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PtdIns(3,4,5)P3 is a regulator of myosin-X localization and filopodia formation
Laure Plantard, Antti Arjonen, John G. Lock, Ghasem Nurani, Johanna Ivaska, Staffan Strömblad
Journal of Cell Science 2010 123: 3525-3534; doi: 10.1242/jcs.069609
Laure Plantard
Antti Arjonen
John G. Lock
Ghasem Nurani
Johanna Ivaska
Staffan Strömblad
For correspondence: staffan.stromblad@ki.se
Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] is a key regulator of cell signaling that acts by recruiting proteins to the cell membrane, such as at the leading edge during cell migration. Here, we show that PtdIns (3,4,5)P3 plays a central role in filopodia formation via the binding of myosin-X (Myo10), a potent promoter of filopodia. We found that the second pleckstrin homology domain (Myo10-PH2) of Myo10 specifically binds to PtdIns(3,4,5)P3, and that disruption of this binding led to impairment of filopodia and partial re-localization of Myo10 to microtubule-associated Rab7-positive endosomal vesicles. Given that the localization of Myo10 was dynamically restored to filopodia upon reinstatement of PtdIns(3,4,5)P3-binding, our results indicate that PtdIns(3,4,5)P3 binding to the Myo10-PH2 domain is involved in Myo10 trafficking and regulation of filopodia dynamics.
Migrating cells display polarized structures such as lamellipodia and filopodia at their leading edge. The formation of lamellipodia requires phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] (Oikawa et al., 2004), a membrane lipid that localizes to the leading edge of migrating cells in a tightly regulated manner and is linked to actin cytoskeleton remodeling (Chen et al., 2003; Insall and Weiner, 2001; Nishio et al., 2007). PtdIns(3,4,5)P3 was also reported to be enriched at the tip of dendritic filopodia (Luikart et al., 2008). The role of PtdIns(3,4,5)P3 in cell migration is evident because disruption of PtdIns(3,4,5)P3 metabolism impairs motility, potentially by inhibiting the specific recruitment of proteins to the leading edge (Nishio et al., 2007). Conversely, the role of phosphoinositides (PIs) in filopodia formation has remained unclear, although filopodia are functionally involved in cell migration and in important physiological events such as angiogenesis, neuronal growth cone formation and phagocytosis (Cox et al., 2002; Dent et al., 2007; Gerhardt et al., 2003). PtdIns(3,4,5)P3 participates in the membrane recruitment of proteins via interaction with PI-binding domains such as pleckstrin homology (PH) domains (Halet, 2005). Although most PH domains exhibit unspecific binding properties to PIs (Lemmon and Ferguson, 2000), some are described as being highly specific (Lemmon et al., 1995; Varnai et al., 1999). For example, PLCδ1 and Btk PH domains specifically bind to PtdIns(4,5)P2 and PtdIns(3,4,5)P3, respectively. A few PH domains are also involved in protein–protein interactions (Cohen et al., 2007; Zhu et al., 2007a).
Myosin-X (Myo10) is an actin binding motor possessing three PH domains (Berg et al., 2000). Myo10 also associates with regions of dynamic actin (Berg et al., 2000), is recruited to phagocytic cups in a phosphoinositide 3-kinase (PI3K)-dependent manner (Cox et al., 2002), and plays a role in spindle formation during cell division (Toyoshima and Nishida, 2007; Weber et al., 2004; Woolner et al., 2008). Myo10 localizes to the tip of filopodia, at least in part owing to its ability to specifically bind bundled actin, and undergoes intrafilopodial motility both towards and from the filopodia tips (Berg and Cheney, 2002; Kerber et al., 2009; Nagy et al., 2008). Myo10 also promotes filopodia formation and/or stabilization (Bohil et al., 2006; Zhang et al., 2004) and conveys β-integrins and netrin (Zhang et al., 2004; Zhu et al., 2007b) to the tip of filopodia. However, it remains unclear by which mechanisms Myo10 can promote filopodia. Although dimerization of the Myo10 head, neck and coiled-coil domains has been suggested to be required for Myo10-induced filopodia promotion (Tokuo et al., 2007), it is still not clear whether the full-length Myo10 protein actually dimerizes (Knight et al., 2005).
Myo10 contains three distinct PH domains, but the potential specificity of Myo10-PH-domain-binding to PIs and the potential role of PI-binding in Myo10 function are not known in detail. Two recent screenings aiming at isolating PH domains that bind to PI3K products [PtdIns(3,4)P2 and PtdIns(3,4,5)P3] identified Myo10 as one of the proteins binding to PI3K products, although it was not clear whether Myo10 might bind PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3 in these screens (Isakoff et al., 1998; Park et al., 2008). Here, we show that PtdIns(3,4,5)P3 plays an important role in filopodia induction via binding to Myo10. We demonstrate that the second PH domain of Myo10 (Myo10-PH2) specifically binds to PtdIns(3,4,5)P3 and no other PI, although Myo10-PH1 and Myo10-PH3 showed no binding to any tested lipids. Upon disruption of Myo10 binding to PtdIns(3,4,5)P3, either by mutation of Myo10 or by inhibition of PtdIns(3,4,5)P3 synthesis, Myo10 partially re-localized from filopodia tips to Rab7-positive vesicles moving along microtubules. Notably, Myo10 localization was reverted from vesicles to filopodia upon restoration of PtdIns(3,4,5)P3 synthesis, suggesting that PtdIns(3,4,5)P3 might regulate Myo10 intracellular trafficking. Myo10 binding to PtdIns(3,4,5)P3 also contributes to Myo10-induced increases in filopodia number. Taken together, these results indicate that PtdIns(3,4,5)P3 binding regulates Myo10 trafficking and filopodia dynamics.
Myo10 intracellular localization is influenced by its PH domains
The Myo10 domain structure is highly conserved, and includes three PH domains (Fig. 1A; supplementary material Fig. S1). Interestingly, although a construct of mCherry fused to wild-type Myo10 (mCherry–Myo10WT) localized at filopodia tips, deletion of all three Myo10 PH domains caused the partial re-localization of Myo10 to cytosolic puncta (Fig. 1B). Confocal microscopy showed that these puncta were indeed intracellular and not dorsal or ventral filopodia (Fig. 1B). This suggests that the PH domains of Myo10 are important in regulating Myo10 localization.
PtdIns(3,4,5)P3 binds to Myo10-PH2 domain
To determine the lipid-binding capacity of individual Myo10 PH domains, we purified each of the three Myo10 PH domains fused to maltose binding protein (MBP) (Fig. 2A; supplementary material Fig. S1) and subjected them to a lipid pull-down assay against eight different PIs. MBP-PH2 bound specifically to PtdIns(3,4,5)P3, although MBP-PH1, MBP-PH3 and MBP control showed no binding to any of the PIs (Fig. 2B,C). The Myo10 tail (Myo10Tail), which includes all three PH domains, also bound to PtdIns(3,4,5)P3 but this binding was lost upon deletion of PH2 (Fig. 2D,E).
PtdIns(3,4,5)P3 binding regulates Myo10 localization
Upon observing that Myo10 deleted of its three PH domains was re-localized, we mutated conserved PH domain-residues (which in other PH domains are crucial for lipid binding) in each of the three PH domains of Myo10 (Fig. 3A; supplementary material Fig. S1 and Table S1). Expression levels of the different constructs were comparable (supplementary material Fig. S2A). Mutagenesis of the conserved residues in Myo10-PH1 (K1179A) and Myo10-PH3 (K1395A) did not result in altered Myo10 localization (Fig. 3A,B). However, Myo10-KK1215/6AA (Myo10PH2pm; bearing point mutations in Myo10-PH2) as well as Myo10 with deleted PH2 (Myo10ΔPH2), showed a similar punctate localization to that observed upon deletion of all three Myo10 PH domains (Fig. 3B; supplementary material Movies 1, 2). Coincident with the punctuate staining, Myo10-PH2 mutants were also targeted significantly less to the tips of filopodia (Fig. 3B,C). Thus, Myo10 localization at the tip of filopodia is regulated by the Myo10-PH2 domain.
We then replaced the Myo10-PH2 domain with well-characterized PH domains from Btk, PLCδ1 and TAPP1, which bind specifically to PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3,4)P2, respectively (Halet, 2005; Varnai and Balla, 2006) (Fig. 3A). Although replacement of Myo10-PH2 with PtdIns(3,4,5)P3- or PtdIns(4,5)P2-binding PH domains restored the localization from intracellular puncta to filopodial tips, introduction of a PtdIns(3,4)P2-binding PH domain did not correct the mislocalization of Myo10ΔPH2 (Fig. 3D,E). This shows that binding to either PtdIns(3,4,5)P3 or to PtdIns(4,5)P2 can promote Myo10 localization to the tips of filopodia.
