Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Representative images of aggregates ( green ) observed after transfection of TDP-43 216-414 (panel A), HTT-Q74 (panel B) , and GFP-HTT-Q74 (panel C) in both HEK and Neuro2A cells. Panels D-E show the expression pattern of FLAG-tagged proSAAS (panel D) and FLAG-tagged clusterin (panel E) in HEK and Neuro2A cells, both shown in red . Images were taken at 40X 24h after transient transfection. Scale bar 10 μm. Magnified images on the right correspond to 10 μm. Arrow in panel D indicates the spherical structures in HEK cells transfected with proSAAS cDNA.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Transfection, Expressing

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: Co-expression of proSAAS ( upper row ) and clusterin ( lower row ) with TDP-43 216-414 in HEK cells, showing aggregate morphology. Panel B: Effect of 12.5 nM thapsigargin in HEK cells after expression of TDP-43 216-414 alone ( upper row ) or in combination with proSAAS ( middle row ) or clusterin ( lower row ). Images were taken at 40X. Cells were transfected for 24 h following overnight treatment with thapsigargin (12.5 nM). Scale bar 10 μm. Magnified images correspond to 10 μm. Panels C and D represent quantitation of 80 cells taken from 3 experiments; the mean + SE is given. *, p<0.05.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Expressing, Transfection, Quantitation Assay

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: Cytoplasmic aggregates are present following co-expression of TDP-43 216-414 with proSAAS ( red ) ( upper row ) and clusterin ( red ) ( lower row ). Panel B: Thapsigargin treatment increases the co-localization of TDP-43 216-414 aggregates ( green ) with proSAAS in a subpopulation of Neuro2A cells which exhibit a differentiated morphology. Images were taken at 40X. Cells were transfected for 24 h following overnight treatment with thapsigargin (12.5 nM). Scale bar 10 μm. Magnified inserts correspond to 10 μm. Arrows indicate colocalization of proSAAS and TDP-43 216-414 .

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: Representative confocal images of Neuro2A cells transfected with cyto-proSAAS cDNA for either 24 h (left) or 48 h (right) showing characteristic proSAAS-immunoreactive spheres (shown enlarged at right). Panel B: TDP-43 216-414 aggregates ( upper row ), but not HTT-Q74 aggregates ( middle row ) are found inside cyto-proSAAS spheres ( red ) when both are co-expressed in Neuro2A cells. The lower row shows the characteristic cytosolic fluorescence of the control protein mCherry ( red ), which is not accumulated within the cyto-proSAAS spheres (green) when both are co-expressed in Neuro2A cells. Images taken at 40X, 24 h after transfection. Scale bar, 10 μm. Magnified inserts correspond to 10 μm.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Transfection, Fluorescence

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: cyto-proSAAS expression alone. Panel B : TDP-43 216-414 aggregates, but not HTT-Q74 aggregates (panel C) nor GFP-HTT-Q74 aggregates (panel D) are found inside cyto-proSAAS spheres ( red ) in HEK cells. Panel E shows the characteristic cytosolic fluorescence of dispersed mCherry ( red ) aggregates that do not become accumulated within the cyto-proSAAS spheres ( green ). Images were taken 24 h after transfection at 40X. Scale bar 10 μm. Magnified images correspond to 10 μm.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Expressing, Fluorescence, Transfection

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: Schematic view of the TDP-43 constructs used. TDP-43 86-414 ( panel B ) and TDP-43 170-414 ( panel C ) formed aggregates when expressed alone in HEK cells, and these were efficiently incorporated into cyto-proSAAS spheres when this protein was co-expressed. PrLD, prion-like domain; RRM, RNA binding domain; NES, nuclear export signal; NLS, nuclear localization signal. Panel D-F , Quantification of 3-dimensional Z-stacks for the size, dimensions, and intensity of TDP-43 214-416 aggregates present either inside cyto-proSAAS spheres (blue circles), or outside cyto-proSAAS spheres ( red squares). Panel D: Ratio of the total surface area of TDP-43 214-416 to the total volume of the same objects. Panel E: The mean pixel intensity for each group. Panel F: The total signal intensity of each group, determined by the sum of signal intensity of all pixels in that group. (n=12 cells, two-tailed paired t-test *p<0.05, **p<0.01, ***p<0.001). Panel G: Transfection of TDP-43 216-414 cDNA, followed 24 h later by transfection of cyto-proSAAS cDNA, also results in the detection of TDP-43 216-414 aggregates inside sphere cores, supporting the idea that the TDP-43 216-414 core can be formed later than the spheres. Images taken at 40X. Scale bar 10 μm. Magnified inserts correspond to 10 μm.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Construct, RNA Binding Assay, Two Tailed Test, Transfection

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panels A-D: Confocal images of HEK cells co-transfected with TDP-43 216-414 and cyto-proSAAS cDNAs. The great majority of transfected cells (93%) are similar to the cell shown in Panel A , with all aggregates present within the core of the cyto-proSAAS sphere. The remainder of the transfected cells (7%) exhibit the other types of aggregate distribution depicted in Panels B-D . In Panel E, a 3D visualization of the spherical cyto-proSAAS structures is shown. Panel F depicts electron microscopy images of HEK cells transfected only with TDP-43 216-414 . The characteristic TDP-43 216-414 aggregates are indicated with arrows, and a magnified image is shown at right . Panel G: Electron microscopy images of HEK cells expressing TDP-43 216-414 and cyto-proSAAS, showing the characteristic dense spheres. Scale bars of 10 μm are indicated in the images. Red, cyto-proSAAS, green , GFP-TDP-43 216-414 .

