Ars.els-cdn.com
Supplementary MaterialMaterials and MethodsDrosophila strainsThe following stocks were used: en-Gal4, da-Gal4, Oregon R, Jupiter-GFP, UAS-khc-RNAi, UAS-Rab5SN and UAS-shibireDN (Bloomington Stock Center); UAS-DE-cad-GFP and UAS-α-catenin-GFP (DGRC), UAS-ed-RNAi and UAS-α-adaptin-RNAi (VDRC); UAS-Ed-GFP (this study), UAS-Ed-mCherry (this study), UAS-DE-cad-mCherry (this study), UAS-mCherry-Rab5 (this study), UAS-mCherry-Rab11 (this study), UAS-LifeAct-mRFP (this study), shgg317 PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UZXBhc3M8L0F1dGhvcj48WWVhcj4xOTk2PC9ZZWFyPjxS
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ADDIN EN.CITE.DATA [23], UAS-Rab11DN ADDIN EN.CITE <EndNote><Cite><Author>Satoh</Author><Year>2005</Year><RecNum>1929</RecNum><DisplayText>[24]</DisplayText><record><rec-number>1929</rec-number><foreign-keys><key app="EN" db-id="war2dtppvtxr9ie9pxsvdtw2px9dtt92tzt9">1929</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Satoh, A. K.</author><author>O'Tousa, J. E.</author><author>Ozaki, K.</author><author>Ready, D. F.</author></authors></contributors><auth-address>Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.</auth-address><titles><title>Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors</title><secondary-title>Development</secondary-title></titles><periodical><full-title>Development</full-title></periodical><pages>1487-97</pages><volume>132</volume><number>7</number><edition>2005/02/25</edition><keywords><keyword>Animals</keyword><keyword>Animals, Genetically Modified</keyword><keyword>Calcium Channels/biosynthesis/genetics</keyword><keyword>Drosophila/genetics/*metabolism</keyword><keyword>Drosophila Proteins/biosynthesis/genetics</keyword><keyword>Endocytosis/physiology</keyword><keyword>Immunohistochemistry</keyword><keyword>Microscopy, Electron</keyword><keyword>Photoreceptor Cells, Invertebrate/*metabolism</keyword><keyword>Protein Transport/physiology</keyword><keyword>Rhodopsin/*metabolism</keyword><keyword>Transient Receptor Potential Channels</keyword><keyword>rab GTP-Binding Proteins/*metabolism</keyword></keywords><dates><year>2005</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>0950-1991 (Print)
0950-1991 (Linking)</isbn><accession-num>15728675</accession-num><work-type>Research Support, Non-U.S. Gov't
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ADDIN EN.CITE.DATA [27], and UAS-spastin-EGFP ADDIN EN.CITE <EndNote><Cite><Author>Jankovics</Author><Year>2006</Year><RecNum>1662</RecNum><DisplayText>[17]</DisplayText><record><rec-number>1662</rec-number><foreign-keys><key app="EN" db-id="war2dtppvtxr9ie9pxsvdtw2px9dtt92tzt9">1662</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Jankovics, F.</author><author>Brunner, D.</author></authors></contributors><auth-address>Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.</auth-address><titles><title>Transiently reorganized microtubules are essential for zippering during dorsal closure in Drosophila melanogaster</title><secondary-title>Dev Cell</secondary-title><alt-title>Developmental cell</alt-title></titles><periodical><full-title>Dev Cell</full-title><abbr-1>Developmental cell</abbr-1></periodical><alt-periodical><full-title>Dev Cell</full-title><abbr-1>Developmental cell</abbr-1></alt-periodical><pages>375-85</pages><volume>11</volume><number>3</number><edition>2006/08/16</edition><keywords><keyword>Animals</keyword><keyword>*Body Patterning</keyword><keyword>Centrosome/metabolism</keyword><keyword>Drosophila melanogaster/*embryology</keyword><keyword>Embryo, Nonmammalian</keyword><keyword>*Embryonic Development</keyword><keyword>Epithelium/embryology/metabolism</keyword><keyword>Microscopy, Fluorescence</keyword><keyword>Microtubules/*physiology</keyword><keyword>*Morphogenesis</keyword></keywords><dates><year>2006</year><pub-dates><date>Sep</date></pub-dates></dates><isbn>1534-5807 (Print)
