Supplementary Figures 1-4 from Sunitinib and SU11652 Inhibit Acid Sphingomyelinase, Destabilize Lysosomes, and Inhibit Multidrug Resistance

PDF - 207KB, Supplementary Figure S1: Screen for small molecules that exhibit non-apoptotic cytotoxicity. Supplementary Figure S2: Kinetics of SU11652- and sunitinib-induced cytotoxicity. Supplementary Figure S3: SU11652 and sunitinib accumulate rapidly in lysosomes of MCF7 cells. Supplementary Figure S4: SU11652 does not alter the ultrastructure of mitochondria or ER.</p

Rabip4’ localization does not require AP-3.

<p>VSVG-rabip4’-expressing HeLa cells were treated with 5 µg/ml BFA for 15 min at 37°C and stained with a rabbit antibody against rabip4’ and mouse anti-δ-adaptin or mouse anti-TfR, followed by Alexa568-anti-rabbit and Alexa488-anti-mouse IgG. Rabip4’ overexpression did not affect AP-3 sensitivity to BFA. Lower row represents insets of boxed areas. Arrows point to BFA-induced tubulation of rabip4’ and TfR (<b>A</b>). Rabip4s-directed siRNA oligos were transfected in HeLa cells for 3 days. AP-3 distribution was similar in both siRNA-transfected and control cells. Scale bar is 10 µm. Silencing was monitored by Western blotting and gave routinely 80–85% reduction of both rabip4’ and rabip4 isoforms (<b>B</b>). AP-3 siRNA oligos were transfected in HeLa cells for 3 days. Two days after siRNA treatment, cells were transfected with VSVG-rabip4’ for another day and labeled for δ-adaptin and rabip4’. Rabip4’ distribution did not depend on AP-3. Scale bar is 10 µm. Western blots were probed with antibodies against δ-adaptin. The level of δ-adaptin in siRNA-transfected cells was quantified and expressed as % of control (<b>C</b>).</p

An optimized protocol for immuno-electron microscopy of endogenous LC3

MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.</p

AP-3 and Rabip4’ Coordinately Regulate Spatial Distribution of Lysosomes

<div><p>The RUN and FYVE domain proteins rabip4 and rabip4’ are encoded by <em>RUFY1</em> and differ in a 108 amino acid N-terminal extension in rabip4’. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing <em>RUFY1</em> gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4’ yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4’. Rabip4’ colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4’ in regulating lysosome positioning through an interorganellar pathway.</p> </div

Rabip4’ and AP-3 cooperate in lysosome positioning.

<p>HEK293T cells were depleted of rabip4s or AP-3 and then labeled for immunofluorescence with mouse antibodies against CD63 and TfR (green) or with rabbit antisera specific for cathepsin D and Ti-VAMP (red) (<b>A</b>). HEK293T cells were processed as above and co-labeled with anti-cathepsin D (red) and anti-tubulin (green) antibodies or with anti-cathepsin D antibody (red) and Alexa-488-conjugated phalloidin for actin staining. Nuclei were stained with DAPI. (<b>B</b>). Images represent projections of confocal Z-stacks. Scale bar, 10 µm. Depletion of rabip4s and AP-3 selectively redistributed the lysosomal markers CD63, Cathepsin D, and Ti-VAMP to cellular protrusions (arrows).</p

Rabip4’ interacts specifically and directly with AP-3.

<p>Immobilized GST-rabip4’ (aa 299–708) was incubated with brain cytosol. Bound proteins were resolved by SDS-PAGE and analyzed by tandem mass spectrometry, which yielded β3-adaptin as binding partner (<b>A</b>). Eluates were probed with antibodies against subunits of adaptor complexes showing specificity for AP-3 (<b>B</b>). GST-rabip4’ beads were incubated with detergent extracts from rescued <i>mocha</i> cells and bound proteins were analyzed by Western blot for the indicated AP-3 and AP-1 subunits. The ubiquitous AP-3 specifically interacted with rabip4’ (<b>C</b>). AP-3 was immunoprecipitated from lysates of HeLa cells expressing VSVG-rabip4’ and analyzed by Western blot with antibodies against VSVG and AP-3 subunits. Immunoprecipitation of AP-1 or a monoclonal antibody against HA (control IgG) did not co-immunoprecipitate VSVG-rabip4’ (<b>D</b>). GST-rabip4’ was immobilized on GSH beads and incubated with ³⁵S-labeled AP-3 subunits. Rabip4’ interacted directly with AP-3 through the β3 subunit (<b>E</b>).</p

Rabip4’ functions in distribution of lysosomes.

