QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online July 10, 2003
doi: 10.1242/10.1242/jcs.00637

This Article Summary Full Text Full Text (PDF) Supplemental Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guha, A. Articles by Mayor, S. Search for Related Content PubMed PubMed Citation Articles by Guha, A. Articles by Mayor, S.

shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes

A. Guha^1,2,*, V. Sriram^1,*, K. S. Krishnan^2, and S. Mayor^1,

¹ National Center for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bangalore 560 065, India
² Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhaba Road, Bombay 400 005, India

View larger version (46K): [in a new window]   Fig. 1. Embryonic macrophages and larval hemocytes in primary culture bind and internalize Cy3mBSA via endogenous scavenger receptors. Embryonic macrophages (A, arrows) and larval hemocytes (C, arrows) in culture are the only population of cells from wild-type animals to internalize Cy3mBSA. The fluorescence image of cells (outlined in A and C) indicates that Cy3mBSA uptake is specifically mediated by ALBR endogenously expressed in embryonic macrophages (B) and larval hemocytes (D). Inset in panel D shows that binding and uptake of Cy3mBSA (D, arrows) can be competed by excess unlabeled mBSA. Histogram in panel E shows the relative amount of Cy3mBSA internalized via ALBR in hemocytes incubated in the absence (Control) or presence of excess unlabeled mBSA, fucoidan or LPS, expressed as a fraction of total endocytosed Cy3mBSA internalized in the absence of any other competing ligand. Bar, 5 µm.

View larger version (84K): [in a new window]   Fig. 2. Endocytosed F-Dex and Cy3mBSA colocalize in Rab7-containing endosomes at late times but are present in Rab-7-negative structures at early times. (A-C) Hemocytes incubated with F-Dex (A, green in C) and Cy3mBSA (B, red in C) for 5 minutes and chased for 2 minutes, and imaged on a wide-field microscope, colocalize in large centrally located endosomes (C, arrows). (D-H) Confocal microscopy shows that at 5 minutes the majority of F-Dex-containing endosomes (D, arrows; green in F) colocalize with Rab7 (red in F), whereas in a 45 seconds pulse of F-Dex (H, green; arrowheads) and Alexa488 mBSA (G, green; small arrows) do not colocalize with anti-Rab7 (red in G and H, arrows). Insets show magnified view of the area marked by an asterisk. Bar, 5 µm; inset, 1 µm.

View larger version (40K): [in a new window]   Fig. 3. B-Cy3mBSA and F-Dex are internalized into distinct endosomal compartments at very early times. Hemocytes from CS animals were incubated at room temperature with B-Cy3mBSA (A,E; red in D,H) and F-Dex (C,G; green in D,H) for 45 seconds either without (A-D) or with (E-H) a pre-pulse of B-Cy3mBSA for 3 minutes. Cells were then immediately chilled on ice and Cy5-SA (B,F; blue in D,H) was added on ice to detect surface-accessible receptors, prior to fixation and imaging on wide-field microscope. Note that after 45 seconds a significant fraction of the cell-associated B-Cy3mBSA is distributed in endosomes inaccessible to Cy5-SA (A,D and E,H; arrows). Most of the brightest F-Dex-labeled structures (C,D and G,H; arrowheads) do not colocalize with B-Cy3mBSA. A large fraction of the peripherally distributed B-Cy3mBSA is colocalized with Cy5-SA, remaining surface accessible at both times (A,B,D, and E,F,H; small arrowheads). Insets show magnified view of the area marked by an asterisk. Bar, 5 µm; inset, 1 µm.