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ADDIN EN.CITE.DATA [15]), mouse anti-Discs large (1:125; Hybridoma Bank), Alexa 594-phalloidin (1:200; Invitrogen), mouse anti-α-tubulin (1:200; Sigma), Cy3- and Cy5-conjugated secondary IgGs (1:250; Jackson ImmunoResearch Laboratories). After staining samples were mounted in 90% glycerol and nail polish was used to seal the slices. Images were acquired using 63x NA1.4 Oil Plan-Apochroma objectives on a confocal microscope (LSM510, Carl Zeiss, Inc.). Live imaging, 3D reconstruction and quantificationEmbryos of stage 14/15 were dechorionated in 50% bleach, mounted in Halocarbon oil 700 (Sigma) and imaged with a confocal microscope (LSM510 or LSM780, Carl Zeiss, Inc.). We observed that Ed-GFP and DE-cad-GFP labeled both AJs and endocytosed vesicles (Figs. 1B and 4A), while Ed-mCherry and DE-cad-mCherry only weakly labeled AJs but strongly labeled endocytosed vesicles (Figs. 2A and 4B). The reason for this differential labeling of mCherry is unclear. However, the relatively weak labeling of Ed-mCherry and DE-cad-mCherry at AJs, together with the fact that mCherry signals were easily quenched upon excitation when compared to GFP, led to the loss of Ed-mCherry/DE-cad-mCherry signals from AJs, although signal remained detectable in the vesicles. We took advantage of this to differentially label vesicles with Ed-mCherry or DE-cad-mCherry. For 3D reconstruction, a single section of 0.4 ?m was captured with 0.4 seconds per frame. Z-stacks were then assembled and analyzed using ZEN (Carl Zeiss, Inc.). Lattice light sheet microscopy 3D imaging were acquired as in Chen et al. 2014 PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5DaGVuPC9BdXRob3I+PFllYXI+MjAxNDwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA [28], using a massively parallel array of coherently interfering beams comprising a non-diiffracting 2D optical lattice as light sheet illumination focused by 0.65 NA objective for excitation (Special Optics). This creates a coherent structured light sheet that can be dithered to create uniform excitation in a 400 nm thick plane across the entire field of view determined by the length of the light sheet. In order to obtain the array of lattice light sheet, a binary spatial light modulator (SXGA-3DM, Forth Dimension Displays) is placed conjugate to the sample plane, and a binarized version of the desired structured pattern at the sample is projected on the display. For imaging, the fluorescence generated within the specimen is collected by a detection objective (Nikon, CFI Apo LWD 25XW, 1.1 NA, 2 mm WD) whose focal plane is co-incident with the light sheet. Four laser of excitation wavelength 488 nm was used. A filter (Semrock, FF03-525/50-25) is placed before a CMOS camera (ORCA-flash4.0, Hamamatsu) to filter the excitation wavelengths. The obtained 3D imaging data were deconvolved using a Richardson-Lucy algorithm. For microinjection, embryos were injected posteriorly with CellMask Deep Red (Invitrogen) or cytochalasin D (Sigma) (3 mg/ml in DMSO) with FemtoJet (Eppendorf). Photobleaching was performed at full laser power (argon laser, 488 nm) over an ROI of 3?m diameter using an LSM510 confocal microscope. The frequency of cell-to-cell movement of Ed-GFP or DE-cad-GFP vesicles was calculated by dividing the number of intercellular movements by the cell number and the time. Electron microscopyEmbryos from Drosophila melanogaster (Oregon R) flies were?collected at 25 °C on yeasted grape juice/agar plates at timed intervals, rinsed off plates and dechorionated by standard methods.?Dechorionated embryos were transferred to agar pads and selected for dorsal closure stage.? Embryos were prepared for transmission electron microscopy (TEM) by high-pressure rapid freezing and then by freeze substitution? ADDIN EN.CITE <EndNote><Cite><Author>McDonald</Author><Year>2000</Year><RecNum>3081</RecNum><DisplayText>[29]</DisplayText><record><rec-number>3081</rec-number><foreign-keys><key app="EN" db-id="war2dtppvtxr9ie9pxsvdtw2px9dtt92tzt9">3081</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>McDonald, K.</author><author>Sharp, D.J.</author><author>Rickoll, W.L.