Re-localization of Myo10 in the absence of PH domains. (A) Structure of constructs of Myo10WT and Myo10 with deleted PH domains, showing the different domains of the protein. (B) Mislocalization of Myo10 upon deletion of the three Myo10 PH domains. COS-7 cells transfected with mCherry–Myo10WT (red) and mCherry–Myo10ΔPH (red) were stained with Alexa-Fluor-488–Phalloidin (green). Single-colour images are shown as inverted contrast images. Top views are shown of the bottom and a mid plane, and side views of a single x–y slice, with individual channels and channels merged. Scale bars: 10 μm.
Complex formation containing more than one Myo10 molecule
We then examined whether different molecules of Myo10 could form complexes. We co-transfected COS-7 cells with either EGFP–Myo10WT and mCherry–Myo10WT or with EGFP–Myo10PH2pm and mCherry–Myo10ΔPH2 (Fig. 4A) and performed a co-immunoprecipitation with antibodies specific for either EGFP or mCherry (supplementary material Fig. S2B,C). The full-length Myo10WT co-immunoprecipitated with mCherry–Myo10WT (Fig. 4B, upper panel). In addition, mutation of the Myo10-PH2 domain had no effect on this association, because the two Myo10-PH2 mutant proteins also co-immunoprecipitated (Fig. 4B, lower panel). Furthermore, the presence of Myo10WT affected the localization of Myo10ΔPH2, because mCherry–Myo10ΔPH2 was localized at tips of filopodia to a higher degree when co-transfected with EGFP–Myo10WT than when co-transfected with EGFP (Fig. 4A,C). This indicates that different molecules of Myo10 do associate and that Myo10WT can bring the mutant Myo10ΔPH2 to filopodia tips. Notably, this might explain the detectable though substantially weaker localization of Myo10ΔPH2 to filopodia tips in singly transfected cells, because endogenous Myo10 is also present in these cells. Thus, the observed localization of Myo10 PH2 mutants in filopodia might result from association with endogenous Myo10 rather than from direct independent targeting.
Inhibition of PI3K reversibly re-localizes Myo10 to cytoplasmic puncta
Given the Myo10 re-localization upon mutation of its PtdIns(3,4,5)P3 binding site, we next inhibited the production of PtdIns(3,4,5)P3 using the PI3K inhibitors LY294002 and wortmannin in cells where filopodia were induced by overexpression of Myo10, and analyzed Myo10 localization (Fig. 5A,C,D; supplementary material Fig. S4, Movie 3). Efficient PI3K inhibition was controlled by Akt phosphorylation activity (Fig. 5B). After PI3K inhibition, mCherry–Myo10WT partially re-localized from the tips of filopodia to cytoplasmic puncta, similar to those labeled by mCherry–Myo10ΔPH2 (Fig. 5A,C,D). This effect was independent of the mCherry tag because the same effect was seen in cells transfected with EGFP–Myo10WT (supplementary material Movies 4, 5). The effects of the PI3K inhibitors on Myo10 localization were less prominent than those observed with the PH2-mutant forms of Myo10. These differences might arise because filopodia are formed in the presence of the Myo10-PH2 mutant under one experimental condition, although the effects on pre-existing filopodia are characterized in the case of short-term treatment with PI3K inhibitor. Nonetheless, wash-out of the PI3K inhibitor effectively restored Myo10 filopodial localization (Fig. 5E), suggesting that the puncta represent a transient re-localization of Myo10 in the absence of PtdIns(3,4,5)P3 binding.
Myo10 localizes to endosomal vesicles in the absence of PtdIns(3,4,5)P3 binding
We next performed more detailed analyses of the localization of Myo10-PH2 mutants as well as of Myo10WT in cells treated with PI3K inhibitors. First we noted that the Myo10-PH2 mutants were more soluble in cold Triton than PtdIns(3,4,5)P3 binding Myo10 (supplementary material Fig. S3). Triton insolubility is frequently an indicator of cytoskeletal association (Fox, 1985; Quinn et al., 1989), and therefore this result suggests that PtdIns(3,4,5)P3 binding to Myo10WT supports Myo10 binding to F-actin. We also found that the puncta of Myo10-PH2 mutants moved within the cell cytoplasm with top speeds of 0.33±0.05 μm/second, (Fig. 6A; supplementary material Fig. S4, Movies 6, 7), which correlates with previously published endosome velocities (Tacon et al., 2004). This movement was abolished by Nocodazole treatment (Fig. 6A). These results indicate that in the absence of PtdIns(3,4,5)P3 binding, Myo10 localizes to vesicles moving along microtubules.
To characterize Myo10-positive vesicles, we co-labeled Myo10-expressing cells with endosomal and lysosomal markers (Fig. 6B; supplementary material Fig. S5). Interestingly, Myo10-KK1215/6AA PH2 colocalized partially with Rab7 and LAMP1 (Fig. 6B; supplementary material Fig. S5, Movie 8), but not with Rab4a, Rab5, Rab11 or Rab21 (supplementary material Fig. S5A), suggesting that Myo10 localized to late endosomal/lysosomal vesicles. Furthermore, overexpression of Rab7 together with Myo10WT increased Myo10 localization to vesicles (Fig. 6B). In addition, endogenous Myo10 was localized in Rab7-positive vesicles, both in the presence of the PI3K inhibitor LY294002 and to a lower extent in its absence (supplementary material Fig. S5B). However, endogenous Myo10 was not found in LAMP1- or Rab21-positive vesicles (Fig. 6B; supplementary material Fig. S5) and therefore we cannot exclude the possibility that the observed localization of the mCherry–Myo10-PH2 mutant to lysosomes might be an artifact of protein overexpression. We also found that Myo10 and Rab7 double-positive vesicles undergo dynamic movements close to the cell membrane, as shown by total internal reflection fluorescence microscopy (TIRFM) (Fig. 6C; supplementary material Movie 9). This is in line with a previous report describing Rab7 recruitment to clathrin-coated structures at the plasma membrane (Barroso-Gonzalez et al., 2009). Thus, in the absence of PtdIns(3,4,5)P3 binding, Myo10 predominantly localizes to endosomal vesicles trafficking close to the plasma membrane, suggesting that Myo10 might be transported to the plasma membrane via Rab7-containing vesicles.
Myo10 PH2 domain binds specifically to PtdIns(3,4,5)P3. (A) Constructs allowing the expression of different domains of Myo10 fused to MBP for protein purification. (B) Purified MBP, MBP-PH1, MBP-PH2 and MBP-PH3 were subjected to a lipid pull-down assay with different PIs. After pull-down, immunoblot detected Myo10. The numbers below the gels correspond to the numbers in C. (C) Quantification of the binding of isolated PH1, PH2 and PH3 domains of Myo10 to lipids in a lipid pull-down assay. Comparison by Student's t-test (***P<0.001) to the MBP control. (D) MBP–Myo10TailWT but not MBP–Myo10TailΔPH2 binds to PtdIns(3,4,5)P3. MBP–Myo10TailWT or MBP–Myo10TailΔPH2 were purified and subjected to immunoblotting after a PtdIns(3,4,5)P3 pull-down. (E) The Myo10 tail binds to PtdIns(3,4,5)P3 via its second PH domain (PH2). Quantification of the binding of MBP–Myo10TailWT and MBP–Myo10TailΔPH2 to PtdIns(3,4,5)P3 in a lipid pull-down assay. Comparison by Student's t-test (*P<0.05). Graphs show means + s.e.m. of three independent experiments.
PtdIns(3,4,5)P3 regulates Myo10 localization. (A) mCherry–Myo10 constructs used in these experiments. (B) Mutations in the Myo10-PH2 domain lead to partial mislocalization of Myo10. HeLa cells transfected with mCherry–Myo10 constructs (red) and stained with FITC–phalloidin (green) and Hoechst 33342 (blue). Inverted contrast images for each of the channels are shown below each colour image in B and D. (C) The proportion of cells with Myo10 present in puncta and the proportion of filopodia with Myo10 at the tip were quantified. Analysis of 34–49 cells per condition in three independent experiments. (D) Binding to PtdIns(3,4,5)P3 or PtdIns(4,5)P2 can localize Myo10 at the tip of filopodia. COS-7 cells were transfected with mCherry, mCherry–Myo10WT, mCherry–Myo10ΔPH2, mCherry–Myo10Btk, mCherry–Myo10PLCδ1 or mCherry–Myo10TAPP1 (all red) and stained with FITC–phalloidin (green). (E) The proportion of cells with Myo10 present in puncta and the proportion of filopodia with Myo10 at the tip were quantified. Analysis of 42–68 cells per condition in three independent experiments. Graphs show means + s.e.m. Comparison by Student's t-test (ns, not significant; *P<0.05, **P<0.01, ***P< 0.001) to the mCherry control as indicated on top of bars and to mCherry–Myo10WT as indicated by brackets. Scale bars: 10 μm.
