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First published online 9 December 2008
doi: 10.1242/jcs.036673

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Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

Department of Biological Sciences, Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN 37235, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. RBO facilitates FM1-43-dye endocytosis at the neuromuscular synapse. (A) Schematic of dye-loading, temperature and imaging protocols used at the larval NMJ. In protocol 1, FM1-43 was loaded for 2 minutes with 60 mM [K+] at 25°C and then unloaded for 2 minutes with 60 mM [K+] at 37°C. In protocol 2, following a 10-minute incubation at 37°C, dye was loaded for 2 minutes at 37°C and then unloaded for 2 minutes at 25°C. (B) Representative images of wild type (OR), syx3-69 and rbots single mutants, and the rbots;syx3-69 double mutant following protocol 1. (C) Top three panels show representative images comparing FM1-43 loading and unloading following protocol 2. Bottom panel shows rbots;syx3-69 37°C unloading after 5 minutes of loading at 25°C. Scale bars: 10 µm. (D) Quantification of loading (L) and unloading (UL) with both protocols. Error bars show mean ± s.e.m. ***Significance of P<0.001.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Endocytic defects in rbots mutants are fully rescued by the wild-type rbo gene. (A) Representative images of rbots and rbots/rbo2; rbo-egfp/rbo-egfp (rescue) larval NMJs loaded with FM1-43 dye after 10 minutes at 37°C (see Fig. 1, protocol 2). Scale bar: 10 µm. (B) Magnified loaded synaptic boutons in both genotypes. The wild-type rbo transgene rescues the rbots dye-loading defect. Scale bar: 5 µm. (C) Quantification of dye loading in wild-type (OR) and rbots compared with rescue animals.

View larger version (90K): [in this window] [in a new window]   Fig. 3. RBO localizes to synapses in central brain primary neuron cultures. Representative images of neuron cultures of rbo2/rbo2; rbo-egfp/rbo-egfp. (A) RBO-GFP (green) at 6 DIV, double-labeled with anti-BRP (red) marking active zones. Scale bar: 10 µm. (B) Area in boxed area in A magnified to show RBO-GFP and BRP colocalization in axonal synaptic varicosities. Scale bar: 5 µm. (C) RBO-GFP (green) double-labeled with anti-CSP (red) marking synaptic punctae. Scale bar: 10 µm. (D) Area in boxed area in C magnified to show RBO-GFP and CSP colocalization in synaptic punctae. Scale bar: 5 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 4. RBO localizes to functional synapses with cycling SVs. (A) Cultured neurons containing elav-GAL4 driven UAS-CD8::GFP (green) doubled-labeled with anti-Synapsin (red) to show axonal varicosities containing the presynaptic marker. Scale bar: 2 µm. (B) FM1-43 labeling with 60 mM [K+] for 45 seconds loads synaptic varicosities (left). A shorter, 30 second, exposure to 60 mM [K+] partially unloads dye (middle). Nomarski DIC image showing neuronal structure (right). Scale bar: 20 µm. (C) In rbo2/rbo2; rbo-egfp/rbo-egfp 6-DIV cultures, loaded FM1-43 dye (white) colocalizes with RBO-GFP (green) and the synaptic marker anti-CSP (red). Scale bar: 5 µm. (D) Quantification of the percentage of colocalization of BRP and CSP with RBO-GFP punctae.

View larger version (34K): [in this window] [in a new window]   Fig. 5. RBO facilitates FM1-43-dye endocytosis in central brain synapses. Dye labeling in cultured neurons from wild type (OR) and rbots after 10 minutes at 37°C. Left panels show lower-magnification fields and Nomarski DIC images; right panels show higher-magnification images of FM1-43-labeled synapses (arrows). (A-D) Shown is the range of loading in wild type with more intense (A) and less intense (B) labeling, and in rbots with more intense (C) and less intense (D) labeling. (E) Rescue of loading in rbots/rbo2; rbo-egfp/rbo-egfp neurons. Scale bars: 10 µm. (F) Quantification of FM1-43 loading. The number of loaded varicosities as a percentage of the total varicosities from DIC images, per 20 µm of axon length. Error bars show mean ± s.e.m. for four independent trials for each genotype. ***Significance of P<0.001.

View larger version (28K): [in this window] [in a new window]   Fig. 6. RBO is required for Texas-red–avidin-tracer endocytosis in Garland cells. (A) RBO-GFP in rbo2/rbo2; rbo-egfp/rbo-egfp Garland cells is strongly expressed at the periphery. (B) Most RBO-GFP is tightly associated with the plasma membrane, but some RBO associates with internal organelles (arrows). (C) Wild-type (OR) cells loaded at 25°C (left) or following 10 minutes at 37°C (right). (D) rbots at 25°C (left) is indistinguishable from control, but at 37°C (right) shows no dye internalization. (E) rbots;syx3-69 also shows a complete block of uptake at 37°C. (F) As positive control, shibirets shows a block at 37°C (right); this phenotype is indistinguishable from rbots. Scale bars: 5 µm. (G) Quantification of fluorescence intensity underlying the plasma membrane.