</author></authors></contributors><titles><title>Preparation of thin sections of Drosophila for examination by transmission electron microscopy</title><secondary-title>W. Sullivan, M. Ashburner, and R.S. Hawley (Eds.), Drosophila Protocols, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY</secondary-title></titles><periodical><full-title>W. Sullivan, M. Ashburner, and R.S. Hawley (Eds.), Drosophila Protocols, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY</full-title></periodical><pages>245-271</pages><dates><year>2000</year></dates><urls></urls></record></Cite></EndNote>[29]. Thin sections were mounted on uncoated 200 mesh grids, and stained with uranyl acetate and?bismuth?subnitrate.?Supplementary Figures and Figure LegendsFig. S1. Hypothetical models illustrating cell-to-cell movement. Ed vesicle (red), AJs (green) and SJs (blue) are indicated. The focal plane (yellow) of the confocal microscope was 0.7 μm thick. (A) Schematic showing an Ed vesicle, within the focal plane, moving from donor cell, on average 3 μm, into the recipient cells. The angles between AJs and SJs of lateral epithelial cells are about 180?, which agrees with what we observed experimentally. (B) Schematic showing an out-of-focus Ed vesicle moving slightly basal to AJs but remaining within the donor cell. For this Ed vesicle to move 3 μm at the level of the SJ, the angles between the AJ and SJ would have to be significantly less than 180?. (C) Schematic showing an out-of-focus Ed vesicle moving apical to AJs in the perivitelline space. (D) Schematic showing an Ed vesicle, within the focal plane, moving through filopodia extending from AJs.Fig. S2. Ed vesicle did not move apical to AJs in the extracellular space. (A) Serial confocal z sections (from apical to basal) of en>Ed-GFP embryos showing that Ed-GFP particles (arrowheads) first appeared at the level of Ed-GFP-labeled AJs. (B) Side view maximal intensity projections of the distribution of Ed-GFP vesicles in en>Ed-GFP embryos using lattice light sheet microscopy, extracting from a 4D data set covering 225 time points at 1 second intervals. Ed-GFP particles (arrowheads) never appeared above Ed-GFP-labeled AJs. Bars, 5μm.Fig. S3. Ed is endocytosed via an AP-2-, dynamin-, Rab5- and Rab11-dependent pathway. Increased accumulation of membrane-associated Ed-GFP was detected when α-adaptin RNAi (B) or a dominant negative form of dynamin (shibireDN) (C) was overexpressed. Increased cytosolic accumulation of Ed-GFP was detected when a dominant negative form of Rab5 (D) or Rab11 (F), but not Rab4 (E), was co-expressed. Bar, 5 ?m.Fig. S4. Cell-to-cell movement of Ed-containing endosomes. Movie sequences from a live imaging of en>Ed-GFP+mCherry-Rab5 embryo (A) and en>Ed-GFP+mCherry-Rab11 embryo (B) showing that Ed-GFP vesicle (green arrowhead) co-localized and moved with mCherry-Rab5 (red arrowhead in A) and mCherry-Rab11 (red arrowhead in B) during cell-to-cell movement. Summary of Ed-GFP vesicles trajectories are shown, with the white dot indicating the starting position of each trajectory. Bars, 5 ?m.Fig. S5. MTs and cell-to-cell transport. (A) MTs (green) were localized to actin (red) enriched apical region of the prism-shaped lateral epithelial cells, shown in xy and xz planes. (B) These apical MTs (green) were localized to Ed-labeled AJs (red), shown in xz plane. (C) Movie sequences from a live imaging of Jupiter-GFP+en>Ed-mCherry embryo showing that after crossing the CellMask Deep Red-labeled cell membrane (red), the Ed-mCherry vesicle (white) changed direction when it fell off from the Jupiter-GFP labeled MT arrays (green). Arrowhead indicates the position of Ed-GFP vesicle, arrows indicate MT arrays, and dashed line indicates cell-cell boundary. Summary of Ed-GFP vesicle trajectory is shown, with the white dot indicating the starting position of trajectory. Bars, 5 ?m (A,B) and 2 ?m (C).Legends for Supplementary VideosVideo S1. Cell-to-cell movement of Ed-GFP vesicles through AJs. (A) Live imaging (time interval: 1.6 seconds) of en>Ed-GFP embryo. One cell receives two Ed-GFP vesicles at different AJ locations from different donor cells. Arrowheads (red and blue) indicate the Ed-GFP vesicles undergoing cell-to-cell movement. (B) Live imaging (time interval: 3.2 seconds) of en>Ed-GFP embryo. An Ed-GFP vesicle (indicated by arrowhead) crosses the plasma membrane and fuses with another Ed-GFP vesicle. (C) Live imaging (time interval: 1.6 seconds) of Ed-YFP embryo. Cell-to-cell movement of Ed-YFP vesicle (indicated by arrowhead) can be detected in an ed protein trap line (Ed-YFP).Video S2. The trajectories of an Ed-mCherry (red) vesicle crossing the DE-cad-GFP-labeled AJs (green) into recipient