Intracellular complex formation of more than one Myo10 molecule. (A) EGFP–Myo10 constructs. (B) Different full-length Myo10 molecules co-immunoprecipitate independently of PtdIns(3,4,5)P3 binding. Co-immunoprecipitations were performed with anti-EGFP antibodies using COS-7 cells co-transfected with either mCherry–Myo10WT and EGFP–Myo10WT or with mCherry–Myo10ΔPH2 and GFP–Myo10PH2pm. Immunoprecipitates were then resolved by SDS-PAGE and analyzed by detecting mCherry with an anti-RFP antibody. The signal intensity was measured along three lines per lane on the western blot and plotted as mean + s.d. The gray and black lines represent, respectively, the IgG control and the precipitation by the anti-GFP antibody. (C) Myo10WT can relocate Myo10ΔPH2 to tips of filopodia. COS-7 cells were transfected with mCherry, mCherry–Myo10WT or mCherry–Myo10ΔPH2 in the presence of either EGFP or EGFP–Myo10WT. The proportion of filopodia with mCherry at their tip was quantified. Analysis of 43–52 cells per condition in three independent experiments. Graph shows means + s.e.m. Comparison by Student's t-test (ns, not significant; *P<0.05, **P<0.01, ***P<0.001) to the EGFP control as indicated on top of bars or to mCherry–Myo10WT as indicated by brackets.
PtdIns(3,4,5)P3 binding is necessary for Myo10 promotion of filopodia
Myo10 induces filopodia in various mammalian cells (Berg and Cheney, 2002; Bohil et al., 2006; Zhang et al., 2004). Because Myo10 defective in PtdIns(3,4,5)P3 binding was partially re-localized from filopodia, we analyzed whether the binding of PtdIns(3,4,5)P3 to Myo10 affected its function in filopodia promotion. Although mCherry–Myo10WT, mCherry–Myo10-K1179A (PH1 mutant) and mCherry–Myo10-K1395A (PH3 mutant) transfected cells displayed a large increase in filopodia number in comparison to cells expressing mCherry control, the mCherry–Myo10-KK1215/6AA (PH2 mutant) and mCherry–Myo10ΔPH did not (Fig. 7A,B). mCherry–Myo10Btk and mCherry–Myo10PLCδ1 also promoted filopodia number although mCherry–Myo10ΔPH2 and mCherry–Myo10TAPP1 failed to induce filopodia (Fig. 7C). Thus, Myo10 promoted filopodia only when it was able to bind PtdIns(3,4,5)P3 or PtdIns(4,5)P2. In addition, a short treatment with PI3K inhibitor was able to decrease the number of filopodia in mCherry–Myo10WT transfected cells (Fig. 7D). Expression of mCherry–Myo10WT also caused an increase in filopodia length, although expression of mCherry–Myo10ΔPH2 did not (Fig. 7E). In conclusion, disruption of Myo10–PtdIns(3,4,5)P3 binding impaired the capacity of Myo10 to promote filopodia number and length.
We demonstrate here that the Myo10-PH2 domain binds specifically to PtdIns(3,4,5)P3 and that this binding is important for Myo10 localization and function. Myo10 localization to the tip of filopodia is dependent on its head, neck and coiled-coil domains (HMM construct), with the motor domain binding to actin bundles and allowing the protein to reach the tip of filopodia (Berg and Cheney, 2002). Myo10 dimerization is also required for the motor domain to get to the tip of filopodia (Tokuo et al., 2007). These conclusions on the role for the Myo10 HMM region in Myo10 localization are based upon the use of Myo10 constructs lacking the tail (PH, MyTH4 and FERM domains). Although the Myo10 head, neck and coiled-coil domains are sufficient for filopodial tip localization, the full-length Myo10 constructs possessed additional domains that obviously contribute additional localization-regulating properties. Indeed, a construct corresponding to the three PH domains of Myo10 has also been indicated to localize at the plasma membrane (Mashanov et al., 2004).We therefore conclude that the C-terminal tail of Myo10 also contributes to Myo10 localization by binding to the membrane lipid PtdIns(3,4,5)P3.
We demonstrate that different molecules of full-length Myo10 can associate, which is consistent with dimerization of Myo10 (Tokuo et al., 2007). Myo10 dimerization might be required for its motor activity (Tokuo et al., 2007). Although Myo10 in solution appears to be mainly monomeric (Knight et al., 2005), the head domain has been reported to dimerize when two molecules are in close vicinity (Sun et al., 2010). One hypothesis concerning the regulation of Myo10 dimerization is that Myo10 might dimerize upon cargo binding, just like MyoVI (Yu et al., 2009). However, another motor protein, KIF1A, dimerizes only upon binding to PtdIns(4,5)P2 (Klopfenstein et al., 2002). Our results are consistent with the dimerization of Myo10 being regulated independently of its PtdIns(3,4,5)P3 binding.
Myo10 is reversibly re-localized upon inhibition of PI3K. (A) Myo10WT re-localization from filopodia tips to cytoplasmic puncta upon PI3K inhibition. COS-7 cells expressing mCherry–Myo10WT (red) were treated with DMSO, LY294002 or wortmannin and stained with FITC–phalloidin (green) and Hoechst 33342 (blue). Inverted contrast images for each of the channels are shown below each double channel image. (B) PI3K inhibition was assessed by checking Akt phosphorylation using immunoblotting. (C) The proportion of cells with Myo10 present in puncta was quantified. Analysis of 58–90 cells per condition in three independent experiments.(D) The proportion of filopodia with Myo10 at the tip was quantified. Analysis of 58–90 cells per condition in three independent experiments. (E) Myo10 mislocalization was rescued by wash-out after PI3K inhibitor treatment. COS-7 cells were transfected with mCherry–Myo10WT and treated with the PI3K inhibitor LY294002 for 1 hour, and the inhibitor was then washed out. Bar graph shows the proportion of cells with Myo10 in puncta. Analysis of 50–58 cells per condition in three separate experiments. Graphs show means + s.e.m. Comparison by Student's t-test (ns, not significant; *P<0.05, **P<0.01, ***P<0.001) to the mCherry control as indicated on top of the bars or as indicated by brackets. Scale bars: 10 μm.
In addition, we reveal that Myo10, when unable to bind to PtdIns(3,4,5)P3, is localized in Rab-7-positive endosomal vesicles. This is consistent with earlier observations of a small amount of Myo10WT being located in intracellular puncta (Berg et al., 2000). Because we showed that this vesicular localization was reversible (Fig. 5E), and that the vesicle motion was microtubule-dependent, the endosomal localization of Myo10 might therefore be part of Myo10 trafficking or recycling pathways. The endocytic pathway has recently been described as an assembly platform implicated in actin dynamics and directed cell motility (Gould and Lippincott-Schwartz, 2009; Palamidessi et al., 2008). In a similar way, a machinery responsible for filopodia formation might be assembled on Rab-7-positive vesicles and might be activated upon PtdIns(3,4,5)P3–Myo10 binding. Rab7 has been shown to be involved in phagocytosis and in phagosome fusion with late endosomes (Harrison et al., 2003). The formation of filopodia is a key step during phagocytosis, and Myo10 as well as PtdIns(3,4,5)P3 have been shown to be important in phagocytosis (Cox et al., 2002; Niedergang and Chavrier, 2004). An interesting hypothesis is therefore that the Rab7-positive vesicles might be responsible for targeting Myo10 to sites of filopodia and/or phagocytotic cup formation.
We have also demonstrated that Myo10 unable to bind to PtdIns(3,4,5)P3 was incapable of promoting filopodia, as is HMM–Myo10 (Berg and Cheney, 2002). The presence of the Myo10 tail is therefore required to increase the number and length of filopodia. Interestingly, a decrease in filopodia number has previously been reported in T cells treated by the PI3K inhibitor wortmannin (Johnson et al., 2008). In addition, as in our experiments, a recovery in filopodia number was observed after wortmannin washout (Johnson et al., 2008). Furthermore, inhibition of PI3K by LY294002 reduced filopodial motility in dendritic growth cones (Luikart et al., 2008). A decrease in filopodia merging rate has also been reported in a melanoma cell line treated with Nocodazole, which suggests that Nocodazole makes filopodia dynamically more stable (Schober et al., 2007). These reports are consistent with our findings on a role for PI3K in Myo10 trafficking and filopodia dynamics, and a role for microtubules in Myo10-labeled vesicle trafficking.
