NBR1 enables autophagy-dependent focal adhesion turnover.

Cell culture

MCF10A and MCF10A-Ras mammary epithelial cells were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin, penicillin, and streptomycin.

PyMT mammary carcinoma cells

(R221A clone; Martin et al., 2008 ; gift from B. Fingleton, Vanderbilt University, Nashville, TN) and HEK-293T cells were cultured in DMEM with 10% FBS, penicillin, and streptomycin. iBMK cells were a gift from E. White (Rutgers University, New Brunswick, NJ) and are now commercially available from Applied Biological Materials (T0082 and T3027). They were originally derived via stable transformation of primary kidney epithelial cells isolated from Atg5 +/+ and Atg5 −/− isogenic newborn littermates (postnatal day 1, C57BL/6 genetic background) with adenovirus E1A and dominant negative p53 (p53DD; Mathew et al., 2008 ). They were cultured in DMEM with 10% FBS, penicillin, and streptomycin. For experiments, MCF10A-Ras cells were routinely incubated in assay media (DMEM/F12, 2% horse serum, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin, penicillin, and streptomycin; Debnath et al., 2003 ); 20 mM Hepes was added to the culture medium during live-cell imaging experiments. cDNA constructs, retroviral and lentiviral vectors, and generation of stable cell lines We previously generated pBabeneo-HRas V12 , pBabepuro-GFP-LC3 (22405; Addgene), pBabehygro-GFP-LC3, and pLenti6blast-paxillin-mCherry; our subcloning strategy for these plasmids has been described elsewhere ( Hu et al., 2007 ; Fung et al., 2008 ; Lock et al., 2014 ; Stehbens et al., 2014 ). pMXs-IP-GFP-NBR1 and pMRX-IP-Venus-LC3 were gifts from N. Mizushima (University of Tokyo, Tokyo, Japan; 38283 and 58740; Addgene; Itakura and Mizushima, 2011 ; Koyama-Honda et al., 2013 ). pHIV-ZsGreen was provided by B. Welm (University of Utah School of Medicine, Salt Lake City, Utah; 18121; Addgene; Welm et al., 2008 ). pDest-mCherry-NBR1 was a gift from P. Kim (University of Toronto, Toronto, Ontario, Canada) and T. Johansen (University of Tromsø, Tromsø, Norway; Kirkin et al., 2009 ; Deosaran et al., 2013 ). Paxillin-mTurquoise, mCherry-vinculin, and zyxin-mCherry were from M. Davidson (Florida State University, Tallahassee, FL; 55573, 55159, and 55166; Addgene), and pLentiblast-GFP was provided by E. Campeau (University of Massachusetts Medical School, Worcester, MA; 19069; Addgene; Campeau et al., 2009 ). Lentiviral packaging and envelope plasmids pRSV-REV, pMD2Lg/pRRE, and pMD2.g were gifts from D. Trono (École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; 12253, 12251, and 12259; Addgene). To generate pMXspuro-GFP, pMXspuro-GFP-NBR1, pMXspuro-GFP-NBR1 ΔLIR, and pMXspuro-GFP-NBR1 ΔUBA, GFP alone and GFP-NBR1 were first amplified from pMXs-IP-GFP-NBR1 and subcloned into pcDNA3 (Life Technologies). Site-directed mutagenesis was then performed for deletion of aa 727–738 in NBR1 (NBR1 ΔLIR) or to change aa 877 to a premature stop (NBR1 ΔUBA); GFP, GFP-NBR1, GFP-NBR1 ΔLIR, and GFP-NBR1 ΔUBA were subsequently subcloned into BamHI–XhoI sites of pMXspuro for retroviral expression ( Kitamura et al., 2003 ). mCherry-NBR1 was PCR amplified from pDest-mCherry-NBR1 and subcloned into the BamHI–XhoI sites of pMXspuro to generate pMXspuro-mCherry-NBR1 for retroviral expression. To generate pLentiblast-paxillin-mTurquoise, paxillin-mTurquoise was PCR amplified and subcloned into the BamHI–SalI sites of pLentiblast-GFP for lentiviral expression. Vesicular stomatitus virus G pseudotyped retroviruses were produced by transfection of a 293GPG retrovirus producer cell line ( Ory et al., 1996 ) with 15 µg of expression cDNA using Lipofectamine 2000 (Life Technologies). Retroviral supernatants were collected at days 5–7 after transfection, filtered through a 0.45-µm filter, and stored at –80°C. Lentivirus was made using a four-plasmid, third-generation producer system ( Dull et al., 1998 ) by cotransfecting HEK-293T cells with packaging and envelope vectors (2 µg of pRSV-REV, 2 µg of pMD2Lg/pRRE, and 4 µg of pMD2.g) and 12 µg of expression cDNA using Lipofectamine LTX/PLUS (Life Technologies). Lentiviral supernatants were collected 48 h after transfection, filtered through a 0.45-µm filter, and stored at –80°C. For infection with retroviruses and lentiviruses, 100,000 cells were seeded per well in six-well dishes the day before infection and incubated overnight with virus-containing supernatants supplemented with 8 µg/ml polybrene (Sigma-Aldrich). Stable cell lines were obtained by selecting with 200 µg/ml G418 (Sigma-Aldrich), 0.5–2 µg/ml puromycin (Sigma-Aldrich), 6 µg/ml blasticidin (Life Technologies), or 200 µg/ml hygromycin (Life Technologies). After stable pools were obtained, cells were cultured in the absence of selection agents.