View larger version (47K): [in a new window]   Fig. 4. Cy3mBSA is internalized into GFP-Rab5-positive early endosomes distinct from LR-Dex early endosomes that are devoid of GFP-Rab5. Hemocytes from Collagen-Gal4;UAS-GFPdRab5 larvae were incubated for 45 seconds with Cy3mBSA (A-C) or LR-Dex (D-F) prior to fixation, permeabilization and imaging on wide-field microscope. At 45 seconds, a fraction of Cy3mBSA-labeled structures (A, red in C; small arrows) are also positive for GFP-dRab5 (B, green in C; small arrows). Several peripherally distributed Cy3mBSA-labeled structures (A, red in C; small arrowheads) are negative for GFP-dRab5 (B, green in C) and are likely to be surface accessible (see Fig. 3). LR-Dex-containing endosomes (D, red in F; arrowheads) do not colocalize with GFP-dRab5-positive structures (E; green in F; small arrows). Insets show magnified view of the area marked by an asterisk. Bar, 5 µm; inset, 1 µm.

View larger version (61K): [in a new window]   Fig. 5. GFP-GPI is internalized into LR-Dex early endosomes and Cy5mBSA early endosomes. Hemocytes from Collagen-Gal4;UAS-GFP-GPI (A-D) were incubated with Fl-anti-GFP (pseudo-colored green; Cy5-anti-GFP in A,C and Alexa568-anti-GFP in B,D) at room temperature for the indicated times in the presence of LR-Dex (A,C; red) or Cy5mBSA (B,D, red). Note that Fl-anti-GFP is partially colocalized with LR-Dex (A, arrowheads; C, arrows; insets) and with Cy5mBSA (B, small arrows; D, arrows; insets) at these times. However, at late times, all three probes colocalize in late endosomes (arrows, C,D). Insets (red, top; green, middle; merge, bottom) show the area marked by the asterisk. Bar, 5 µm; inset, 1 µm.

View larger version (58K): [in a new window]   Fig. 6. Endocytosis of Cy3mBSA is perturbed in shits cells at a restrictive temperature while F-Dex uptake appears unaffected. (A) Schematic of Drosophila dynamin (PH, plextrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain) showing the locations of the shits2 (G141S) and shits1 mutations (G267D). (B-J) Cells from wild-type CS animals (B,E,H) internalize Cy3mBSA (red) and F-Dex (green) into endosomes following a pulse for 5 minutes at 21°C (B, arrows) and 31°C (E, arrows). Cells from shits1 (C,F,I) and shits2 (D,G,J) animals internalize Cy3mBSA and F-Dex at 21°C (C,D, arrows). At 31°C in cells from shits animals (F,G), Cy3mBSA remains peripherally distributed in punctate (small arrowheads) and hazy distribution, whereas F-Dex continues to be internalized into large endosomes (arrows). Cells labeled with Cy3mBSA for 20 minutes on ice (H,I,J) show a distribution similar to shits cells at 31°C (small arrowheads). Bar, 5 µm.

View larger version (17K): [in a new window]   Fig. 7. Cells from shits animals arrest mBSA at the plasma membrane in cell-surface-accessible structures. (A) Cartoon depicting the scheme adopted for detecting surface accessibility of mBSA bound to ALBR using Cy5-SA and B-Cy3mBSA. (B) Histogram shows the ratio of Cy5 to Cy3 fluorescence obtained for shits1 cells at the temperatures and conditions indicated. The cartoons indicate the expected surface-accessible or -inaccessible status of ALBR ligand (B-Cy3mBSA) as probed using Cy5-SA. (C) Histogram shows the average extent of B-Cy3mBSA bound to the cells that remains accessible to Cy5-SA (Cy5/Cy3 ratio) after a pulse for 5 minutes at 21°C and 31°C in the indicated alleles, relative to the total accessible pool of receptors (Cy5/Cy3 ratio determined for Ice condition; arrow). Bars represent mean±s.e. obtained from three experiments, each with at least 75 cells from two dishes in each condition Note that both shits alleles quantitatively arrest Cy3mBSA internalization at this temperature.