Absence of PtdIns(3,4,5)P3 binding localizes Myo10 to Rab7-positive vesicles. (A) Myo10-KK1215/6AA PH2 shows microtubule-dependent vesicular movement in the cell body. Upper: mCherry–Myo10-KK1215/6AA PH2 transfected COS-7 cells with or without Nocodazol treatment were imaged over time, and trajectories of vesicle tracks are shown in black lines. The cell edge is shown by the red perimeter line. Lower: Bar graphs show the vesicle top speed measured by tracking. Graphs show means + s.e.m. Comparison by Student's t-test (***P<0.001). (B) Myo10-KK1215/6AA PH2 colocalizes with Rab7. COS-7 cells were transfected with mCherry–Myo10-KK1215/6AA PH2 or mCherry–Myo10WT and EGFP-Rab7 or were stained for endogenous Myo10 and Rab7 as indicated. Images were acquired with a confocal microscope using 0.7 μm optical slices. In close-ups, regions of interest (ROI) are shown and a pixel line profile is drawn over merged images to illustrate colocalization. Maximum intensity projections are shown. Myo10 in the filopodia tips show no colocalization with Rab7 (yellow arrowheads). (C) Myo10WT is transported close to the plasma membrane in Rab7-positive vesicles. TIRFM imaging shows time-lapse series of COS-7 cells co-transfected with EGFP–Rab7 and mCherry–Myo10WT. Cells were treated with LY294002 to induce the vesicular phenotype of Myo10WT. Three channels were acquired: red shows Myo10 in widefield microscopy, green shows Rab7 in widefield, and blue shows Rab7 in the TIRFM channel (<100 nm proximity to glass surface). An ROI is shown in the panel of images where a vesicle positive for both Myo10 and Rab7 is tracked over time. Vesicle appearance in the TIRFM channel (asterisk) indicates close proximity of the vesicle to the membrane. Scale bars: 10 μm.
PtdIns(3,4,5)P3 has been implicated in the regulation of actin polymerization (Chen et al., 2003; Insall and Weiner, 2001), which is required at the tip of filopodia for their formation. PtdIns(3,4,5)P3 was also reported to be enriched in dendritic filopodia (Luikart et al., 2008). The mechanism by which PtdIns(3,4,5)P3 regulates Myo10 function remains unclear. However, our data support the possibility that PtdIns(3,4,5)P3 provides an anchor to the membrane (Manna et al., 2008; Oikawa et al., 2004) that recruits Myo10 to the plasma membrane. Thereafter, Myo10 might be involved in transporting PtdIns(3,4,5)P3 to filopodia tips where it might serve to promote actin polymerization. We also found that Myo10 mutants binding PtdIns(4,5)P2 were correctly localized and could induce filopodia, suggesting that for filopodia dynamics, binding of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 to Myo10 might be exchangeable. Membrane anchoring and actin remodeling are roles that both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 can sustain.
We found no binding partners for Myo10-PH1 or Myo10-PH3 domains amongst the PIs tested. PH domains might have other binding targets in addition to PIs, including protein partners (Cohen et al., 2007; Lemmon, 2004; van Rossum et al., 2005; Zhu et al., 2007a). However, no particular phenotype was seen upon expression of Myo10-PH1 or Myo10-PH3 mutants in mammalian cells. The role of Myo10-PH1 and Myo10-PH3 domains therefore remains unclear. However, Myo10 has several roles in cells; not only is Myo10 implicated in filopodia formation, but also in phagocytosis (Cox et al., 2002) and in cell division (Toyoshima and Nishida, 2007; Weber et al., 2004; Woolner et al., 2008), and it is possible that PH1 and/or PH3 might have roles in these or other Myo10-driven events.
In conclusion, we have demonstrated that Myo10 binds specifically to PtdIns(3,4,5)P3 through its second PH domain. Our results also provide new insights into Myo10 trafficking by showing that Myo10 can be localized to endosomal vesicles moving along microtubules. Overall, it is now clear that PtdIns(3,4,5)P3 binding regulates not only Myo10 localization and trafficking but also the frequency and structure of filopodia.
Myo10-PH2 binding to PtdIns(3,4,5)P3 is required for Myo10 function in filopodia. (A) Deletion of the three PH domains of Myo10 impairs filopodia. Cos-7 cells were transfected with mCherry–Myo10WT and mCherry–Myo10ΔPH. Bar graph shows the mean number of filopodia per cell normalized to Myo10WT transfected cells (n=3). (B) Mutation of Myo10-PH2 impairs filopodia. HeLa cells were transfected with mCherry, mCherry–Myo10WT, mCherry–Myo10-K1179A PH1, mCherry–Myo10-KK1215/6AA PH2 or mCherry–Myo10-K1395A PH3. Bar graph shows the number of filopodia per cell in 34–49 cells per condition in three experiments. (C) Myo10 binding to PtdIns(3,4,5)P3 or PtdIns(4,5)P2 promotes filopodia. COS-7 cells were transfected as in Fig. 2B prior to quantification of the number of filopodia per cell in 42–68 cells per condition in three experiments. (D) Inhibition of PI3K inhibits Myo10-induced filopodia. COS-7 cells expressing either mCherry, mCherry–Myo10WT or mCherry–Myo10ΔPH2 were treated for 1 hour with either DMSO, LY294002 or wortmannin. Quantification of filopodia number per cell was performed in 58–90 cells per condition in three experiments. (E) PtdIns(3,4,5)P3 binding is necessary for Myo10-induced increase in filopodia length. Filopodia length was measured in COS-7 cells expressing mCherry, mCherry–Myo10WT or mCherry–Myo10ΔPH2. Bar graph displays the proportion of filopodia shorter than 1.5 μm, between 1.5 μm and 2.5 μm, or longer than 2.5 μm, based on seven independent experiments. Graphs display means + s.e.m. Comparison by Student's t-test (ns, not significant; *P<0.05, **P<0.01, ***P<0.001) to the mCherry-control as indicated on top of the bars or as indicated by brackets.
DNA constructs
Myo10-PH2 (E1206-A1304), Myo10-PH3 (E1386-D1491) and Myo10 tail (R1160-R2052) were amplified by PCR (supplementary material Table S1 and Fig. S1) and cloned into pMalC2E (New England Bioloabs) within XbaI and HindIII restriction sites to fuse them in N-terminal with MBP. Myo10-PH1 was cloned by deleting PH2 (E1206-A1304) from a Myo10-PH1+PH2 construct (P1170-G1378) (supplementary material Fig. S1).
pmCherry–Myo10WT was constructed by substituting EGFP from pEGFP–Myo10 (Berg and Cheney, 2002) using mCherry amplified by PCR from pR-SETBmCherry (Shaner et al., 2004). Mutations in these constructs were created using Quickchange XL site-directed mutagenesis kit (Stratagene). Point mutations of Myo10 PH domains (Myo10-K1179A PH1, Myo10-KK1215/6AA PH2 and Myo10-K1395A PH3) were made thanks to primers introducing one or two point mutations into the lipid binding site (supplementary material Table S1 and Fig. S1). Primers were also designed to delete the second PH domain of Myo10 (E1206-A1304) while inserting a SpeI restriction site (supplementary material Table S1 and Fig. S1). The three PH domains (mCherry–Myo10ΔPH) were deleted (S1131-Y1472) by adding an AgeI site by site-directed mutagenesis (see supplementary material Table S1) followed by digestion and re-ligation of the construct. Btk, PLCδ1 and TAPP1 PH domains were amplified by PCR (supplementary material Table S1) and introduced in the mCherry–Myo10ΔPH2 construct in place of PH2, creating three chimeras: mCherry–Myo10Btk, mCherry–Myo10PLCδ1 and mCherry–Myo10TAPP1. EGFP–Myo10-KK1215/6AA PH2 was constructed by introducing the KK1215/6AA mutations into EGFP–Myo10WT with the same primers as used for pmCherry–Myo10-KK1215/6AA PH2.
Plasmids encoding EGFP–Rab4, EGFP–Rab5a, EGFP–Rab7, EGFP–Rab11, EGFP–Rab21 and EGFP–TAPP1PH have been previously described (Barbero et al., 2002; Gomes et al., 2003; Lebrand et al., 2002; Marshall et al., 2002; Pellinen et al., 2006; Rzomp et al., 2003; Wilcke et al., 2000). Plamids encoding EGFP–BtkPH and EGFP–PLCδ1PH were kind gifts from Matthias Wymann (University of Basel, Switzerland). All primers are listed in supplementary material Table S1. All plasmid sequences were verified by sequencing prior to use. Nucleotide alignment shown in supplementary material Fig. S1 was performed using Clustal W and Clustral X (Larkin et al., 2007).
HeLa carcinoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) with 5% FBS. COS-7 African green monkey kidney cells and MDA-MB-231 breast adenocarcinoma cells were cultured in DMEM with 10% FBS. Mouse aortic endothelial (MAE) cells were cultured in RPMI (Invitrogen) with 10% FBS. All DNA constructs were transfected using Lipofectamine 2000 (Invitrogen) or Fugene6 (Roche) according to the manufacturer's protocol.