RNA interference For stable

RNA interference, pLKO.1puro lentiviral plasmids with nontargeting shRNA or shRNA against ATG7 (human: NM_006395 , mouse: NM_028835 ), human ATG12 ( NM_004707 ), and human NBR1 ( NM_031858 ) were purchased from Sigma-Aldrich. The target sequence for shRNA against human ATG7 (TRCN0000007587) is 5′-CCCAGCTATTGGAACACTGTA-3′, against human ATG12 (TRCN0000007394) is 5′-TGGAACTCTCTATGAGTGTTT-3′, against mouse ATG7 (TRCN0000092163) is 5′-CCAGCTCTGAACTCAATAATA-3′, and against human NBR1 (TRCN0000123161) is 5′-GCCAGGAACCAAGTTTATCAA-3′. The sequence of the nontargeting shRNA (SHC002; Sigma-Aldrich), which targets no known mammalian genes, is 5′-CAACAAGATGAAGAGCACCAA-3′. shRNA lentivirus was prepared as described above for lentiviral vectors. In brief, HEK-293T cells were cotransfected with packaging and envelope vectors and pLKO.1 shRNA expression plasmids. Virus was collected 48 h after transfection, filtered through a 0.45-µm filter, and stored at –80°C. Cells were seeded in six-well dishes and infected as described for generation of stable cell lines. Stable pools of knockdown cells were obtained by selecting with 2 µg/ml puromycin for 48 h. Cells were used within one to two passages after selection for experiments. New stable pools were generated for each experimental repeat, and knockdown was verified by immunoblotting for each individual experiment. For siRNA-mediated knockdown, ON-TARGETplus SMARTpool siRNAs were purchased from Dharmacon. The sense sequences of the individual duplexes comprising each pool are as follows: human p62/SQSTM1 (L-010230-00): 5′-GAACAGAUGGAGUCGGAUA-3′, 5′-GCAUUGAAGUUGAUAUCGA-3′, 5′-CCACAGGGCUGAAGGAAGC-3′, and 5′-GGACCCAUCUGUCUUCAAA-3′; human NBR1 (L-010522-00): 5′-GAGAACAAGUGGUUAACGA-3′, 5′-CCACAUGACAGUCCUUUAA-3′, 5′-GAACGUAUACUUCCCAUUG-3′, and 5′-AGAAGCCACUUGCACAUUA-3′; human OPTN (L-016269-00): 5′-GGGCUCAGAUGGAAGUUUA-3′, 5′-CCAUGAAGCUAAAUAAUCA-3′, 5′-CUUCGAACAUGAGGAGUUA-3′, and 5′-CUAAUGGCCUUGAGUCAUG-3′; human CALCOCO2/NDP52 (L-010637-00): 5′-GGAAACCCAUAUUCUGGUA-3′, 5′-GGAUUGGAUUGGCAUCUUU-3′, 5′-GGACGUCACAUGUCAUUAU-3′, and 5′-GGAAGACAACCCGUGAGUA-3′; and nontargeting control (D-001810-10): 5′-UGGUUUACAUGUCGACUAA-3′, 5′-UGGUUUACAUGUUGUGUGA-3′, 5′-UGGUUUACAUGUUUUCUGA-3′, and 5′-UGGUUUACAUGUUUUCCUA-3′. The Amaxa Nucleofector device (Lonza) was used to electroporate cells using program T-024 and nucleofector kit V according to manufacturer’s instructions. Antibodies, immunoblotting, immunoprecipitation, and immunofluorescence The following antibodies were used for immunoblotting: goat anti-ATG7 for human (sc-8668, 1:200; Santa Cruz Biotechnology, Inc.), rabbit anti-ATG12 for human (2010, 1:500; Cell Signaling), rabbit anti-ATG7 for mouse (2631, 1:500; Cell Signaling), rabbit anti-ATG12 for mouse (2011, 1:500; Cell Signaling), guinea pig anti-p62/SQSTM1 (GP62-C, 1:1,000; Progen Biotechnik), mouse anti-NBR1 (H00004077-A01, 1:500; Abnova), rabbit anti-OPTN (ab23666, 1:1,000; Abcam), rabbit anti-CALCOCO2/NDP52 (ab68588, 1:500; Abcam), mouse anti-GFP (390394, 1:500; Santa Cruz