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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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Rabankyrin-5 knock-down drives the elongation of apical endocytic tubes.(a) Rabankyrin-5 knock-down results in the elongation of endocytic tubules along the apico-basal axis. Single-plane two-photon optical cross-section of a Rabankyrin-5 siRNA embryo expressing sec::GFP showing a typical, elongated, sec::GFP-positive tubule (red arrow). Scale bar, 5 μm. (b) Ultrastructural characterization of an elongated endocytic tubule in a Rabankyrin-5 siRNA embryo. A single section from an EM tomogram of a Rabankyrin-5 siRNA embryo showing a typical elongated tubule that extends past the base of the nucleus (blue arrow indicates a nuclear pore at the base of the nucleus). Scale bar, 500 nm. (c–f) Ultrastructural characterization of elongated endocytic tubules in Rabankyrin-5 siRNA embryos. (c–e) Correlative light-EM of a basal sec::GFP structure in direct continuity with the elongated tubule shown in panel b. Fluorescence image of an EM section showing sec::GFP fluorescence (green) (c), a fluorescence/EM overlay (d) and a section from an EM tomogram of the same volume (e). Scale bar, 1,000 nm. (f) High-magnification tomographic section corresponding to panel e showing interconnected vesicular varicosities (red arrows that extend from the base of the elongated tubule shown in panel b. Vesicular varicosities of the elongated tubule exist in close proximity to microtubules (green arrow). Note, the sec::GFP-positive vesicular structure marked by the red arrows in c–e correspond to the basal vesicular varicosity marked by the red arrow in panel b. Scale bar, 200 nm.
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f6: Rabankyrin-5 knock-down drives the elongation of apical endocytic tubes.(a) Rabankyrin-5 knock-down results in the elongation of endocytic tubules along the apico-basal axis. Single-plane two-photon optical cross-section of a Rabankyrin-5 siRNA embryo expressing sec::GFP showing a typical, elongated, sec::GFP-positive tubule (red arrow). Scale bar, 5 μm. (b) Ultrastructural characterization of an elongated endocytic tubule in a Rabankyrin-5 siRNA embryo. A single section from an EM tomogram of a Rabankyrin-5 siRNA embryo showing a typical elongated tubule that extends past the base of the nucleus (blue arrow indicates a nuclear pore at the base of the nucleus). Scale bar, 500 nm. (c–f) Ultrastructural characterization of elongated endocytic tubules in Rabankyrin-5 siRNA embryos. (c–e) Correlative light-EM of a basal sec::GFP structure in direct continuity with the elongated tubule shown in panel b. Fluorescence image of an EM section showing sec::GFP fluorescence (green) (c), a fluorescence/EM overlay (d) and a section from an EM tomogram of the same volume (e). Scale bar, 1,000 nm. (f) High-magnification tomographic section corresponding to panel e showing interconnected vesicular varicosities (red arrows that extend from the base of the elongated tubule shown in panel b. Vesicular varicosities of the elongated tubule exist in close proximity to microtubules (green arrow). Note, the sec::GFP-positive vesicular structure marked by the red arrows in c–e correspond to the basal vesicular varicosity marked by the red arrow in panel b. Scale bar, 200 nm.

