MOLECULAR DETERMINANTS OF SELECTIVE CLEARANCE OF PROTEIN INCLUSIONS BY AUTOPHAGY

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1 Nature Communications Supporting Information MOLECULAR DETERMINANTS OF SELECTIVE CLEARANCE OF PROTEIN INCLUSIONS BY AUTOPHAGY Esther Wong, Eloy Bejarano, Moumita Rakshit, Karen Lee, Hugo H. Hanson, Nava Zaarur, Greg R. Phillips, Michael Y. Sherman, and Ana Maria Cuervo Supplementary Figures 1-12 Supplementary Methods

2 Supplementary Figure S1. Sph1 contains molecular determinants for aggresome and aggregate formation. (a) Schematic diagram depicting different molecular domains in the full-length and various truncated Sph1 proteins used in this study. (b) Protein expression levels of various GFP-Sph1 proteins in SY5Y cells in the absence or presence of proteasome inhibitor, lactacystin. (-) indicates untransfected control. (c) Parameters utilized throughout this work for distinguishing aggresome (Agm) from aggregates (Agg). Representative examples using FL GFP-Sph1 protein are shown on the right. (d) Representative fluorescence images of Agm and Agg formed by various GFP-Sph1 proteins in SY5Y cells under proteasomal inhibition. (e) Relative propensity of the full-length and various truncated Sph1 proteins to form Agm and Agg. Graphs reflect the percentage of transfected cells containing either Agm or Agg and are mean + SE of 3 different experiments. 2

3 Supplementary Figure S2. Effect of modulating autophagy on the characteristics of aggregates formed by different Sph1 variants. Autophagy was blocked (by treatment with 3-methyladenine; 3-MA) or upregulated (by removal of serum; S-) in SY5Y cells expressing the indicated forms of Sph1 proteins. Graphs depict the number of aggregates per cell profile (a), percentage of cellular area occupied by the aggregates (b) and the average size of the aggregates (c). Values are mean + SE of the quantification of >10 cells in 2 different experiments. (*) Significant versus basal serum supplemented condition (S+) for P= 0.01 (t-test). 3

Supplementary Figure S3. Cells transfected with different GFP-Sph1 variants show similar levels of viability as untransfected cells under various treatment conditions used in this study.

4 Supplementary Figure S3. Cells transfected with different GFP-Sph1 variants show similar levels of viability as untransfected cells under various treatment conditions used in this study. Viability of SY5Y cells untransfected or transfected with the indicated Sph1 constructs maintained in serumsupplemented (serum+) or serum-free (serum-) media in the absence or presence of lactacystin (Lacta) or combination of lactacystin and 3-methyladenine (Lacta/3-MA). Values are mean + SE of 3 different experiments. 4

$Supplementary Figure S4. Only ANK1 containing GFP-Sph1 variants accumulate in the insoluble pellet fraction upon inhibition of autophagy.$

5 Supplementary Figure S4. Only ANK1 containing GFP-Sph1 variants accumulate in the insoluble pellet fraction upon inhibition of autophagy. Left: Immunoblots for GFP and actin of cell extracts sequentially prepared with Triton X-100 (Soluble) and SDS (Pellet) from SY5Y cells transfected with ANK1-containing GFP-Sph1 ACA (a) and NA (b) or GFP-Sph1 CACT lacking the ANK1 domain (c) and maintained in serum-supplemented (serum+) or serum-free (serum-) media in the absence or presence of 3-methyladenine (3-MA). Right: Densitometric quantification of blots. Values are expressed relative to the levels of the proteins in cells maintained in the presence of serum. Values are mean + SE of 3 different experiments. 5

6 Supplementary Figure S5. Effect of latrunculin A on aggresome formation and basal autophagy. (a-b) SY5Y cells expressing the indicated Sph1 proteins were treated as in Fig. 1a, and then incubated in serum+ media in the absence or presence of 3-MA or 0.2µM latrunculin A (Lat A). (a) Prolonged 17h Lat A treatment perturbs formation of FL Sph1 Agm and induces accumulation of FL Sph1 Agg. (b) Short 6h Lat A treatment did not inhibit Agm formation but we observed significant accumulation of ANK1-containing Agg (right column) similar to when autophagy was blocked. Values are expressed as folds the amount of cells positive for Agm or Agg in untreated cells which were given an arbitrary value of 1. Values are mean + SE of 3 different experiments. (*) Significant versus untreated. P= 0.03 (t-test). 6