Biotechnology, Inc.), mouse anti-paxillin (610051, 1:1,000; BD Biosciences), mouse anti-FAK (610087, 1:500; BD Biosciences), mouse anti-vinculin (V9131, 1:1,000; Sigma-Aldrich), mouse anti-zyxin (H00007791-M01, 1:500; Abnova), mouse anti-TUBA (T6199, 1:5,000; Sigma Aldrich), and mouse anti-GAPDH (AB2302, 1:5,000; Millipore). A rabbit polyclonal antibody against MAP1LC3 has been previously described ( Fung et al., 2008 ) and is now commercially available (ABC232, 1:1,000; Millipore). For immunoblot analysis, cells were lysed in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 25 mM Tris, pH 7.6, and 150 mM NaCl) plus protease inhibitor cocktail (Sigma-Aldrich), 10 mM NaF, 10 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 10 nM calyculin A, 0.5 mM PMSF, 10 µg/ml E64d, and 10 µg/ml pepstatin A. Lysates were freeze-thawed at –80°C, cleared by centrifugation for 30 min at 4°C, boiled in sample buffer, resolved by SDS-PAGE, and transferred to polyvinylidene fluoride membrane. Membranes were blocked for 1 h in 5% milk in PBS with 0.1% Tween 20, incubated in primary antibody overnight at 4°C, washed, incubated for 1 h at RT with HRP-conjugated goat secondary antibodies (1:5,000; Jackson ImmunoResearch Laboratories), washed, and visualized via enhanced chemiluminescence (Thermo Fisher Scientific). For immunoprecipitation, cells expressing GFP or GFP-NBR1 were sparsely plated on fibronectin-coated (10 µg/ml in PBS) dishes and were lysed in nondenaturing lysis buffer (1% Triton X-100, 25 mM Tris HCl, pH 7.4, and 150 mM NaCl) plus protease inhibitor cocktail (Sigma-Aldrich), 10 mM N-ethylmaleimide, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 10 nM calyculin A, 0.5 mM PMSF, 10 µg/ml E64d, and 10 µg/ml pepstatin A. Lysates were precleared with protein A/G beads (Santa Cruz Biotechnology, Inc.) and normal rabbit IgG (Santa Cruz Biotechnology, Inc.) at 4°C and incubated overnight with rabbit anti-GFP primary antibody (ab6556; Abcam; 1 µg/200–300 µg lysate) at 4°C. Immune complexes were captured by incubation with protein A/G beads for 4 h at 4°C and then washed six times with PBS plus inhibitors, eluted with sample buffer, and analyzed by immunoblotting. For immunoblot analysis of paxillin from anti-GFP immunoprecipitates, rat anti–mouse HRP TrueBlot (18-8817-33, 1:1,000; Rockland Immunochemicals) was used. For immunofluorescence, mouse anti-paxillin (610619, 1:200; BD Biosciences) and rabbit anti-NBR1 (71703, 1:200; Novus) were used. Alexa Fluor 488–conjugated phalloidin (A12379, 1:200; Life Technologies) was used for imaging the F-actin cytoskeleton. Cells were fixed with 4% PFA for 20 min at RT, permeabilized with 0.5% Triton X-100 in PBS, rinsed with PBS-glycine, and blocked overnight at 4°C in blocking buffer (10% goat serum and 0.2% Triton X-100 in PBS). Cells were incubated with primary antibodies for 1 h at RT, washed, incubated with Alexa Fluor 488 or 594 goat secondary antibodies (1:500; Life Technologies) for 40 min at RT, washed, and mounted using Prolong Gold Anti-Fade mounting medium (Life Technologies).