View larger version (185K): [in this window] [in a new window]   Fig. 7. Ultrastructure of Garland cells in wild type and in rbots mutants. (A) Transmission electron microscopy (TEM) images of wild-type and rbots Garland cells at 25°C. The cortical cell region extends 3 µm, with many labyrinthine channels (thin black arrows). Numerous vacuoles (a), longitudinal (white arrows) and transverse (thick black arrows) tubular elements, and coated vesicles and/or pits (arrowheads) are present in all section planes. Many sections also contain mitochondria (M) and β vacuoles (b). Wild type and rbots appear indistinguishable at 25°C. (B) Following 10 minutes at 37°C, the number of vacuoles in rbots cells is reduced, with remnant vacuoles being larger and more irregular. Labyrinthine channels swell and distend with many vacuolar structures (*). Scale bar: 1 µm. (C) Higher-magnification images at 37°C. In rbots, very elongated labyrinthine channels extend into the cell; these channels contain many vacuolar structures (*). Thin black arrows show the origin of the labyrinthine channels at the basement membrane. Scale bar: 250 nm.

View larger version (111K): [in this window] [in a new window]   Fig. 8. Block in HRP endocytosis occurs in Garland cells in the absence of RBO. (A) Garland-cell endocytotic activity visualized using HRP uptake in wild type (OR) and rbots after 10 minutes at 37°C. Many coated profiles of vacuoles (a) are labeled in wild type (left), among pre-existing unlabeled vacuoles (arrows). The mutant (right) shows no HRP uptake into vacuoles (a). N, nucleus. Scale bar: 1 µm. (B) Higher-magnification images of vacuoles (a) and labyrinthine channels (arrows). The mutant (right) shows an absence of labeling, with distended and swollen labyrinthine channels. Scale bar: 250 nm. (C) Tannic-acid impregnation shows labyrinthine channels that are continuous with the extracellular space. In rbots, the channels are fused with many vacuolar inclusions. Scale bar: 250 nm. (D) Quantification of cell area. No change was observed in rbots at 25°C compared with wild type, but a highly significant increase in cell area was seen at 37°C. (E) Quantification of loaded endosomes per section. A significant reduction in the number of loaded endosomes was observed in rbots at 25°C compared with wild type, with near complete loss at 37°C.

View larger version (108K): [in this window] [in a new window]   Fig. 9. RBO function is specific for bulk endocytosis in neuromuscular synapses. (A) Wild type (OR) and rbots after 10 minutes at 37°C display similar NMJ synaptic ultrastructures. Boutons contain plentiful SVs, especially clustered near active zones (thick arrows), many mitochondria (M) and a few larger (>60 nm) cisternae (thin arrows). (B) Terminals challenged for 10 minutes with depolarizing 60 mM [K+] saline show activity-dependent changes. In wild type (left), the primary ultrastructure change is a massive production of cisternae (thin arrows). In rbots (right), new cisternae conspicuously fail to form. Scale bar: 250 nm. (C) Quantification of SV number in conditions of rest and high-[K+] stimulation (stim). (D) The number of cisternae (>60 nm in diameter) is the same at rest, but very significantly increased by activity in wild type only.

View larger version (49K): [in this window] [in a new window]   Fig. 10. RBO is required for activity-dependent endosome formation in synapses. FM1-43 photoconversion generates an electron-dense marker that is clearly visible by electron microscopy. Dye was incorporated into NMJ synapses after a 2-minute high-[K+] stimulation at 37°C, photoconverted and examined in wild type (OR) and rbots. (A) Inset shows a light-microscopy image of muscle 6/7 NMJ as photoconverted, ready to be sectioned at the electron-microscopic level. Top panels show representative images of control and mutant bouton profiles. Many FM-labeled vesicles and endosomes can be seen throughout the boutons. Asterisks (*) mark labeled endosomes in wild type; note the absence of label in rbots. Lower panels show higher-magnification images of endosomes (*) and SVs (thin arrows). In rbots, no labeling of endosomes was observed (*), but abundant SVs were labeled. Scale bars: 250 nm. (B) Quantification of FM-labeled SVs per bouton section. (C) Quantification of FM-labeled endosomes per bouton profile. Significance indicated: **P<0.01, ***P<0.001.

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