7 Figure S6. Effect of expression of GFP tagged p38 and ANK1-p38 on cellular autophagic activity. (a) GFP and p38 immunoblots of GFP-p38 (clones #3, 4 and 5) and GFP-ANK1-p38 (clones #9, 29, 31) fusion proteins expressed in SY5Y cells. Lane (-) represents untransfected control. (b) LC3 immunoblots of SY5Y cells transfected with an empty vector, p38 or ANK1-p38 in serum+ or serum- conditions treated or not with lysosomal inhibitors NH 4 CI and leupeptin (N/L). (c) Autophagic flux calculated as the increase in the amount of LC3-II upon blockage of lysosomal degradation in each condition. 7

then immunostained for the lysosomal marker LAMP1.

8 Supplementary Figure S7. Effect of different Sph1 proteins on the association of LAMP1 with p38 aggresomes. SY5Y cells expressing HA-p38 together with the indicated Sph1 protein variants were maintained in the absence of serum and then immunostained for the lysosomal marker LAMP1. The Individual channels, merge of the three channels and the negative black and white images of the LAMP1 channel are shown. Yellow and black arrows indicate the association of Agm with LAMP1 in each image. 8

9 Supplementary Figure S8. FRAP analysis of the dynamics of mcherry-p62 in different types of aggresomes. Representative images before (prebleach) and after photobleaching to show the dynamics of mcherryp62 in the p62 sequestosome (control) and in Agm formed by the indicated aggregation-prone proteins. Time courses and mean values of diffusing fraction and t 1/2 are shown in Fig. 5b. 9

(b-e) Comparison of autophagic activity in control and p62 KD SY5Y cells (b), WT and p62 KO MEFs (c), control and NBR1 KD SY5Y cells (d), and control and p62/nbr1 double KD cells (e) by measuring LC3

10 Figure S9. Autophagic activity remains intact in various p62 and NBR1 KO or KD cells. (a) Formation of p38 and ANK1-p38 Agm under proteasome inhibition is not significantly affected in p62 KD cells compared to control cells. (b-e) Comparison of autophagic activity in control and p62 KD SY5Y cells (b), WT and p62 KO MEFs (c), control and NBR1 KD SY5Y cells (d), and control and p62/nbr1 double KD cells (e) by measuring LC3 flux. Left: Representative immunoblots of LC3-II and actin in total cell lysates from the respective cells treated or not with lysosomal inhibitors NH 4 CI and leupeptin (N/L) maintained in the presence (serum+) or absence of serum (serum-) for 4h. Right: Net LC3-II autophagic flux in the control and various KO or KD cells calculated as the increase in the amount of LC3-II upon blockage of lysosomal degradation under serum+ and serum- conditions and expressed as times the values of the control cells under serum+ condition. Values are mean + SE of 3 different experiments and significance calculated using t-test. 10

decrease in p62 and NBR1 protein levels.

(Ctr), p62 knockdown (p62 KD), NBR1 KD and p62/nbr1 double KD SY5Y cells in

(d) Relative fold difference in the number of FL Agg under basal serum+

11 Supplementary Figure S10. Autophagic clearance of ANK1 containing Sph1 Agm and Agg is not affected by decrease in p62 and NBR1 protein levels. (a-c) Autophagic clearance of ANK1-containing Sph1 Agm and Agg in control (Ctr), p62 knockdown (p62 KD), NBR1 KD and p62/nbr1 double KD SY5Y cells in response to serum removal. (d) Relative fold difference in the number of FL Agg under basal serum+ conditions treated or not with 3-MA. Values are mean + SE (n=3). (*) Significant versus serum+ condition. P= 0.04 (t-test) (e) Fluorescence images showing colocalization patterns of FL Agg (green) with endogenous p62 and NBR1 (red). 11