Antibodies and reagents
The following antibodies and dyes were used: anti-RFP (PM005, MBL), anti-α-tubulin (12G10, Developmental Studies Hybridoma Bank, Iowa), anti-actin (JLA20, Developmental Studies Hybridoma Bank, Iowa), anti-LAMP1 (Santa Cruz Biotechnology), MBP-Probe (N17, Santa Cruz Biotechnology), anti-GFP (Clontech), anti-Akt (Cell Signaling), anti-phospho-Akt (Ser473) (Cell Signaling), anti-Myo10 (2243.00.02, SDIX) and anti-Rab7 (Rab7-117, Abcam), rabbit IgG (Sigma), FITC–Phalloidin (Invitrogen), Hoechst 33342 (Sigma). Alexa-Fluor-680 and Alexa-Fluor-700 secondary antibodies (Invitrogen) were used in LI-COR's Odyssey Infrared Imaging System and horse radish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were used for detection by chemiluminescence. Nocodazole (10 μM, 1 hour at 37°C) was used to depolymerize microtubules. LY294002 (Sigma) (10–20 μM, 1 hour at 37°C) and wortmannin (Sigma) (0.15 μM, 1 hour at 37°C) were used to inhibit PI3K.
Protein purification and lipid pull-down assay
Proteins were produced with pMalC2E constructs. Plasmid expression was induced by IPTG and the bacteria were grown for 1 hour at 37°C prior to lysis. The proteins were then purified on an Amylose column (New England Biolabs) in accordance with the manufacturer's protocol.
Lipid pull-down assay was performed with PIP beads (Echelon Biosciences) according to the manufacturer's protocol. Briefly, 25 μl of PIP bead slurry at 10 nM was incubated with 0.04 μg/μl of purified Myo10 PH domains for 3 hours at 4°C, and then washed in 10 mM HEPES pH 7.4, 150 mM NaCl and 0.25% NP-40. Proteins bound to the beads were eluted in SDS-sample buffer and resolved by SDS-PAGE. Immunoblotting was analyzed with a VersaDoc Imaging System (Bio-Rad). Binding, defined as the ratio between the intensity of the bound fraction and the intensity of the input, was calculated.
Microscopy techniques
For imaging of fixed cells, cells were plated on Ibitreat μ-Dish dishes (Integrrated Biodiagnostics) one day before transfection. Transfected cells were fixed 12–24 hours after transfection with 2% paraformaldehyde, permeabilized with Triton-X100 and blocked with 2% BSA/PBS. Alternatively, cells were replated 24 hours after transfection on glass coverslips and allowed to attach for 2 hours prior to fixation.
Primary antibodies were used with predetermined optimal concentrations of 5–10 μg/ml. The concentration of Alexa-Fluor-conjugated secondary antibodies (Invitrogen) was 5 μg/ml. Alexa-Fluor-conjugated and FITC-conjugated phalloidins (Invitrogen) were used for F-actin detection. Cells were imaged on an Olympus IX71 microscope with Olympus 63× oil/1.4NA objective and a CCD Hamamatsu camera.
In live-cell experiments, cells were plated on Ibitreat μ-Dish dishes (Integrrated Biodiagnostics) one day before transfection. Transfected cells were imaged 12–24 hours after transfection. To maintain optimal conditions, a cell culture dish heater, objective heater at 37°C and 4.8% CO2 support were used. Confocal three-dimensional images were taken using Zeiss Axiovert 200M with spinning disc confocal unit Yokogawa CSU22 and Zeiss Plan-Neofluar 63× oil/1.4 NA objective. Z-stacks with 1 airy unit (approx. 0.7 μm) optical slices were acquired with a step size of 0.3–0.7 μm between slices.
In TIRFM, cells were plated as above on Mattek glass bottom dishes. An Olympus IX71 MT20 CellR system with 60× oil/1.45NA TIRFM objective, MWB2 TIRF/FITC filter block and a 488 nm laser line was used to acquire total internal reflection images and widefield epifluorescence images sequentially (University of Turku, Department of Cell Biology and Anatomy). The maximum intensity projections were created with SlideBook 4.2.0.7 software and NIH ImageJ. QuickTime movies from time-lapse experiments were created using NIH ImageJ software.
In live-cell experiments, transfected cells were imaged 12–24 hours after transfection. Confocal three-dimensional images were taken using Zeiss Axiovert 200M. For PtdIns(3,4,5)P3 localization experiments, cells were imaged with a Zeiss LSM 510.
ImageJ plugin Particle Detector and Tracker (Sbalzarini and Koumoutsakos, 2005) was used to automatically detect, track and visualize vesicle trajectories in time-lapse movies. Also, manual tracking and plugin MTrackJ (Erik Meijering, University Medical Center, Rotterdam, The Netherlands) were used in tracking vesicles. Cell Counter (Kurt De Vos, University of Sheffield, Sheffield, UK) and Manual Tracking (Fabrice P. Cordelières, Institut Curie, Orsay, France) plugins were used to count filopodia and track cells, respectively. Standard ImageJ tools were used to draw line profiles and measure filopodia length as well as signal intensities. Quantitative analysis of colocalization was done with the help of ImageJ plugin JACoP (Bolte and Cordelieres, 2006). Costes' approach was used to calculate Pearson correlation coefficients in the whole cell level for endogenous Myo10, Rab7 and Rab21 (Costes et al., 2004).
Cold Triton fractionation
Cold Triton fractionation protocol was modified from a published method (Ding et al., 1996). Transfected COS-7 cells were washed and scraped in ice-cold cytoskeleton-stabilizing buffer (CSK buffer) containing: 250 mM sucrose, 3 mM MgCl2, 150 mM KCl, 1 mM EGTA and 1 mM PMSF in 10 mM PIPES, pH 6.8. After spinning-down at 7000 g for 5minutes at +4°C, the pellet was resuspended in ice-cold 0.2% Triton X-100 (Sigma) in CSK buffer with protease inhibitor cocktail (Complete, Roche) and incubated on ice for 20 minutes. After centrifugation at 15,000 g for 10 minutes, the supernatant was considered as a soluble fraction and the pellet was considered as an non-soluble fraction. Supernatant and pellet were analyzed with standard western blotting techniques and detected using the LI-COR Odyssey Infrared Imaging System. The protein load was balanced to the tubulin signal.
Co-immunoprecipitation
At 24 hours after transfection, cell lysates were prepared in RIPA buffer from COS-7 cells co-transfected either with EGFP–Myo10WT and mcherry–Myo10WT or with EGFP–Myo10-KK1215/6AA PH2 and mCherry–Myo10ΔPH2. Cell lysates were incubated in the presence of anti-GFP antibody. Protein G beads (Santa Cruz Biotechnology) were used to capture the beads to which the antibodies and proteins were bound. Samples extracts were resolved by SDS-PAGE and detected by immunoblotting with an anti-RFP antibody (MBL, Japan).
We thank Matthias P. Wymann (University of Basel, Switzerland) for the EGFP–BtkPH and EGFP–PLCδ1PH constructs; Richard E. Cheney (University of North Carolina at Chapel Hill, NC) for EGFP–bovMyo10; Aaron J. Marshall (University of Manitoba, Winnipeg, Canada) for EGFP–TAPP1PH; and Andew D. Paterson for assistance with microscopy. S.S. was supported by grants from the Swedish Cancer Society, the Swedish Research Council, and the Center for Biosciences at Karolinska Institutet. J.I. was supported by an ERC starting grant, Academy of Finland, Sigrid Juselius Foundation, Finnish Cancer Organizations and EMBO Young Investigator Program. A.A. is supported by the Emil Aaltonen foundation, Pertelin Aaltonen foundation, Maud Kuistila foundation and TUBS Graduate School. J.G.L is supported by a fellowship from the Swedish Cancer Society. This study was in part performed at the Live Cell Imaging Unit, Department of Biosciences and Nutrition at Karolinska Institutet, supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Center for Biosciences.
↵* These authors contributed equally to this work
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.069609/-/DC1
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You are going to email the following PtdIns(3,4,5)P3 is a regulator of myosin-X localization and filopodia formation
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This module is one of the compulsory modules on your programme that provide you with a deeper engagement with the academic and professional skills to support the preparation of your Final Project work. The module encourages you to engage directly with the range of physical and online library resources necessary to promote advanced level research skills. The module will provide opportunities to develop your academic literacy and proposal writing skills through planned workshops/lectures/seminars. Additionally the teaching helps you develop the personal and professional organisational and self-management attributes that are associated with professional practice. You will be able to participate in arranged English language sessions, as part of the University's mission to enhance academic student support.
• to develop your academic literacy skills; • to advance your understanding of research design, with appropriate methodologies aligned to your discipline; • to enable you to demonstrate a range of skills appropriate to professional situations.
contemporary issues relevant to the creative industries.
conduct research and enquiry applying appropriate research methods to data collection and reporting.
design and deliver work of a professional standard that considers relevant ethical obligations.