Microscopy

For all live-cell imaging and static images of cells during wound healing, cells were imaged in the assay media described for scratch wound-healing migration assay. For cells expressing paxillin-mTurquoise, phenol red-free DMEM/F12 was used to prepare assay media. Fixed specimens for immunofluorescence and SIM were mounted with hard-set Prolong Gold Anti-Fade mounting medium for imaging. Fixed cells for cell spreading assays were imaged in PBS. Static phase contrast and epifluorescence images were obtained at ambient temperature using an Axiovert 200 microscope (Carl Zeiss) with a 10× (NA, 0.25) or 20× (NA, 0.4) objective, Spot RT camera (Diagnostic Instruments), and mercury lamp (for ZsGreen). Images were acquired using MetaMorph software (v6.0; Molecular Devices). Phase-contrast microscopy time-lapse sequences were acquired on a TE 2000–inverted microscope stand (Nikon) with a CoolSNAP HQ2 scientific grade interline charge coupled device camera (Photometrics) and a 40× (NA, 0.6) objective (CFI Plan Fluor ELWD DM; Nikon) housed in an environmentally controlled chamber at 37°C. Microscope hardware and image acquisition were controlled by NIS-Elements software (Nikon). Spinning disk confocal imaging was performed as previously described ( Stehbens et al., 2014 ; Stehbens and Wittmann, 2014 ) using an environmentally controlled TI-inverted microscope stand (Nikon) equipped with a Borealis-modified Yokogawa CSU-X1 confocal head (Spectral Applied Research), solid-state 442-nm (for mTurquoise), 488-nm (for GFP, ZsGreen, and Alexa Fluor 488), 515-nm (for Venus), and 561-nm (for mCherry and Alexa Fluor 594) lasers, and a Clara cooled scientific-grade interline CCD camera (Andor) or a CoolSNAP MYO cooled scientific-grade CCD camera (Photometrics). Intracellular fluorescent-tagged protein dynamics in live cells were imaged at 37°C using a 60× (NA, 1.49; oil) objective (CFI Apochromat TIRF; Nikon), and immunofluorescence images were acquired at 37°C using a 100× (NA, 1.49; oil) objective (CFI Aprochromat; Nikon). Live-cell spreading of ZsGreen cells was imaged at 37°C using a 10× (NA, 0.45) objective (CFI Plan Apochromat; Nikon). TIRF microscopy of live cells was performed at 37°C on the same Nikon TI-inverted microscope stand equipped with a motorized TIRF illuminator (Nikon) and an iXon electron-multiplying CCD camera (Andor). TIRF images were acquired using a 100× objective (NA, 1.49; oil), with 1.5× intermediate magnification. Microscope hardware was controlled with NIS-Elements. Super-resolution SIM was performed on a Ti-E–inverted microscope stand (Nikon) with 488-nm (for GFP) and 561-nm (for mCherry) lasers and an iXon DU-897 electron multiplying CCD camera (Andor). Imaging was performed on fixed samples at ambient temperature using a 100× (NA, 1.49; oil) objective (CFI Aprochromat; Nikon). Microscope hardware was controlled with NIS-Elements. For analysis of all microscopy images, raw image data were used. Details of image analysis for each experimental technique and image processing for presentation are described in the following sections.