<p>The indicated cell lines were screened for expression of rabip4s by Western blot (<b>A</b>). HEK293T cells were transfected with siRNA against rabip4s and three days later processed for Western blot with antibodies against rabip4s and actin as a loading control, followed by Alexa680-conjugated secondary antibody. siRNA induced an >90% reduction of both rabip4’ and rabip4 isoforms (<b>B</b>). In parallel, cells were labeled for immunoflourescence with monoclonal antibodies against CD63, LAMP-1, CI-MPR, and TfR. LAMP-1 was counterstained with Alexa 594-, while CD63, CI-MPR, and TfR with Alexa 488-labeled secondary antibodies. Nuclei were stained with DAPI. Images represent projections of confocal Z-stacks. Asterisks denote plasma membrane protrusions, enriched in CD63 and LAMP-1, induced by depletion of rabip4s. Scale bar, 10 µm (<b>C</b>).</p

Rab5 is essential for endosome localization of rabip4’.

<p>HeLa cells expressing VSVG-rabip4’ and the indicated GFP/YFP-tagged rab5 and rab4 constructs were treated with wortmannin, and labeled for VSVG-rabip4’ and EEA1. Rabip4’ relocalized into the cytoplasm in cells expressing the GFP-rab5S34N mutant (asterisks). Arrows indicate colocalization between rabip4’, EEA1, and either of the GTPases (<b>A</b>). HeLa cells stably expressing VSVG-rabip4’ΔFYVE (aa 1–636) were transfected with the indicated GFP-rab5 and YFP-rab4 constructs, and labeled as above. Rab5 rescued the ΔFYVE mutant from the cytoplasm and relocalized it to endosomes that contained (arrows) or were devoid (arrowheads) of EEA1 (<b>B</b>). HeLa cells expressing VSVG-rabip4’ or VSVG-rabip4’ΔCC3 were labeled with antibodies against rabip4’ (rabbit) and EEA1 (mouse). The secondary antibodies were Alexa488-goat anti-rabbit and Cy3-goat anti-mouse. Scale bar is 10 µm (<b>C</b>).</p

Rabip4’ and AP-3 colocalize on endosomes.

<p>HeLa cells were labeled with antibodies against δ-adaptin (<b>A</b>) or δ-adaptin (red) and endogenous rabip4s (green) (<b>B</b>). Scale bar is 10 µm (<b>A</b>) and 2.5 µm (<b>B</b>). HeLa cells expressing VSVG-rabip4’ were labeled with a rabbit antibody against rabip4’ (green) and monoclonal antibodies against the δ subunit of AP-3, the γ1 subunit of AP-1, and the α subunit of AP-2 (all in red). Scale bar is 10 µm. (<b>C</b>). Arrows in insets indicate structures on which AP-3 and rabip4s or VSVG-rabip4’ colocalize (<b>B and C</b>). Extent of colocalization between rabip4’ and the adaptor complexes (<b>D</b>).</p

Rabip4’, EEA1, and Hrs partially colocalize on endosomes.

<p>HeLa cells were labeled for endogenous rabip4s and EEA1. Arrows denote co-distribution of the two FYVE domain-proteins. Bottom row represents a blow up of indicated areas. The dashed line marks the contour of cells. Scale bar is 10 µm (<b>A</b>). Ultrathin cryosections of HeLa cells expressing VSVG-rabip4’ were immunogold labeled with polyclonal anti-VSVG (10 nm gold particles). VSVG-rabip4’ localized to the endosomal vacuole (E), as well as to surrounding tubular-vesicular membrane profiles characteristic for recycling tubules (arrows). P is plasma membrane. Bar, 200 nm (<b>B</b>). HeLa cells transfected with GFP-rabip4’ and HA-Hrs were labeled for EEA1 (blue) and HA-Hrs (red), while rabip4’ was visualized by EGFP fluorescence. Bottom row represents an enlargement of the indicated areas. Arrows point to endosomes that contain rabip4’, EEA1, and Hrs, and arrowheads indicate endosomes devoid of rabip4’. Scale bar is 10 µm (<b>C</b>).</p