Cottrell, S. (2013). The Study Skills Handbook.
Easterby-Smith, M., Thorpe, R. and Jackson P. R. (2015). Management & Business Research.
Cottrell, S. (2015). Skills For Success: Personal Development And Employability.
Gray, C, & Malins, J. (2004). Visualizing Research: A Guide To The Research Process In Art And Design.
Bilton, C. and Cummings, S (2014). Handbook of Management and Creativity.


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Crank things up a notch to Adobe Premiere Pro CC and get an all-singing all-dancing video editor that's used by multitudes of industry professionals. And it's easy to see why it's so popular for Windows 10 users – it can handle an uncapped amount of video tracks, which can be imported from pretty much any source you can think of: files, tapes, cameras of all standards, and even VR. The automatic sync is a gem when you have multi-angle shots, and it's hard to fault the fine-tuning tools that really make your video stand out from the crowd.
We evaluated all of the software based on functionality, ease of use, performance, sharing options and new features in the latest versions. We also judged the packages based on real-life uses for a number of categories, including best overall, best free app, best for beginners, best for YouTube maestros, best for Mac users and best for Windows platforms.
Another feature that's new to me coming from a T3i that I love is the grid that you can have showing in the view-finder. Before you had to buy a replacement viewfinder eye-piece to get a grid and they didn't offer one for the T3i. Now it's done digitally and it's awesome. My only gripe is that you can't actually customize what grid pattern you want. But it's definitely a step in the right direction.
SaaS is straightforward to subscribe to. All you need to do is go the website and buy a suitable plan for the required number of users. For this reason, many firms pay attention only to the price of the package and not to infrastructure considerations. They may also think that careful evaluation is not necessary since it is so easy to get started quickly.
Building a DVD from the ground up can be difficult and time-consuming, and the best DVD creators makes it as easy as possible to use their features and tools. Some programs build tutorials directly into their interfaces, while others have free lessons, guides and forums on their official websites. Additionally, many of the programs we reviewed have free trials, which you can use to determine if the application suits your needs before you pay.
Other recent features include a basic audio editor, the option to make previews for apps you've developed, and the even ability to make your own movie trailers complete with transitions and end credits. It's not a tour de force in terms of video editing, but it's perfectly suitable for home videos and minor projects. Consider combining it with free audio recording software!
Video editing software can be used by amateurs and professionals alike to edit video files for fun or commercial purposes. There are numerous free and paid platforms in this category available in the market. Each offers its own set of features that can be easy or difficult to use depending on your level of expertise. So, how do you do knowledgeable comparisons to select the best video editing software for your specific needs? We help you in this task by providing details on the standard features you can expect in this type of system and the other relevant factors you should consider while searching.
4k and gif support are boilerplate features for most video editing products today, but one thing Filmora does particularly well is titles. Title tools are trending in video software, and while Filmora's doesn't have the functionality of say, an Apple Final Cut Pro X, which can superimpose 3D titles over your videos and rotate them on three axes, it nonetheless has some snazzy titling features for the money you're spending.
True. The 80D "only" shoots up to 1080p HD. If you want 4K, look elsewhere — if you're into landscapes or travel videography, this may matter to you. The world though is still mostly operating in 1080p. Keep in mind, 4K will multiply (significantly) your storage requirements, in addition to processing power needed to edit and render. Only you can decide if this is the time to make the jump (I still think mainstream 4K adoption is 2+ years away). I love my 4K computer monitors because fonts are razor sharp. Yet, I don't see substantive different between 80D images and those, say, from a Panasonic G7. The latter looks somewhat digital to my eyes, though it's still a fine little camera.
For the first time ever… You'll learn how to use Instagram to create a viral buzz for your products that are people who are going to go crazy for… See Instagram followers differ to Facebook users, since they will see every single post you write, bringing in traffic, and when you master a few golden never before seen secrets, you'll be MILES AHEAD of the competition
The official price for his product is $21.95 for now. Moreover, if you get this now, you can receive attractive and valuable bonuses from the vendors. So, you'd better hurry up because this deal will soon be expired once the launching days end. And the app will be live launched on 27th April, 2017, so I want to make sure in my Intro Video Creator Review that you don't miss this chance. Hurry up!
Since 2008, Top Ten Reviews has reviewed, tested and ranked the best DVD authoring software. In that decade, we've clocked hundreds of hours editing video, building discs and watching the end results. At the end of our most recent evaluation, PowerDirector was our top choice – this wasn't a surprise since it has been our favorite since we started reviewing this type of software. The program offers great tools for both beginners and veteran users. It's important to understand that this application is primarily a video editor, but it's disc authoring tools are as good as or better than those found in programs exclusively designed for burning DVDs.
FinancesOnline is available for free for all our business professionals interested in an efficient way to find top-notch SaaS solutions. We are able to keep our service free of charge thanks to cooperation with some of the vendors, who are willing to pay us for traffic and sales opportunities provided by our website. Please note, that FinancesOnline lists all vendors, we're not limited only to the ones that pay us, and all software providers have an equal opportunity to get featured in our rankings and comparisons, win awards, gather user reviews, all in our effort to give you reliable advice that will enable you to make well-informed purchase decisions.
First, I already kind of mentioned, but the grid view inside the viewfinder, they should give you options and the "thirds" grid should definitely be an option as the "rule of thirds" is a very good guide to follow in the absence of a clearer way to frame a photo. I hope in their future cameras they will have this. Or if somehow a firmware upgrade could add this, I'm not sure how hard-wired this grid is in the viewfinder if moving the lines would even be possible through software...
One of the things I really like about working in Movie Maker is that most of the effects, transitions, and themes preview automatically when you just hover the mouse cursor over their buttons. Another plus is the undo and redo buttons are right up top—video editing is a very trial-and-error process—but I suppose it's too much to ask for a history window in such a simple application.
That's assuming you're just exporting files. You may want to burn a DVD or Blu-ray disc, or upload your videos directly to YouTube or Facebook. Each of these comes with its own set of necessary features that some apps have and others don't. For disc burning, you need not only support for the right formats, but DVD menu authoring tools so viewers can navigate what you're presenting. On the social side, it's much easier if the application syncs up directly with your account online and allows you to enter metadata like a description, tags, and privacy settings.
You'll find the familiar timeline/storyboard workflow, along with other basic tools such as titles and credits, effects and transitions, and a chroma-key (green screen). It also has some, but not all, of the advanced tools you need to make an outstanding video. You'll get a video stabilizer to smooth out shaky footage, 4K compatibility for footage from the newest cameras, 3D editing, motion tracking and more. These are all great tools, and it's a great bang for your buck. But there are some significant trade-offs. For example, there are no themes or templates. So, you'll need to build every project from scratch – not very appealing to beginners. It also lacks an automatic video creator and slideshow creator, which are also great for novices. This program lacks multi-cam and 360-degree editing features that you would find on more expensive programs. If you want the newest, greatest effects and tools, this is not the program for you. In addition, the program's interface is in bad need of a face-lift, and has been for years. It's kind of hard to get around, and it will require working through a few projects before you can use it effectively.
Other measures of performance include startup time and simple stability. Again, video editing is a taxing activity for any computer, involving many components. In the past, video editing programs took longer than most other apps to start up, and unexpected shutdowns were unfortunately common, even in top apps from top developers such as Adobe and Apple. The stability situation has greatly improved, but the complexity of the process, which increases as more powerful effects are added, means crashes will likely never be fully eliminated, and they often raise their ugly heads after a program update, as I found with the latest version of Pinnacle Studio.
Several of the products here (Adobe Premiere Elements is a notable exception) still support 3D video editing if that's your thing, though the this has been replaced by 360-degree VR footage like that shot by the Samsung Gear 360 as the current home-theater fad. As is often the case, our Editors' Choice, CyberLink PowerDirector was the first product in this group to offer support for this new kind of video media.
I have used Movie Maker mostly for small projects, prototypes and home movies. It is a great and simple tool that meets 75% of most users needs. It is not advanced, so not many professional movie maker options are available (like color correction, white balancing, etc.) However, for basic video that you'll probably want to upload to you tube, this is a pretty good tool. Love the simple and easy to use interface.
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If you had access to the video editing pro level, then you must try DaVinci Resolve 14. Except for multi-camera editing, 3D editing, motion blur effects, and spatial noise reduction which are only available on the paid version DaVinci Resolve Studio, you can almost do any professional video/audio editing and color correction with DaVinci Resolve 14.
