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Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila.

Fabrowski P, Necakov AS, Mumbauer S, Loeser E, Reversi A, Streichan S, Briggs JA, De Renzis S - Nat Commun (2013)

Bottom Line: This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes.We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening.These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, Heidelberg 69117, Germany.

ABSTRACT
During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development.

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Subcellular Tracking of apical Rab5-positive endosomes reveals prominent apical biogenesis and subsequent basal descent.(a,b) Tracking analysis was performed on spinning disc confocal time-lapse series of YFP::Rab5-positive endosomes during the late phase of cellularization. All Rab5-positive structures, shown as segmented puncta in white, with an origin within 5 μm of the apical surface, and that could be detected for a minimum duration of six frames were tracked in space over time. (a) All identified tracks are shown, coded in colour by time, with the position of the furrow shown at the beginning (blue bar), middle (green bar) and end (red bar) of late/fast phase cellularization. (b) The extent of displacement from the apical surface for all tracks during the beginning, middle and end of cellularization. Coloured arrows represent the displacement of individual tracks, and point in the direction of travel over time. Blue, green and red arrows represent tracks that occur during the beginning, middle or end of late/fast phase cellularization, respectively. Yellow tracks represent Rab5-positive endosomes that persist at the apical surface. Furrow positions during the beginning, middle and end of late/fast phase cellularization are shown as blue, green and red bars, respectively. Scale bar, 20 μm. (c) Quantification of the length of apical to basal travel with respect to furrow position. The mean track displacement, in microns, for apically formed, basally descending Rab5 endosomes is shown during the beginning (blue), middle (green) and end (red) of the late/fast phase of cellularization. The mean track displacement, in microns, for apically persistent Rab5 endosomes is shown in yellow. Error bars represent the standard deviation of the mean track displacement. The blue line represents furrow position, in microns, with respect to the apical surface over time. n=17 cells.
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f4: Subcellular Tracking of apical Rab5-positive endosomes reveals prominent apical biogenesis and subsequent basal descent.(a,b) Tracking analysis was performed on spinning disc confocal time-lapse series of YFP::Rab5-positive endosomes during the late phase of cellularization. All Rab5-positive structures, shown as segmented puncta in white, with an origin within 5 μm of the apical surface, and that could be detected for a minimum duration of six frames were tracked in space over time. (a) All identified tracks are shown, coded in colour by time, with the position of the furrow shown at the beginning (blue bar), middle (green bar) and end (red bar) of late/fast phase cellularization. (b) The extent of displacement from the apical surface for all tracks during the beginning, middle and end of cellularization. Coloured arrows represent the displacement of individual tracks, and point in the direction of travel over time. Blue, green and red arrows represent tracks that occur during the beginning, middle or end of late/fast phase cellularization, respectively. Yellow tracks represent Rab5-positive endosomes that persist at the apical surface. Furrow positions during the beginning, middle and end of late/fast phase cellularization are shown as blue, green and red bars, respectively. Scale bar, 20 μm. (c) Quantification of the length of apical to basal travel with respect to furrow position. The mean track displacement, in microns, for apically formed, basally descending Rab5 endosomes is shown during the beginning (blue), middle (green) and end (red) of the late/fast phase of cellularization. The mean track displacement, in microns, for apically persistent Rab5 endosomes is shown in yellow. Error bars represent the standard deviation of the mean track displacement. The blue line represents furrow position, in microns, with respect to the apical surface over time. n=17 cells.

Mentions: Live imaging further demonstrated that upon leaving from the apical cytoplasm, Rab5 apical endosomes travel basally for ~15 μm, reaching the base of the nuclei in close proximity to the invaginating furrow (Fig. 4a,c and Supplementary Movie 7). However, as cellularization proceeds, the maximal displacement length of individual Rab5 endosomes did not increase, and, in fact, these endosomes form a stable population below the base of the nuclei (Fig. 4c). Thus, the production of Rab5 endosomes does not seem to be directly linked with furrow ingression but rather correlates with flattening of the apical surface.


Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila.