View larger version (33K): [in a new window]   Fig. 8. Cells from shits animals reversibly arrest mBSA uptake at the cell surface. (A) Protocol designed to test the reversibility of the temperature-sensitive shibire inhibition of endocytosis and the nature of fluid-phase endosomes formed at the restrictive temperature (31°C). (B) Cells derived from the indicated alleles were incubated as in panel A, fixed and imaged on a wide-field microscope. Cy3-mBSA (red in merge) and Cy5-mBSA (blue in merge) colocalize (arrows) in a population of F-Dex (FDx)-containing endosomes (green in merge) that were formed at 31°C, confirming that F-Dex-containing endosomes formed in shits cells at 31°C are capable of mixing with surface-arrested Cy3mBSA when the cells are returned to permissive temperatures. (C) Protocols designed to test the kinetics of sequestration of B-Cy3mBSA when cells from shits1 animals were pre-incubated at 21°C (left panel) or 32°C (right panel) and then rapidly transferred to ice. The percentage of cellular B-Cy3mBSA accessible to Cy5-SA in the depicted cell is indicated at the top right in each image. Note that after transfer from 32°C to ice, much of the B-Cy3mBSA fluorescence remains in peripheral endosomes (arrowheads) inaccessible to Cy-SA. (D) Histogram showing the relative accessibility of B-Cy3mBSA incubated with shits1 cells at the indicated temperatures (white bars) and then transferred to ice (0°C, blue bars), relative to cells directly labeled on ice (arrow). Data shown are mean±s.e.m. obtained from one experiment with duplicate dishes with at least 30 cells in each dish. Similar results were obtained in two independent experiments with cells from of shits1and shits2 animals. Bar, 5 µm.

View larger version (44K): [in a new window]   Fig. 9. ALBR is localized to GFP-clathrin light chain marked sites at the plasma membrane. Hemocytes from Collagen-Gal4;UAS-GFP-Clc (A-C) or shits2;Collagen-Gal4;UAS-GFP-Clc animals (D-F) were incubated with Cy3mBSA (A,D; red in C,F) on ice (0°C; A-C) or at a restrictive temperature (31°C; D-F) for 5 minutes prior to fixation, permeabilization and imaging on wide-field microscope. On ice, the peripherally distributed Cy3mBSA-positive structures (A, red in C; small arrowheads) are positive for GFP-Clc (B, green in C; small arrowheads). At 31°C in cells from shits2 animals, Cy3mBSA remains peripherally distributed in punctate (D, small arrowheads) structures also positive for GFP-Clc (E, small arrowheads). This distribution in shits2 cells at 31°C is similar to cells labeled with Cy3mBSA on ice (small arrowheads). Insets show magnified view of the area marked by an asterisk. Bar, 5 µm; inset, 1 µm.

View larger version (37K): [in a new window]   Fig. 10. Fluid-phase and GPI-AP uptake is unperturbed at restrictive temperatures in cells from mutant alleles of shi. Histogram shows the average amount of F-Dex (FDx; ±s.d.) internalized per cell in the indicated alleles and temperatures in an incubation period of 5 minutes. The similarity in uptake across all alleles, even at restrictive temperatures, demonstrates that mutations at the shi locus do not perturb the uptake of the fluid phase. Data shown are from three experiments each with at least 75 cells. (B,C) Hemocytes from shits2;Collagen-Gal4;UAS-GFP-GPI animals were incubated with Fl-anti-GFP (green) at 31°C for 15 minutes in the presence of LR-Dex (B, red) or Cy5mBSA (C, red) prior to fixation and imaging on a wide-field microscope. Endocytosis of Fl-anti-GFP into LR-Dex (B, arrows)-containing endosomes is unaffected, whereas Cy5mBSA is blocked at the cell surface (C, small arrowheads). Insets (red, top; green, middle; merge, bottom) show the area marked by the asterisk. Bar, 5 µm; inset, 1 µm. (D) Model depicts the existence of dDyn-independent endocytic pathways for the fluid phase (yellow) and GPI-APs (green) in larval hemocytes. ALBR ligands (red) are endocytosed via clathrin and dDyn-dependent pathways into Rab5-positive early endosomes (EE), whereas the fluid phase (yellow) marks a separate Rab5-negative EE, before finally coming together in Rab7-positive late endosomes (LE).