts of the host galaxy, whereas molecular gas was found only around its centre. This is the first time that such a distribution among GRB has been revealed.

"We didn't expect that GRBs would occur in such a dusty environment with a low ratio of molecular gas to dust. This indicates that the GRB occurred in an environment quite different from a typical star-forming region," says Hatsukade. This suggests that massive stars that die as GRBs change the environment in their star-forming region before they explode.

The research team believes that a possible explanation for the high proportion of dust compared to molecular gas at the GRB site is the difference in their reactions to . Since the bonds between atoms which make up molecules are easily broken by ultraviolet radiation, cannot survive in an environment exposed to the strong ultraviolet radiation produced by the hot, massive stars in its star-forming region, including the one that would eventually explode as the observed GRB. Although a similar distribution is also seen in GRB 051022, this has yet to be confirmed due to the lack of resolution (as the GRB 051022 host is located further away than the GRB 020819B host). In any case, these ALMA observations support the hypothesis that it is dust that absorbs the afterglow radiation, causing the dark gamma- ray bursts.

More information: This research was presented in a paper in Nature (12 June 2014) as an article titled "Two gamma-ray bursts from dusty regions with little molecular gas", by B. Hatsukade et al. www.nature.com/nature/journal/v510/n7504/full/nature13325.html

Provided by ESO

"Gigantic explosions buried in dust: ALMA probes environment around dark gamma-ray bursts" June 11, 2014 http://phys.org/news/2014-06-gigantic-explosions-alma-probes-environment.html


Lost star Jorge Garcia has posted a message for the 9,000 people who recently won the lottery using the mysterious numbers from the show.

On Tuesday night, Mega Millions Lottery winners who picked the numbers 4, 8, 15, 16, 23 and 42 - the "cursed" numbers that Garcia's character used in Lost to win millions - scooped $150 (Â£97) each for matching four of the six winning jackpot numbers.

In a post on his blog, titled "When will you people learn?", Garcia wrote: "The numbers are bad," and posted a picture of a $100 and a $50 bill with the word "Cursed" stamped over it.

Commenting on the lottery story, Lost executive producer Damon Lindeof tweeted: "9,078 people played Hurley's numbers in the Mega Millions tonight, each winning $150."


1901 Queen Wilhelmina of the Netherlands

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A strong earthquake in the Gulf of Alaska this morning did not generate a tsunami, officials said.

The quake, which struck at 10:42 a.m., had a magnitude of 6.4 and was centered 349 miles southeast of Anchorage at a depth of 34 miles, according to the U.S. Geological Survey.

The Pacific Tsunami Warning Center in Ewa Beach said there was no tsunami threat. The West Coast and Alaska Tsunami Warning Center also said there was no threat to that region.

She said today's earthquake wasn't of the kind that would generate tsunami concerns, as it was relatively small and involved two sides of the fault moving horizontally - not vertical motion. Ruppert said the earthquake was not directly related to the magnitude 7.7 quake that struck off the west coast of Canada last month, though she said the two are in same plate boundary system.

Louise Petersen, who works at the Glass Door Bar in Yakutat, said items on the wall moved a little, but the quake didn't feel very strong.

She said it wasn't unusual, "but we don't get them too often."