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Ena drives invasive macrophage migration in Drosophila embryos.

Tucker PK, Evans IR, Wood W - Dis Model Mech (2010)

Bottom Line: Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia.However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena.We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.

ABSTRACT
It is seldom the primary tumour that proves fatal in cancer, with metastasis the fundamental pathological process for disease progression. Upregulation of Mena, a member of the evolutionarily conserved Ena/VASP family of actin cytoskeletal regulators, promotes metastasis and invasive motility of breast cancer cells in vivo. To complement in vitro studies of Ena/VASP function in fibroblasts, we manipulated levels of Ena, the Drosophila homologue of Mena, in migrating embryonic macrophages (haemocytes). Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia. However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena. We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

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Related in: MedlinePlus

Ena localises to the tips of filopodia and the leading edge of lamellipodia in vivo. Haemocytes co-expressing Ena-GFP (green) and mCherry-Moesin (red) were imaged at stage 15 of embryonic development. (A) Ena-GFP localises to the tips of filopodia (arrowhead) and the leading edge of lamellipodia (bracket) in haemocytes at the midline. (B) Ena-GFP at a filopodial tip during extension and retraction (see supplementary material Movie 1 for an example of Ena-GFP being lost during retraction). (C′) Kymograph generated along the axis of protrusion (solid line in C), demonstrating the presence of Ena-GFP at the leading edge during lamellipodial extension and its loss from this site upon retraction (arrowheads); the dotted line indicates the time point in the kymograph corresponding to the still image in C. (D) Ena-GFP is present at the lamellipodial leading edge during lateral migration (see supplementary material Movie 2 for corresponding time-lapse). Dots and lines reveal progress of the haemocyte when moving from the midline to the edge of the VNC. (E) Images showing the GFP channel of the haemocyte from D; in these images Ena-GFP is clearly localised at the lamellipodial leading edge (arrowheads). Scale bars in C’ represent 2 μm (vertical) and 60 seconds (horizontal); all other scale bars represent 10 μm.
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f1-0040126: Ena localises to the tips of filopodia and the leading edge of lamellipodia in vivo. Haemocytes co-expressing Ena-GFP (green) and mCherry-Moesin (red) were imaged at stage 15 of embryonic development. (A) Ena-GFP localises to the tips of filopodia (arrowhead) and the leading edge of lamellipodia (bracket) in haemocytes at the midline. (B) Ena-GFP at a filopodial tip during extension and retraction (see supplementary material Movie 1 for an example of Ena-GFP being lost during retraction). (C′) Kymograph generated along the axis of protrusion (solid line in C), demonstrating the presence of Ena-GFP at the leading edge during lamellipodial extension and its loss from this site upon retraction (arrowheads); the dotted line indicates the time point in the kymograph corresponding to the still image in C. (D) Ena-GFP is present at the lamellipodial leading edge during lateral migration (see supplementary material Movie 2 for corresponding time-lapse). Dots and lines reveal progress of the haemocyte when moving from the midline to the edge of the VNC. (E) Images showing the GFP channel of the haemocyte from D; in these images Ena-GFP is clearly localised at the lamellipodial leading edge (arrowheads). Scale bars in C’ represent 2 μm (vertical) and 60 seconds (horizontal); all other scale bars represent 10 μm.

Mentions: Mena, a mammalian homologue of Drosophila Ena, typically localises to the tips of filopodia and leading edges of lamellipodia in cells such as fibroblasts and neurons in vitro (Gertler et al., 1996; Lanier et al., 1999). Ena itself is widely expressed within Drosophila embryos; levels are particularly high within the central nervous system (CNS) (Gertler et al., 1995) and the protein is also present within haemocytes (supplementary material Fig. S1A–C). To determine Ena localisation in vivo, Ena-GFP and mCherry-Moesin were co-expressed in Drosophila haemocytes using the GAL4-UAS system (Brand and Perrimon, 1993). At stage 15 of embryonic development Ena was enriched at the tips of filopodia and lamellipodia in haemocytes (Fig. 1A; supplementary material Movie 1). Time-lapse movies of filopodial extension and retraction revealed Ena at the tips of filopodia during extension, with retention at the tip in 25% of cases (n=77) during retraction (Fig. 1B); in the remainder Ena was lost from filopodial tips upon retraction (supplementary material Movie 1). Kymographic analysis of haemocytes in the embryo indicated that Ena was enriched at lamellipodial leading edges during extension but upon retraction this localisation was lost (Fig. 1C).