Another impressive effect that has made its way into consumer-level video editing software is motion tracking, which lets you attach an object or effect to something moving in your video. You might use it to put a blur over the face of someone you don't want to show up in your video. You specify the target face, and the app takes care of the rest, tracking the face and moving the effect to follow it. This used to be the sole province of special effects software such as Adobe After Effects. Corel VideoStudio was the first of the consumer products to include motion tracking, and it still leads the pack in the depth and usability of its motion-tracking tool, though several others now include the capability.
---New Version 2018 Welcome to the first update of 2018. Fixed a bug where app crashed while selecting multi music. We appreciate your feedback sent to us and now we have come up with those changes that you have requested. All the bugs have been fixed and most of the advertisement gone now.! So do take a look at this version and please send us your feedback again! - New and completely refreshed interface now! - .MOV and .WMV support added. - You can merge or join two videos easily from the home page itself. - You can also trim videos, and custom resize and quality options available like low medium and high. - Solved the bug when you save the application. - Individual thumbnails when you trim a video. - You can also remove the trimmed videos and add again. - You can share your saved videos now on Instagram. Bug Fixed: * Bugs fixed while adding text. * Bug Fixed, when after video is trimmed sound of video is disabled * Black Screen bug fixed while adding music in slideshow Please email us your feedback or you would like any other feature to videopixstore@gmail.com
FinancesOnline utilizes our proprietary SmartScore™ method to evaluate all the solutions in the Video Editing Software category in orded to help you decide on the best possible service. It evaluates the following factors: cruscial components, collaboration capabilities, custom elements, available integrations, user friendliness, customer support, security, mobile support, ratings in media. At the moment, Final Cut Pro is the leader in this category and our experts are really satisfied with it. After extensive research and analysis it had the best results among its competitors and our reviewers strongly suggest that you consider it as one of the options for your needs.
I like that Windows Movie Maker makes my life easier and helps me earn a few extra dollars. And how? Well, as it´s super easy to use, I only have to drag and drop a video (with no audio) inside the panel of Movie Maker. After this, I just add a few special effects from the upper menu, and finally, I add audio (that contains subliminal affirmations) in an easy way by using again the drag-and-drop function that this software allows. Everything I´ve just said is what I like about Windows Movie Maker.
BTW, for whatever the reason, Canon still does not include a lens hood with the bundle. A bit odd that. So factor in about $35 for that (note that the hood from the older 18-135mm lens included with the 70D won't work, you'll need the EW-73D). Plus don't forget you should really add a UV filter to protect your lens. I recommend a 67mm B+W (Hoya is good too) which won't break the bank.
Hay un par de cosas que no me gustan tanto: lo primero es que algunos videos cortos (menos de un par de mega bytes) pueden retrasar el software, y te llevará mucho tiempo. para terminar de subirlos a Windows Movie Maker. Por ejemplo, a veces, cargo un video corto para editar, y está listo en unos segundos. Pero luego, cargo otro video más corto para editar, pero esto lleva mucho tiempo para estar listo. Lo último que no me gusta es que el software deja de funcionar algunas veces.
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redpajama_set_name: RedPajamaCommonCrawl


1 The church group East Dallas Persistent Women — Re: "Women dig for health care data," by Jacquielynn Floyd, April 8 Metro & State column.
Kudos to the women's church group East Dallas Persistent Women and Jacquielynn Floyd for bringing the group and its findings to light. One has to admire these competent women for actually studying and reporting on what amounts to the state of Texas providing a bogus health care program for low-income women.
Hopefully, voters will realize and remember Gov. Greg Abbott and his group of lawmakers' feeble attempt to close the gap on low-income women's health care that they created by imposing hospital-standard regulations on Planned Parenthood clinics.
This group's findings highlight the Republicans' view on regulations, birth control and women's rights. Regulations, which they abhor for business, are good when it involves women's health and their bodies. The information on replacement services are full of outdated information regarding contact numbers, services provided and locations. It's disgraceful! Please help hold these lawmakers accountable.
2 Two police officers — My husband and I were having problems with someone parking vehicles in front of my neighbor's house and our house and leaving them for days. I finally decided to call 911 and report the problem. Police officers M. Cantu and M. Evans from the Northwest Substation responded. They were most helpful and understanding of our problem. I also mentioned my elderly widowed neighbor was having a problem with her garage door and could they help. They were more than happy to assist.
Not enough credit is given to all the wonderful things our police officers do that are above and beyond the call of duty and never mentioned.
3 A kind stranger — One Friday during the noon hour I witnessed a "living angel."At the corner of Alpha and Inwood roads there was a young woman helping an injured young man sitting and asking for donations from motorists. On his leg was a bloody bandage.
She was helping him remove his thongs and seeing if his new shoes fit. In her hand was a DSW bag. She had bought new shoes and socks for him, and he was putting them on.
It was a cold day. She saw his need. She went out of her way to help this disadvantaged man. She, a young, pony-tailed brunette, was a picture of compassion, and it brought me to tears. No way to know who she was, but may God bless her.


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Virgin Atlantic Recruiting Portuguese Speaking Cabin Crew (Based in London)
Photo Credit: Virgin Atlantic
French Flight Attendants to Protest in Paris on 16th September Over Pension Reform Plans
Next year, Virgin Atlantic will launch its first-ever route to South America with a daily service to the Brazilian economic powerhouse of São Paulo and in preparation, the airline is now recruiting Portuguese speaking cabin crew. The daily service will be operated by a Boeing 787 Dreamliner configured with 31 upper class, 35 premium and 192 economy seas, and is set to begin operations at some point in the first quarter.
Virgin Atlantic's foray into the South American market was announced earlier this year where executives said they were hoping to tap into Brazil's fast-growing economy with key corporate customers, a healthy cargo demand and a full suite of products from the profit-making side of the business Virgin Holidays.
"São Paulo represents an incredible opportunity for our business, and we're very excited to be flying to a brand new continent for the first time," explained Juha Jarvinen, Virgin's executive vice president of Commercial.
"There are a significant number of corporate customers, and multi-national businesses, located in this Powerhouse of South America who can now enjoy the Virgin Atlantic experience on this busy international route."
Virgin will be going into direct competition with both British Airways and LATAM who both operate daily flights between the two cities.
Cabin crew recruited for this opportunity will mainly work on the 11 hour 55 minute flight between London Heathrow and São Paulo where they'll help break down cultural and language barriers that can sometimes exist. However, as this position is based in London, language speakers may also find themselves operating on any of Virgin Atlantic's route network.
Applications need to be submitted by 26th August. Further information can be found here.
Virgin Atlantic recently closed an opportunity for standard cabin crew and it's expected that successful candidates will be placed on training courses beginning the end of this year and into early 2020. The airline is unlikely to reopen the application window until everyone on the waitlist has completed training.
Mateusz Maszczynski 19th August 2019
Norwegian Gets $246 Million Cash Boost by Selling Stake in Bank


Posted June 11, 2018 by Dave Kachele & filed under Blog, Letting the Living Water Flow.
I came to know the Lord when no one else around but Him and me. The day after hearing His Voice within and giving my life to Jesus without restriction He sent a Christian mailman to my house. I guessed that he was a Christian because he wore a huge cross around his neck. I'd known him as a neighbor from my childhood.
Ed was speaking Christianese, but we finally figured it out and he got me going to a Jesus loving, people loving, Bible centered fellowship. After sharing what had happened to me a few times someone finally told me, "Cool Man! You got saved!" By then, I'd learned enough Christian lingo to know what she meant.
How do you talk to someone who knows nothing or little about Jesus or Christianity? How do you talk with someone who has a lot of misconceptions about the Lord? How do you explain, "If you're born only once you die twice – physical and spiritual death, but if you're born twice you only die once – physical death," without speaking a language they don't understand. Old 'Nick' didn't get it, so Jesus explained in language that he could understand.(John 3:1-21).
John, the Gospel writer was following up on something he began to explain earlier in his record of the life of Jesus.
The Gospel is not easy for people to understand. And even when they do understand, they have a hard time repenting and following this simple formula.
How can you explain to someone that they can be sure that they will only die a physical death in language that they can understand and receive?
First, LISTEN to them. We are often so eager to share Jesus that we just begin rattling away before we know anything about a person's journey, thoughts and/or feelings. Or, we're afraid to share the Gospel at all because we don't know what to say. When God opens a door to witness for Him begin by asking questions and LISTENING to answers. Then, you have a track to run on.
TALK in language that the person can understand. Reply clearly. Do your best to steer clear of Christianese, and explain clearly terms that can't be avoided.
LET your Partner, the Holy Spirit, lead. You will plant a seed, water a seed already there, or reap the harvest. Remember, God is the Master of the Harvest!
COMMIT to the person with whom you are sharing. If you plant a seed or water, you may have other opportunities to share. Don't push for a slam dunk – a new birth. Develop a trusting friendship, and be ready to water some more, or reap the harvest. If the person does receive Jesus they will need nurturing. I have people that I've been mentoring for years, one on one and in groups.