cell, shown in xy and xyz planes. Live imaging (time interval: 2.8 seconds) of en>DE-cad-GFP+Ed-mCherry embryo. Arrowhead indicates the Ed-mCherry vesicle undergoing cell-to-cell movement.Video S3. An Ed-GFP vesicle (indicated by arrowhead) moves directly across the photobleached AJ (indicated by bracket) into the recipient cell. Live imaging (time interval: 1.6 seconds) of en>Ed-GFP embryo.Video S4. An Ed vesicle co-localizes and moves with Rab5 or Rab11 during cell-to-cell movement. (A) Live imaging (time interval: 1.6 seconds) of en>Ed-GFP+mCherry-Rab5 embryo. Arrowhead indicates the Ed-GFP vesicle (green) co-localizing and co-migrating with mCherry-Rab5 (red). (B) Live imaging (time interval: 1.5 seconds) of en>Ed-GFP+mCherry-Rab11 embryo. Arrowhead indicates the Ed-GFP vesicle (green) co-localizing and co-migrating with mCherry-Rab11 (red).Video S5. Ed vesicle (indicated by arrowhead) moves along MT arrays. Live imaging (time interval: 3.1 seconds) of Jupiter-GFP+en>Ed-mCherry embryo. The Ed-mCherry vesicle (white) moves along the Jupiter-GFP–labeled MT arrays (green).Video S6. Ed vesicle moves along the MT arrays and, through MT arrays at the AJ, crosses the cell membrane into the recipient cell. Live imaging (time interval: 3.1 seconds) of Jupiter-GFP+en>Ed-mCherry embryo injected with CellMask Deep Red (red). Arrowhead indicates Ed-mCherry vesicle (white) moving along the Jupiter-GFP–labeled MT arrays (green, arrows) and then crossing the CellMask Deep Red-labeled cell membrane (dashed line) into the recipient cell.Video S7. (A) Live imaging (time interval: 1.0 seconds) of en> Ed-GFP embryos. (B) Live imaging (time interval: 1.6 seconds) of en>Spastin-EGFP+Ed-GFP embryos. Ed-GFP vesicles became largely stationary. (C) Live imaging (time interval: 2.0 seconds) of en>khc-RNAi+Ed-GFP embryos. Ed-GFP vesicles (arrowheads) moved slowly but still can approach AJ membrane.Video S8. Armadillo, Discs large and Lethal giant larvae do not exhibit cell-to-cell movement. Live imaging (time interval: 1.6 seconds, 1.6 seconds, 2.0 seconds) of Armadillo-GFP (left), Discs large-GFP (middle) and en>Lethal giant larvae-mCherry (right) embryos.Video S9. DE-cad or dEGFR vesicle co-localizes and moves with Ed during intercellular movement. (A) Live imaging (time interval: 1.4 seconds) of en>Ed-GFP+DE-cad-mCherry embryo. DE-cad vesicle (red, arrowhead) co-localizes and move with Ed-GFP (green) during intercellular movement. (B) Live imaging (time interval: 1.6 seconds) of en>dEGFR-GFP+Ed-mCherry embryo. dEGFR vesicle (green, arrowhead) co-localizes and moves with Ed-mCherry (red) during intercellular movement.References[23] G. Assaker, D. Ramel, S.K. Wculek, M. Gonzalez-Gaitan and G. Emery, Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration, Proc. Natl. Acad. Sci. U S A 107, 010, 22558–22563.[24] A.K. Satoh, J.E. O'Tousa, K. Ozaki and D.F. Ready, Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors, Development 132, 2005, 1487–1497.[25] T.A. Evans, H. Haridas and J.B. Duffy, Kekkon5 is an extracellular regulator of BMP signaling, Dev. Biol. 326, 2009, 36–46.[26] J. Huang, L. Huang, Y.J. Chen, E. Austin, C.E. Devor, F. Roegiers and Y. Hong,Differential regulation of adherens junction dynamics during apical-basal polarization, J. Cell. Sci. 124, 2011, 4001–4013.[27] F. Wirtz-Peitz, T. Nishimura and J.A. Knoblich, Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization,?Cell?135,?2008,?161–173.[28] B.C. Chen, W.R. Legant, K. Wang, L. Shao, D.E. Milkie, M.W. Davidson, C.Janetopoulos, X.S. Wu, J.A. Hammer, 3rd, Z. Liu, B.P. English, Y. Mimori-Kiyosue, D.P. Romero, A.T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin,R.D. Mullins, D.M. Mitchell, J.N. Bembenek, A.C. Reymann, R. Bohme, S.W.Grill, J.T. Wang, G. Seydoux, U.S. Tulu, D.P. Kiehart and E. Betzig, Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution, Science 346, 2014, 1257998.[29] K. McDonald, D.J. Sharp and W.L. Rickoll, Preparation of thin sections of Drosophila for examination by transmission electron microscopy, In: W.Sullivan, M. Ashburner and R.S. Hawley, (Eds.), Drosophila Protocols, 2000, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY, 245–271 ................
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