Scratch-wound healing migration assay

For assessment of wound closure by different cell types, monolayers were grown to confluency and incubated in the following media: assay media supplemented with 5 ng/ml EGF for wild-type MCF10A cells, DMEM with 2% FBS, 50 ng/ml EGF, penicillin, and streptomycin for PyMT cells, and DMEM supplemented with 2% FBS, penicillin, and streptomycin for iBMK cells. The proliferation inhibitor mitomycin C (1 µg/ml) was added to the cultures during assays. Confluent monolayers were wounded using a 200-µl pipette tip, washed several times to remove cell debris, and imaged at time of wounding (0 h) and the indicated time points. Wound widths were determined using MetaMorph software and were taken as the mean of six to nine measurements across the wound; to account for differences in starting wound width, data are reported as the decrease in wound width calculated by subtracting the final width from the initial width. For live-cell phase contrast imaging of migrating MCF10A-Ras cells, cells were grown to confluency on 3.5-cm glass bottom dishes (Mattek), incubated in assay media, and wounded. Image fields were randomly chosen along the length of the wound, and images were acquired every 3 min for 3 h. Single-cell tracking analysis was performed in NIS-Elements by randomly choosing one to two cells per image field and using the “Tracking” feature to manually track cell nucleoli (discerned as dark spots in the nucleus) for each frame over the time course; time and position data were then used to create single-cell migration paths and calculate migration speed as the total distance traveled divided by total time ( d/t f − t 0 ) for each cell over 3 h.

Cell-spreading assay

Coverslips in 24-well dishes and glass bottom six-well dishes (Mattek) were prepared by coating overnight at 4°C with 10 µg/ml fibronectin in PBS and then blocking with 1% BSA in DMEM/F12 for 30 min at 37°C. Subconfluent monolayers of MCF10A-Ras cells expressing ZsGreen were incubated overnight in assay media, harvested with fresh aliquots of 0.05% trypsin/EDTA diluted 1:1 with PBS, and sparsely plated in assay media. To synchronize attachment and initiation of spreading, plates were spun at 300 rpm in a swinging bucket rotor for 5 min immediately on plating cells. For live-cell confocal imaging of spreading, cells in six-well dishes were imaged every 5 min for 3 h. Cells on coverslips were incubated for 1 h, fixed in 4% PFA for 20 min at RT, and stored in PBS for imaging; epifluorescence images of fixed ZsGreen-expressing cells were used to quantify cell area using ImageJ (National Institutes of Health) by manually outlining cell borders. For each experiment, two coverslips per condition were plated, and four random fields per coverslip were imaged. All cells entirely present in each image field were measured.

FA size analysis

MCF10A-Ras cells were plated at confluency in assay media on coverslips coated with 10 µg/ml fibronectin, wounded, and fixed 4–6 h after wounding for anti-paxillin immunostaining. Confocal images of migrating cells were acquired randomly along the wound edge, and FAs at the leading edge of cells were manually outlined for area measurements using NIS-Elements. FAs from one to three cells were measured in each field, and the mean area of FAs per field was determined and plotted.