Ena drives invasive macrophage migration in Drosophila embryos.

Tucker PK, Evans IR, Wood W - Dis Model Mech (2010)

Ena localises to the tips of filopodia and the leading edge of lamellipodia in vivo. Haemocytes co-expressing Ena-GFP (green) and mCherry-Moesin (red) were imaged at stage 15 of embryonic development. (A) Ena-GFP localises to the tips of filopodia (arrowhead) and the leading edge of lamellipodia (bracket) in haemocytes at the midline. (B) Ena-GFP at a filopodial tip during extension and retraction (see supplementary material Movie 1 for an example of Ena-GFP being lost during retraction). (C′) Kymograph generated along the axis of protrusion (solid line in C), demonstrating the presence of Ena-GFP at the leading edge during lamellipodial extension and its loss from this site upon retraction (arrowheads); the dotted line indicates the time point in the kymograph corresponding to the still image in C. (D) Ena-GFP is present at the lamellipodial leading edge during lateral migration (see supplementary material Movie 2 for corresponding time-lapse). Dots and lines reveal progress of the haemocyte when moving from the midline to the edge of the VNC. (E) Images showing the GFP channel of the haemocyte from D; in these images Ena-GFP is clearly localised at the lamellipodial leading edge (arrowheads). Scale bars in C’ represent 2 μm (vertical) and 60 seconds (horizontal); all other scale bars represent 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3008967&req=5

f1-0040126: Ena localises to the tips of filopodia and the leading edge of lamellipodia in vivo. Haemocytes co-expressing Ena-GFP (green) and mCherry-Moesin (red) were imaged at stage 15 of embryonic development. (A) Ena-GFP localises to the tips of filopodia (arrowhead) and the leading edge of lamellipodia (bracket) in haemocytes at the midline. (B) Ena-GFP at a filopodial tip during extension and retraction (see supplementary material Movie 1 for an example of Ena-GFP being lost during retraction). (C′) Kymograph generated along the axis of protrusion (solid line in C), demonstrating the presence of Ena-GFP at the leading edge during lamellipodial extension and its loss from this site upon retraction (arrowheads); the dotted line indicates the time point in the kymograph corresponding to the still image in C. (D) Ena-GFP is present at the lamellipodial leading edge during lateral migration (see supplementary material Movie 2 for corresponding time-lapse). Dots and lines reveal progress of the haemocyte when moving from the midline to the edge of the VNC. (E) Images showing the GFP channel of the haemocyte from D; in these images Ena-GFP is clearly localised at the lamellipodial leading edge (arrowheads). Scale bars in C’ represent 2 μm (vertical) and 60 seconds (horizontal); all other scale bars represent 10 μm.
Mentions: Mena, a mammalian homologue of Drosophila Ena, typically localises to the tips of filopodia and leading edges of lamellipodia in cells such as fibroblasts and neurons in vitro (Gertler et al., 1996; Lanier et al., 1999). Ena itself is widely expressed within Drosophila embryos; levels are particularly high within the central nervous system (CNS) (Gertler et al., 1995) and the protein is also present within haemocytes (supplementary material Fig. S1A–C). To determine Ena localisation in vivo, Ena-GFP and mCherry-Moesin were co-expressed in Drosophila haemocytes using the GAL4-UAS system (Brand and Perrimon, 1993). At stage 15 of embryonic development Ena was enriched at the tips of filopodia and lamellipodia in haemocytes (Fig. 1A; supplementary material Movie 1). Time-lapse movies of filopodial extension and retraction revealed Ena at the tips of filopodia during extension, with retention at the tip in 25% of cases (n=77) during retraction (Fig. 1B); in the remainder Ena was lost from filopodial tips upon retraction (supplementary material Movie 1). Kymographic analysis of haemocytes in the embryo indicated that Ena was enriched at lamellipodial leading edges during extension but upon retraction this localisation was lost (Fig. 1C).

Bottom Line: Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia.However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena.We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.

ABSTRACT
It is seldom the primary tumour that proves fatal in cancer, with metastasis the fundamental pathological process for disease progression. Upregulation of Mena, a member of the evolutionarily conserved Ena/VASP family of actin cytoskeletal regulators, promotes metastasis and invasive motility of breast cancer cells in vivo. To complement in vitro studies of Ena/VASP function in fibroblasts, we manipulated levels of Ena, the Drosophila homologue of Mena, in migrating embryonic macrophages (haemocytes). Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia. However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena. We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.

Show MeSH
Related in: MedlinePlus