12 Supplementary Figure S11. Autophagic clearance of ANK1 containing Sph1 Agm is not affected in p62 knockout MEF. (a) Representative fluorescence images of Agm and Agg formed by various GFP-Sph1 proteins in p62 KO MEF under proteasomal inhibition. (b) Autophagic clearance of various GFP-Sph1 Agm and Agg in WT and p62 KO MEFs in response to serum removal. Values are mean + SE (n=3). (*) Significant versus serum+ condition. P= (t-test). 12

13 Supplementary Figure S12. FRAP analysis of the dynamics of LysoTracker in different types of Agm. FRAP analysis of the dynamics of LysoTracker in stained lysosomes alone or in lysosomes close to various types of Agm. Values are mean + SE of 10 cells analyzed over 3 different experiments. 13

14 Supplementary Methods Transfections. For microscopy experiments, 2.5 x 10 4 SY5Y cells and MEFs were seeded on coverslips for transient transfection with various expression vectors. 0.25µg DNA was used for all single transfection reactions except for GFP-CACT where 1µg DNA was used. For co-expression of two proteins, 0.25µg of each plasmid DNA was used. For immunoblotting and immunoprecipitation, 2 x 10 5 SY5Y cells were seeded in 6 well plates for transfection with 0.5µg of DNA in single transfection or 0.5µg of each plasmid DNA in co-transfection reactions. Inclusion formation and autophagic clearance. The susceptibility of various inclusions formed upon proteasomal inhibition toward autophagic removal was analyzed as described. 24h post-transfection, cells were first treated with 5μM lactacystin (12h), a proteasome inhibitor, to enhance aggresome-like inclusion formation. To analyze the effect of basal quality control autophagy on the inclusion number, the lactacystin treated cells were allowed to recover in serum containing media either in the presence or absence of autophagy inhibitor, 10mM 3-MA (Sigma) for 24h. Alternatively, basal quality control autophagy in the treated cells was inhibited by 6h treatment with latrunculin A (Invitrogen). To analyze the effect of stimulated autophagy on the inclusion number, a concurrent set of treated cells were washed extensively before incubating in serum free media to stimulate autophagy either in the presence or absence of 3-MA. Thereafter, the cells were fixed in 3% formaldehyde and the number of cells possessing aggresomes or aggregates were visualized using an Axiovert fluorescence microscope (Carl Zeiss Ltd., Thornwood, NY). Random fields of each slide were taken and a minimum of 200 SY5Y cells and 100 MEFs were counted for each experiment. Agm and Agg were scored according to the characteristics described in Fig. S1c. Briefly, an Agm-containing cell is that displaying a distinct large perinuclear structure of > 5μM, whereas Agg-containing cells were considering those containing 2 or more small aggregate structures of < 3μM disperse throughout the cells. For allocation purposes, those cells containing a distinct perinuclear large aggresomal structure, even in the presence of smaller aggregates, were scored as Agm-containing cells. In addition of counting the percent of cells containing Agg or Agm, where indicated, the percentage of cellular area occupied by these structures was calculated, by tracking individual cellular profiles and, after thresholding, comparing the total cellular area and the area of the fluorescent compartment. 14