FA turnover assay and analysis

Analysis of dynamic

FA turnover in live cells was performed as previously described ( Stehbens et al., 2014 ; Stehbens and Wittmann, 2014 ). In brief, MCF10A-Ras cells expressing paxillin-mCherry were plated at confluency in assay media on 3.5-cm glass bottom dishes coated with 10 µg/ml fibronectin and wounded. 15–25 image fields were taken along the wound edge, and cells were imaged every 2–3 min for 1.5–3 h. For analysis, FAs were randomly chosen, and three to five FAs were measured per cell; only FAs that could be tracked completely from their appearance through disappearance were measured. To track FAs, the Bezier ROI tool was used to manually outline individual FAs and was redrawn in each frame as necessary over time if the FA significantly changed in size or location. The “Time Measurement” feature in NIS-Elements was used to generate fluorescence intensity data for each tracked FA, and background intensity was similarly determined using a duplicated region of interest placed adjacent to the FA. Background corrected intensity data were smoothed with a three-frame running mean and plotted against time. Smoothed fluorescence intensity curve plots were used to calculate FA assembly rate constant, disassembly rate constant, and lifetime by curve fitting in Excel (Microsoft). Assembly was determined by curve fitting the initial portion of the intensity plot during which fluorescence intensity steadily increases (when the FA is assembling) with a logistic function ( Fig. 2 B , green line). Disassembly was determined by curve fitting the latter portion of the intensity plot when fluorescence intensity is decreasing (FA disassembly) with an exponential function ( Fig. 2 B , red line). Rate constants for each parameter were obtained from these functions for each FA. The assembly and disassembly curve fits were also used to calculate FA lifetime, the time during which fluorescence intensity remained above half of the maximum intensity value ( Fig. 2 B , double black arrow). Analysis of GFP-LC3 and GFP-NBR1 targeting to FAs in live cells MCF10A-Ras cells coexpressing GFP-LC3 and paxillin-mCherry were imaged every 3 min during migration as described under FA turnover assay and analysis. For enumeration of GFP-LC3 targeting to FAs, 5–10 FAs were first randomly chosen per cell independent of the GFP channel, and then, the number of GFP-LC3 vesicles that associated with each FA was counted. Note that targeting was strictly defined as observable physical contact of GFP-LC3 vesicles with FAs ( Fig. 4 B ). Multiple targeting events were counted if the FA was targeted more than once over its lifetime or by multiple vesicles at the same time. Then, FAs were tracked and measured as described in FA turnover assay and analysis for generation of paxillin-mCherry fluorescence intensity plots to delineate assembly, stability, and disassembly phases of each targeted FA to determine the phase during which GFP-LC3 targeting events occurred. Qualitative live-cell analysis of GFP-LC3 association with dynamic FAs during spreading was performed by imaging cells within 30 min of plating at 1-min intervals. For analysis of enrichment of GFP-LC3 at FAs, three random frames per time lapse were chosen for each cell, and GFP-LC3 puncta at FAs and in non-FA areas were enumerated in the leading edge region. Total FA area and non-FA areas were manually measured for the leading edge, and the number of puncta per square micrometer FA or non-FA area was determined by dividing the total number of puncta for that region by the total area for that region. GFP-NBR1 enrichment at FAs was quantified in the same manner. Localization of mCherry-tagged FA proteins to autophagosomes For two-color imaging of paxillin and autophagosomes, MCF10A-Ras cells stably coexpressing GFP-LC3 and paxillin-mCherry as described for analysis of GFP-LC3 targeting to FAs were used. For two-color imaging of vinculin or zyxin and autophagosomes, MCF10A-Ras cells stably expressing GFP-LC3 were electroporated using the Amaxa nucleofector device as described for RNA interference with mCherry-vinculin or zyxin-mCherry. Cells were plated, wounded, and imaged by spinning disk confocal microscopy as described for FA turnover and analysis, and GFP/mCherry double-positive puncta were identified in migrating cells.

Analysis of Venus-LC3 and mCherry-NBR1 colocalization at FAs

MCF10A-Ras cells stably coexpressing paxillin-mTurquoise, Venus-LC3, and mCherry-NBR1 were plated and imaged by spinning disk confocal microscopy after wounding as described in FA turnover assay and analysis. For analysis, three random frames per time lapse were chosen for each cell. FA-associated Venus-LC3 puncta were identified using the paxillin-mTurquoise and Venus-LC3 channels. Then, the Venus-LC3 and mCherry-NBR1 channels were used to count the number of FA-associated Venus-LC3 puncta that were also mCherry-NBR1 positive.