Stay BIBLE CENTERED. Don't sell a cheap Gospel without repentance (a word you may need to avoid or explain as "to stop doing things that the Bible says are wrong and start doing things that would please God). Jesus preached …Repent and believe the Gospel. (Mark 1:15) Yes, 'God Has a Wonderful Plan for Everyone's Life,' but there is a cross to carry daily. Share in plain language what that means. Don't be afraid to challenge your new believer. People are looking for something bigger than themselves to believe and commit to.
MODEL a fully devoted life. This includes humbly and openly confessing and repenting in the sight of your mentee. How else will they know how?
Remember that Jesus showed clearly that a person who is born twice will only die once. He rose from the grave with an indestructible body and showed Himself alive to hundreds of people over a period of nearly a month and a half! We are not sharing some pipe dream! If you're privileged to lead someone to Christ, your newly born mentee will come by faith, just like you did. But, as you help him or her grow into maturity, his or her faith will grow into an assured knowing! Jesus is returning soon to bring His own home! Let's take some precious people with us!
Thank you Hanan! If you haven't done so yet, please preview the 50 day journey, "THE RELUCTANT EVANGELIST: Embrace Your Gifts and Fulfill God's Purpose." You'll be able to do so, and purchase if you decide, from this website. See you on Facebook!
Thank you Mbere Jacob! Yes, like Nicodemas. If you haven't done so yet, please preview the 50 day journey, "THE RELUCTANT EVANGELIST: Embrace Your Gifts and Fulfill God's Purpose." You'll be able to do so, and purchase if you decide, from this website. See you on Facebook!


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Penguin Computing Launches Consultancy for Piecing AI Strategies Together
AI stands before the HPC industry as a beacon of great expectations, yet market research repeatedly shows that AI adoption is commonly stuck in the talking phase, on the near side of a difficult chasm to cross. In response, many vendors have built integrations of their server and storage platforms with AI hardware – usually Nvidia GPUs – designed to leapfrog the initial steps involved in AI implementations.
Penguin Computing this week announced its own market strategy for helping organizations jumpstart their AI journeys, launching a new AI practice that the company says will operate as a full-service consultancy, providing guidance on system design, building custom technology solutions, and delivering professional services and support.
In so doing, a senior strategist at Penguin Computing also looked ahead to a future in which GPUs may be augmented with special purpose inference processors as chips of choice for AI workloads (see below). He also discussed, in light of the widely shared observation that "AI is the new HPC workload," the significantly different demands AI and traditional HPC workloads place on the processor.
About its new AI consultancy, Penguin Computing CTO Philip Pokorny told HPCwire, "We help customers build networks, rack layouts, and assist with figuring out the best deployment strategy, so they can focus on doing AI and not have to worry about the details."
Since Penguin Computing was acquired by SMART Global Holdings in June, the newly formed subsidiary has been on a mission to double down on AI initiatives, said Pokorny, who leads the new AI consultancy. He added that the acquisition by SMART can provide financial resources that would help grow the Penguin-Computing-on-Demand service, a bare metal HPC cloud that is planned to be part of the menu of offerings provided by the AI practice.
Penguin Computing is known for its versatile product set that includes Linux servers, turnkey HPC clusters, as well as managed and hosted solutions. In addition to the aforementioned Penguin-Computing-on-Demand cloud service, the company also offers a managed service (on-prem at the customer site or collocated in Penguin Computing's datacenters), available through its professional services arm.
"It's that customizability of solutions and access to a broad base of technologies that we think will make our AI practice really valuable to customers who probably don't have a lot of experience – or if they do have the experience, decide their time is better spent focusing on AI," said Pokorny.
"We're not announcing a point product and we're not saying that we have a one-size-fits-all solution. What we're saying is that if you want a solution customized to your needs, we have the expertise to build those customized solutions and we have the menu of capabilities to tailor that to each individual user's needs."
With its 20-year history as a turnkey Linux system builder, Penguin Computing's objective is to set up its HPC and AI customers with the right infrastructure. The AI landscape is evolving fast and there are dozens of frameworks and libraries in the mix. Penguin Computing doesn't want to tell customers what would work best for them or what framework they should optimize around. "We want to emphasize to customers they are free to choose whatever tool chain works for them, but we will have the experience in our AI practice to say, if you've chosen a given framework, then that restricts some choices you have in terms of operating system, fabric, and so on. We have the expertise to say, given your choices, this is what will work best," said Pokorny.
Pokorny said Penguin Computing's more popular hardware configurations for AI workloads include 4-GPU and 8-GPU servers, and it's a certified reseller of the Nvidia DGX-1 system. He noted the company has done remarkably well deploying a large number of Nvidia DGX-1s and has seen its revenue attributable to Nvidia grow significantly over the past few years.
GPUs are currently a primary means of accelerating AI training and inference workloads, and Penguin Computing provides a number of GPU-centered options. But it is also exploring other architectures for AI, such as GraphCore and RISC-V-based silicon. Graphcore's first card, based on its 16nm "Colossus" microarchitecture, is reportedly coming soon and Penguin Computing is in line to receive samples.
RISC-V is an open, free instruction set architecture based on established reduced instruction set computing principles. Penguin Computing bought one of the first stand-alone RISC-V Linux machines from SiFive earlier this year and has put some of them in the hands of its government customers, who are similarly interested in forward-looking CPU architectures. Pokorny sees RISC-V as "an open CPU architecture with the potential to dramatically lower costs and potentially give users access to specialized instructions for novel new architectures that could be enabled by not having a licensing tax."
While these newer architectures don't optimize for double-precision, the mainstay of HPC, that is not a mandate that the AI space shares. "When you look at the direction that tensor cores are going and the way that Nvidia is changing their architectures, double-precision is not where it's at with respect to training, it's all about smaller and smaller data sizes. It's still very important in high-performance computing," said Pokorny. He is open to the possibility, however, that the pattern-matching of AI could potentially replace or augment first principles physics-based simulations.
"There's been a question posed but I don't think it has been answered yet, in the HPC community," said Pokorny, "which is that if you think of a baby, a baby learns about gravity by falling down and watching balls go up in the air and come down and roll down hill, and it doesn't learn about gravity from force equals mass times acceleration and studying physics. So historically, we have tried to figure out what are the equations that underpin physics and then use those equations to build models that allow us to simulate physical phenomena. We do that starting from the ground up and it tends to be very expensive in floating-point cycles and fidelity. So the question that has been posed is, 'Couldn't we train a neural network to discover the rules of combustion or discover the rules of gravity by giving it a bunch of training sets and then asking it to tell us what would happen in a different scenario?'
"If it turns out that that actually works and is actually more efficient, you could imagine that could really alter the way we do HPC in the future. It opens an avenue to being much more energy efficient because it takes fewer computations to get the same fidelity of result. But it would also change to some extent whatever we call determinism. If you ran the same problem twice, you might get two different results due to some small fluctuations, so it's an interesting problem to be stated and answered by the HPC community with regard to artificial intelligence."
Pokorny also holds the view that while current GPUs are a dominant architecture for today's most visible AI workloads, they may not continue that supremacy across the broader swath of AI coming out of the shadows. "If you take the example of say risk assessment – credit card risk and identifying fraudulent transactions – that was to some extent AI before AI was cool and as far as I know that work is done on traditional CPUs," he told us. "You also have things like high-frequency trading which involve machines making decisions about recognizing patterns and then training on those patterns. To some extent that's also AI before AI was cool."
Pokorny is keen on the moves that Intel is making around FPGAs integrating into CPU sockets and points to Microsoft Azure's dramatically speeding up its Bing search engine with FPGAs. "An FPGA has the promise of being a reconfigurable pile of hardware to do exactly what you need it to do very quickly," Pokorny said. "It remains to be seen whether the cost is tractable, whether the performance pays off against the cost. But I also think that we're seeing a global accessibility to ASICs to some extent strangely driven by the bitcoin miners. We're seeing developments around making toolchains more approachable."
Pokorny's takeaway message: AI deserves the hype. "I don't think it's going to be as disillusioning as maybe we've seen in prior generations of artificial intelligence where we thought AI was just around the corner for the last 30 years. I think people are approaching this a lot more realistically. I think we have [done so] at Penguin Computing, and so the net of that is, I think commercial and Fortune 500 companies are going to be making more use of this to improve efficiencies and augment processes and do a better job of suggesting things. And it's going to be really interesting to see in what ways AI transforms a lot of different workflows by being able to recognize patterns that maybe people weren't able to recognize before."
— Doug Black contributed to this report.


Whether you are a vegetarian or a meat lover, we all can agree that grilled food tastes great! Take a moment to browse through our hand selected recipes and try one out next time you fire up your grill. Along with the natural wood flavor of GrillEasy™ FireQube, these easy recipes guarantee mouth watering results.
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