15 Cell viability measurement. Viability of cells transfected with the different GFP-Sph1 variants as well as untransfected SY5Y control cells incubated under the various treatment conditions was measured using CellTiter-Blue Cell Viability Assay according to the manufacturer s instructions (Promega). Immunoblot and immunoprecipitation. Lysates were prepared by suspending the cells in ice cold RIPA lysis buffer. Protein was determined by the Lowry method, using BSA as a standard. 100ug of protein per sample was loaded for LC3-II flux analysis while 50-75µg of protein was used in other immunoblotting. In some cases, sequential fractionation of cell lysates into Triton-X-solubilized (Soluble) and SDS-solubilized (Pellet) fractions was performed as previously described 23. After SDS-PAGE and immunoblotting, the proteins recognized by the specific antibodies were visualized by chemiluminescence methods (Western lightning ECL plus, PerkinElmer). The intensity of the bands was quantified using the ImageJ software (NIH). For immunoprecipitation, cells co-transfected with either pcdna vector or various HA-ubiquitin constructs and the GFP-p38 variants or FL GFP-Sph1 variants were lysed in RIPA lysis buffer containing 6M Urea with brief sonication. Immunoprecipitation (IP) was performed with anti-gfp antibody at 4 C overnight. The IP mixtures were centrifuged at 800g for 5min to remove any precipitates prior to the addition of protein A/G beads (Amersham Biosciences) for further incubation of another 1h. Immunoprecipitates were washed three times with lysis buffer and subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Live cell imaging and photobleaching. SY5Y cells expressing GFP tagged p38, ANK1-38, ANK1-p38 K385R mutant and mcherry-p62 separately or in combination were cultured on glass bottom 35mm culture dishes (MatTek). Cells were maintained in Minimum Essential Medium without phenol red supplemented with 10% FBS, sodium bicarbonate and HEPES at 37ºC for the duration of the experiment. Fluorescence recovery after photobleaching (FRAP) analysis was performed 24h after transfection using either the 488nm or 543nm laser wavelength for GFP and mcherry constructs respectively. Singles nonsaturated images were taken with a 63x 1.4 NA objective and a pinhole diameter equivalent to 4-5 Airy units at 512x512 pixels using a Leica TCS SP5 inverted confocal microscope (Leica Microsystems Inc.). Selective photobleaching of the aggresome area was carried out at % laser power until the fluorescence bleached away from the aggresome. The recovery of fluorescence in the region of interest was monitored by capturing images every 3 seconds at a low power of laser in a time course sequence. 15

16 Before bleaching, 3-4 images at low intensity illumination were taken to establish the fluorescence baseline. FRAP quantification analysis was performed as described previously 37. Average aggresomeassociated fluorescence intensity was determined using Leica Confocal software and the relative fluorescence intensity (RFI) was calculated as the average of at least three data points. RFI values were calculated using the equation (Ne t /N1 t )/(Ne 0 /N1 0 ) where Ne t represents the average intensity of bleachedaggresome at each time point and N1 t is the average intensity of non-bleached area in the neighboring cell at this given point which functions as control for global photobleaching. Ne 0 /N1 0 represents the ratio average intensity of the bleached and non-bleached area before photobleaching. FRAP data were fit using Sigma Plot Software to two-phase exponential method as described for other proteins in aggresomes 37. Same procedures were carried out for FRAP analysis of LysoTracker stained lysosomes. Correlative light and electron microscopy. SY5Y cells expressing GFP tagged NA Sph1, CACT Sph1, p38 and ANK1-p38 proteins grown on grid glass bottom 35mm culture dish were recovered in serum-free medium for 6h after proteasome inhibition. 5µM vinblastine was added for the last 2h of starvation to enhance the presence of autophagosomes. Thereafter the cells were fixed with 4% glutaraldehyde in 0.1M sodium cacodylate buffer with 1mM CaCl 2. Confocal stacks and DIC images of transfected cells were acquired on a Zeiss LSM 510 META microscope. Cells were embedded in Epon and processed as described The imaged cell was relocated in the block face and sectioned through. Sections were contrasted with lead citrate and uranyl acetate and serial sections of the cell of interest were documented at 10,000, 15,000 and 30,000x magnifications on a Hitachi 7000 electron microscope. Confocal, DIC and TEM images were realigned and oriented using nuclear and other morphological landmarks. References 37 Kim, S., Nollen, E. A. A., Kitagawa, K., Bindokas, V. P. & Morimoto, R. I. Polyglutamine protein aggregates are dynamic. Nat. Cell Biol. 4, (2002). 38 Hanson, H. H. et al. LC3-dependent intracellular membrane tubules induced by gammaprotocadherins A3 and B2: A role for intraluminal interactions. J. Biol. Chem. 285, (2010). 16

17 39 Hanson, H. H., Reilly, J. E., Lee, R., Janssen, W. G. & Phillips, G. R. Streamlined embedding of cell monolayers on gridded live imaging dishes for correlative light and electron microscopy. Microsc. Microanal. 20, 1-8 (2010). 17

T H E J O U R N A L O F C E L L B I O L O G Y

T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Rainero et al., http://www.jcb.org/cgi/content/full/jcb.201109112/dc1 Figure S1. The expression of DGK- is reduced upon transfection