Image processing

All image analysis was performed on raw image data; however, for presentation purposes, images were processed using established methods ( Stehbens et al., 2014 ). In NIS-Elements, 14-bit spinning disk confocal microscopy time-lapse sequences of paxillin-mCherry were corrected for photobleaching over time using the “Equalize Intensity in Time” tool, and reduction of pixel noise and enhancement of contrast were performed using a low-pass filter and unsharp mask, respectively. Images were then linearly adjusted as needed for brightness and contrast and converted to 8 bit. “Complement Colors” was used to contrast invert images for visualization of black FAs on a white background. Throughout the figures, leading edge inset areas were cropped and rotated for closer visualization of FA dynamics. To generate two-color black/magenta images as previously described ( Stehbens et al., 2014 ) of GFP-LC3 and paxillin-mCherry or GFP-NBR1 and paxillin-mCherry, the GFP and mCherry channels were first separated and processed individually to 8-bit images as detailed for single-color time-lapse sequences of paxillin-mCherry. Next, the images were added using ND Image Arithmetics: GFP + mCherry, and subsequently combined by merging channels (red: GFP, green: GFP + mCherry (from ND Arithmetics), and blue: GFP). The merged image was then contrast inverted using “Complement Colors” to create black/magenta overlays with magenta FAs and black vesicles on a white background. Two-color immunofluorescence (Alexa Fluor 488/Alexa Fluor 594) and live-cell (GFP/mCherry) images were processed to 8 bit as described for single-color time-lapse sequences of paxillin-mCherry, and green/magenta overlays were created in NIS-Elements using merge channels (red: 561 nm, green: 488 nm, and blue: 561 nm). Three-color (mTurquoise/Venus/mCherry) live-cell images were also processed as described for two-color images in NIS-Elements. Finally, both phase contrast and epifluorescence images were linearly brightness and contrast adjusted to better discern cell bodies and outlines in MetaMorph or ImageJ.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software). The normality of the distribution of datasets was determined by a Shapiro–Wilk normality test (P < 0.05, indicating a non-normal distribution); for datasets in which the sample size was not large enough ( n ≤ 6) for determination of normality, a normal distribution was assumed. For normal distributions, groups were compared using unpaired t test or one-way analysis of variance followed by Tukey post-hoc test for multiple comparisons. For nonparametric statistics, a Mann-Whitney test or Kruskall-Wallis test followed by Dunn’s post-hoc test for multiple comparisons was used. P < 0.05 was considered to be significant for all tests. Online supplemental materials Fig. S1 shows that inhibition of autophagy caused by loss of ATGs impairs migration and leads to enlarged FAs in multiple epithelial cell types. Fig. S2 shows SIM and TIRF imaging of autophagosomes at FAs and presence of mCherry-tagged FA proteins at autophagosomes. Fig. S3 shows validation of RNA interference against autophagy cargo receptors, no changes in basal autophagic flux with NBR1 depletion, and localization of NBR1 to FAs. Fig. S4 shows that loss of NBR1 leads to enhanced cell spreading. Fig. S5 shows interaction of GFP-NBR1 with endogenous FA proteins and representative paxillin-mCherry time-lapse sequences of cells expressing GFP, GFP-NBR1, GFP-NBR1 ΔLIR, or GFP-NBR1 ΔUBA. Video 1 shows migration of autophagy-inhibited cells. Videos 2 and 3 show FA dynamics upon autophagy inhibition in migrating cells expressing paxillin-mCherry. Video 4 shows a migrating cell expressing GFP-LC3 and paxillin-mCherry. Videos 5 and 6 show FA dynamics in a migrating cell expressing GFP-LC3 and paxillin-mCherry. Videos 7 and 8 show FA dynamics upon depletion of NBR1 in migrating cells expressing paxillin-mCherry. Video 9 shows the leading edge of a migrating cell expressing GFP-NBR1 and paxillin-mCherry. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201503075/DC1 .

Online supplemental materials Fig. S1 shows that inhibition of autophagy caused by loss of ATGs impairs migration and leads to enlarged FAs in multiple epithelial cell types. Fig. S2 shows SIM and TIRF imaging of autophagosomes at FAs and presence of mCherry-tagged FA proteins at autophagosomes. Fig. S3 shows validation of RNA interference against autophagy cargo receptors, no changes in basal autophagic flux with NBR1 depletion, and localization of NBR1 to FAs. Fig. S4 shows that loss of NBR1 leads to enhanced cell spreading. Fig. S5 shows interaction of GFP-NBR1 with endogenous FA proteins and representative paxillin-mCherry time-lapse sequences of cells expressing GFP, GFP-NBR1, GFP-NBR1 ΔLIR, or GFP-NBR1 ΔUBA. Video 1 shows migration of autophagy-inhibited cells. Videos 2 and 3 show FA dynamics upon autophagy inhibition in migrating cells expressing paxillin-mCherry. Video 4 shows a migrating cell expressing GFP-LC3 and paxillin-mCherry. Videos 5 and 6 show FA dynamics in a migrating cell expressing GFP-LC3 and paxillin-mCherry. Videos 7 and 8 show FA dynamics upon depletion of NBR1 in migrating cells expressing paxillin-mCherry. Video 9 shows the leading edge of a migrating cell expressing GFP-NBR1 and paxillin-mCherry. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201503075/DC1 .

Supplementary Material Supplemental Materials (PDF) Video 1 Video 3 Video 7 Video 2 Video 4 Video 9 Video 6 Video 5 Video 8 Video 1