Fabrowski P, Necakov AS, Mumbauer S, Loeser E, Reversi A, Streichan S, Briggs JA, De Renzis S - Nat Commun (2013)

Subcellular Tracking of apical Rab5-positive endosomes reveals prominent apical biogenesis and subsequent basal descent.(a,b) Tracking analysis was performed on spinning disc confocal time-lapse series of YFP::Rab5-positive endosomes during the late phase of cellularization. All Rab5-positive structures, shown as segmented puncta in white, with an origin within 5 μm of the apical surface, and that could be detected for a minimum duration of six frames were tracked in space over time. (a) All identified tracks are shown, coded in colour by time, with the position of the furrow shown at the beginning (blue bar), middle (green bar) and end (red bar) of late/fast phase cellularization. (b) The extent of displacement from the apical surface for all tracks during the beginning, middle and end of cellularization. Coloured arrows represent the displacement of individual tracks, and point in the direction of travel over time. Blue, green and red arrows represent tracks that occur during the beginning, middle or end of late/fast phase cellularization, respectively. Yellow tracks represent Rab5-positive endosomes that persist at the apical surface. Furrow positions during the beginning, middle and end of late/fast phase cellularization are shown as blue, green and red bars, respectively. Scale bar, 20 μm. (c) Quantification of the length of apical to basal travel with respect to furrow position. The mean track displacement, in microns, for apically formed, basally descending Rab5 endosomes is shown during the beginning (blue), middle (green) and end (red) of the late/fast phase of cellularization. The mean track displacement, in microns, for apically persistent Rab5 endosomes is shown in yellow. Error bars represent the standard deviation of the mean track displacement. The blue line represents furrow position, in microns, with respect to the apical surface over time. n=17 cells.
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Related In: Results  -  Collection

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f4: Subcellular Tracking of apical Rab5-positive endosomes reveals prominent apical biogenesis and subsequent basal descent.(a,b) Tracking analysis was performed on spinning disc confocal time-lapse series of YFP::Rab5-positive endosomes during the late phase of cellularization. All Rab5-positive structures, shown as segmented puncta in white, with an origin within 5 μm of the apical surface, and that could be detected for a minimum duration of six frames were tracked in space over time. (a) All identified tracks are shown, coded in colour by time, with the position of the furrow shown at the beginning (blue bar), middle (green bar) and end (red bar) of late/fast phase cellularization. (b) The extent of displacement from the apical surface for all tracks during the beginning, middle and end of cellularization. Coloured arrows represent the displacement of individual tracks, and point in the direction of travel over time. Blue, green and red arrows represent tracks that occur during the beginning, middle or end of late/fast phase cellularization, respectively. Yellow tracks represent Rab5-positive endosomes that persist at the apical surface. Furrow positions during the beginning, middle and end of late/fast phase cellularization are shown as blue, green and red bars, respectively. Scale bar, 20 μm. (c) Quantification of the length of apical to basal travel with respect to furrow position. The mean track displacement, in microns, for apically formed, basally descending Rab5 endosomes is shown during the beginning (blue), middle (green) and end (red) of the late/fast phase of cellularization. The mean track displacement, in microns, for apically persistent Rab5 endosomes is shown in yellow. Error bars represent the standard deviation of the mean track displacement. The blue line represents furrow position, in microns, with respect to the apical surface over time. n=17 cells.
Mentions: Live imaging further demonstrated that upon leaving from the apical cytoplasm, Rab5 apical endosomes travel basally for ~15 μm, reaching the base of the nuclei in close proximity to the invaginating furrow (Fig. 4a,c and Supplementary Movie 7). However, as cellularization proceeds, the maximal displacement length of individual Rab5 endosomes did not increase, and, in fact, these endosomes form a stable population below the base of the nuclei (Fig. 4c). Thus, the production of Rab5 endosomes does not seem to be directly linked with furrow ingression but rather correlates with flattening of the apical surface.

Bottom Line: This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes.We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening.These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, European Molecular Biology Laboratory Heidelberg, Meyerhofstrasse 1, Heidelberg 69117, Germany.

ABSTRACT
During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development.

Show MeSH
Related in: MedlinePlus