Mentions: The data presented here demonstrate that apical surface flattening is a dynamin-dependent morphogenetic process associated with an upregulation of tubular-endocytic intermediates and increasing levels of Rab5-positive endosomes. To identify proteins potentially involved in Rab5-positive endosome biogenesis, we carried out large-scale affinity chromatography and purified Rab5-specific effectors operating during these early stages of embryonic development. This experiment led to the identification of several proteins that were bound specifically to Rab5 in its active conformation (Fig. 5a). The most abundant effector identified by this approach was CG41099, the Drosophila homologue of human Rabankyrin-5 (Fig. 5a), a Rab5 effector linked to apical endocytosis in polarized MDCK cells29. In addition, Rabankyrin-5 has been shown to regulate macropinocytosis, a distinct form of endocytosis that involves the formation of large (0.2–10 μm) vesicular/vacuolar structures29. We therefore tested whether tubular endocytosis at the apical surface is regulated by Rabankyrin-5 through its interaction with Rab5. Simultaneous imaging of EGFP::Rabankyrin-5 and sec::mCherry revealed that Rabankyrin-5 associates with apical vacuolar structures positive for sec::mCherry (Fig. 5b–d). This result was confirmed using an antibody against Drosophila Rabankyrin-5 (Supplementary Fig. S4a). Moreover, correlative light-electron microscopy revealed that EGFP::Rabankyrin-5-positive membranes are organized as convoluted tubular structures (Fig. 5e–g). Live imaging of control embryos demonstrated that vacuolar structures formed at the apical plasma membrane and moved towards the basal side of the cell with long-range movements, which are presumably microtubule dependent. Conversely, in embryos expressing short hairpin RNAs against Rabankyrin-5 (the knock-down of Rabankyrin-5 was almost complete at both mRNA and protein levels, Supplementary Fig. S4b,c) this step was impeded resulting in the formation of long tubular membranes extending for over 15 μm along the apico-basal axis of the cell (Fig. 6a and Supplementary Fig. S4d–m and Supplementary Movies 8,9). Using correlative light-electron microscopy, we tracked one of these long tubular-endocytic membranes filled with endocytic cargo (Fig. 6b). This long tubule extended parallel to microtubules and its basal tip appeared composed of multiple varicosities interconnected by a constricted membrane domain (Fig. 6f and the tomogram depicted in Supplementary Movie 10). We speculate that, at the functional level, this terminal region may serve as a platform for the budding and generation of vacuoles, in a process equivalent to the formation of vacuoles from the shorter tubular invaginations seen in wild-type embryos (Fig. 2c–f). In support of this hypothesis, we observed the sequential budding of vacuoles from the tip of elongated tubes by live imaging in Rabankyrin-5-depleted embryos (Supplementary Movie 9). The elongation of tubular-endocytic membranes induced upon Rabankyrin-5 knock-down revealed that one role of the Rab5 machinery in this apical endocytic pathway is to control budding, and that other molecule/s must act upstream of Rabankyrin-5 in the initiation of tubular endocytosis and surface flattening.


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)

Rabankyrin-5 knock-down drives the elongation of apical endocytic tubes.(a) Rabankyrin-5 knock-down results in the elongation of endocytic tubules along the apico-basal axis. Single-plane two-photon optical cross-section of a Rabankyrin-5 siRNA embryo expressing sec::GFP showing a typical, elongated, sec::GFP-positive tubule (red arrow). Scale bar, 5 μm. (b) Ultrastructural characterization of an elongated endocytic tubule in a Rabankyrin-5 siRNA embryo. A single section from an EM tomogram of a Rabankyrin-5 siRNA embryo showing a typical elongated tubule that extends past the base of the nucleus (blue arrow indicates a nuclear pore at the base of the nucleus). Scale bar, 500 nm. (c–f) Ultrastructural characterization of elongated endocytic tubules in Rabankyrin-5 siRNA embryos. (c–e) Correlative light-EM of a basal sec::GFP structure in direct continuity with the elongated tubule shown in panel b. Fluorescence image of an EM section showing sec::GFP fluorescence (green) (c), a fluorescence/EM overlay (d) and a section from an EM tomogram of the same volume (e). Scale bar, 1,000 nm. (f) High-magnification tomographic section corresponding to panel e showing interconnected vesicular varicosities (red arrows that extend from the base of the elongated tubule shown in panel b. Vesicular varicosities of the elongated tubule exist in close proximity to microtubules (green arrow). Note, the sec::GFP-positive vesicular structure marked by the red arrows in c–e correspond to the basal vesicular varicosity marked by the red arrow in panel b. Scale bar, 200 nm.
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Related In: Results  -  Collection

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Show All Figures
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f6: Rabankyrin-5 knock-down drives the elongation of apical endocytic tubes.(a) Rabankyrin-5 knock-down results in the elongation of endocytic tubules along the apico-basal axis. Single-plane two-photon optical cross-section of a Rabankyrin-5 siRNA embryo expressing sec::GFP showing a typical, elongated, sec::GFP-positive tubule (red arrow). Scale bar, 5 μm. (b) Ultrastructural characterization of an elongated endocytic tubule in a Rabankyrin-5 siRNA embryo. A single section from an EM tomogram of a Rabankyrin-5 siRNA embryo showing a typical elongated tubule that extends past the base of the nucleus (blue arrow indicates a nuclear pore at the base of the nucleus). Scale bar, 500 nm. (c–f) Ultrastructural characterization of elongated endocytic tubules in Rabankyrin-5 siRNA embryos. (c–e) Correlative light-EM of a basal sec::GFP structure in direct continuity with the elongated tubule shown in panel b. Fluorescence image of an EM section showing sec::GFP fluorescence (green) (c), a fluorescence/EM overlay (d) and a section from an EM tomogram of the same volume (e). Scale bar, 1,000 nm. (f) High-magnification tomographic section corresponding to panel e showing interconnected vesicular varicosities (red arrows that extend from the base of the elongated tubule shown in panel b. Vesicular varicosities of the elongated tubule exist in close proximity to microtubules (green arrow). Note, the sec::GFP-positive vesicular structure marked by the red arrows in c–e correspond to the basal vesicular varicosity marked by the red arrow in panel b. Scale bar, 200 nm.
Mentions: The data presented here demonstrate that apical surface flattening is a dynamin-dependent morphogenetic process associated with an upregulation of tubular-endocytic intermediates and increasing levels of Rab5-positive endosomes. To identify proteins potentially involved in Rab5-positive endosome biogenesis, we carried out large-scale affinity chromatography and purified Rab5-specific effectors operating during these early stages of embryonic development. This experiment led to the identification of several proteins that were bound specifically to Rab5 in its active conformation (Fig. 5a). The most abundant effector identified by this approach was CG41099, the Drosophila homologue of human Rabankyrin-5 (Fig. 5a), a Rab5 effector linked to apical endocytosis in polarized MDCK cells29. In addition, Rabankyrin-5 has been shown to regulate macropinocytosis, a distinct form of endocytosis that involves the formation of large (0.2–10 μm) vesicular/vacuolar structures29. We therefore tested whether tubular endocytosis at the apical surface is regulated by Rabankyrin-5 through its interaction with Rab5. Simultaneous imaging of EGFP::Rabankyrin-5 and sec::mCherry revealed that Rabankyrin-5 associates with apical vacuolar structures positive for sec::mCherry (Fig. 5b–d). This result was confirmed using an antibody against Drosophila Rabankyrin-5 (Supplementary Fig. S4a). Moreover, correlative light-electron microscopy revealed that EGFP::Rabankyrin-5-positive membranes are organized as convoluted tubular structures (Fig. 5e–g). Live imaging of control embryos demonstrated that vacuolar structures formed at the apical plasma membrane and moved towards the basal side of the cell with long-range movements, which are presumably microtubule dependent. Conversely, in embryos expressing short hairpin RNAs against Rabankyrin-5 (the knock-down of Rabankyrin-5 was almost complete at both mRNA and protein levels, Supplementary Fig. S4b,c) this step was impeded resulting in the formation of long tubular membranes extending for over 15 μm along the apico-basal axis of the cell (Fig. 6a and Supplementary Fig. S4d–m and Supplementary Movies 8,9). Using correlative light-electron microscopy, we tracked one of these long tubular-endocytic membranes filled with endocytic cargo (Fig. 6b). This long tubule extended parallel to microtubules and its basal tip appeared composed of multiple varicosities interconnected by a constricted membrane domain (Fig. 6f and the tomogram depicted in Supplementary Movie 10). We speculate that, at the functional level, this terminal region may serve as a platform for the budding and generation of vacuoles, in a process equivalent to the formation of vacuoles from the shorter tubular invaginations seen in wild-type embryos (Fig. 2c–f). In support of this hypothesis, we observed the sequential budding of vacuoles from the tip of elongated tubes by live imaging in Rabankyrin-5-depleted embryos (Supplementary Movie 9). The elongation of tubular-endocytic membranes induced upon Rabankyrin-5 knock-down revealed that one role of the Rab5 machinery in this apical endocytic pathway is to control budding, and that other molecule/s must act upstream of Rabankyrin-5 in the initiation of tubular endocytosis and surface flattening.

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