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Transfection, Electron Microscopy, Expressing

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: Lack of immunostaining of Neuro2A cells with TDP-43 antibody (C-terminal; in white) after co-expression of cyto-proSAAS ( red ) and TDP-43 216-414 ( green ), shows that this antibody cannot penetrate inside the sphere, supporting core inaccessibility. Arrows indicate fluorescent but immunologically undetected TDP-43 216-414 aggregates inside spheres . Panel B: Cyto-proSAAS spheres are likely not destined for autophagy, as no fluorescent signal was observed inside cyto-proSAAS spheres following triple transfection of Cherry-LC3, cyto-proSAAS and TDP-43 216-414 cDNAs. Panels C-D : Lack of cyto-proSAAS sphere immunostaining with sera against Lamp-1 ( panel C ) giantin ( panel D ) and HSP70 ( panel E ).

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Immunostaining, Expressing, Transfection

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel A: HEK cells transfected with cDNAs encoding TDP-43 216-414 alone, or in combination with cyto-proSAAS or clusterin, were monitored by Western blotting using an anti-GFP antibody (left panels). Quantitation is shown in the right panel; approximately 80% of TDP-43 216-414 disappears after 6 h of cycloheximide treatment when cells are co-transfected with either an empty vector or a clusterin-encoding vector; however, 43% remains in cells co-transfected with cyto-proSAAS (*, p<0.05). Panel B: Confocal images taken prior to (0 h) and following (6 h) cycloheximide treatment confirm reduced cellular quantities of TDP-43 216-414 (left) and clusterin (right) following cycloheximide treatment; in contrast, TDP-43 216-414 fluorescence is retained within cyto-proSAAS spheres (middle). In red , the percentage of cells positive for anti-FLAG clusterin/proSAAS immunostaining (300 cells analyzed, from 4 different fields).

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Transfection, Western Blot, Quantitation Assay, Plasmid Preparation, Fluorescence, Immunostaining

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel 6A. Cyto-proSAAS is not itself ubiquitinated but is associated with highly K48-ubiquitinated TDP-43 216-414 . Representative images of HEK cells expressing UbK48-HA with cyto-proSAAS (top row) or TDP-43 216-414 (middle row) or both (bottom row) . Immunostaining against FLAG and HA shows no colocalization of cyto-proSAAS and UbK48-HA (top row) . UbK48-HA is colocalized with TDP-43 216-414 ( middle row, notched arrowheads ). Increased colocalization of TDP-43 216-414 with UbK48-HA is observed in aggregates clustered proximal to the cyto-proSAAS spheres, suggesting preferential association ( bottom row, notched arrowheads ). Scale bar, 10 μm. Magnified inserts correspond to 10 νm. Panel 6B. Expression of either TDP-43 216-414 or cyto-proSAAS induces stress granule formation; stress granules are proximal to cyto-proSAAS spheres. Representative images of HEK cells expressing PABPC4 alone (top row) ; with TDP-43 216-414 (middle row); or with cyto-proSAAS (bottom row). Anti-HA staining shows diffuse PABPC4-HA-FLAG immunoreactivity when this marker is expressed alone (top row) , but punctate staining colocalized with TDP-43 216-414 in the presence of this protein ( middle row, notched arrowheads ). Immunostaining against HA and proSAAS reveals that PABPC4-HA-FLAG-positive puncta are proximal to cyto-proSAAS spheres (bottom row, empty arrowhead) . Scale bar, 10 μm. Magnified inserts correspond to 10 μm.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Expressing, Immunostaining, Staining, Marker

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Panel 7A. Expression of Cherry-cyto-proSAAS ( red ) generates dispersed cytosolic aggregates in Neuro2A cells ( top row ); co-transfection of Cherry-cyto-proSAAS with a five-fold excess of cyto-proSAAS ( green ) eliminates these dispersed aggregates and restores Cherry-tagged proSAAS fluorescence to the spheres ( bottom row ). Panel 7B. ProSAAS and 7B2 rescue TDP-43 cytotoxicity in a yeast model. Serial-dilution spotting assay of yeast expressing TDP-43 and the indicated chaperone from a galactose-inducible promoter ( left side ). The empty vector is used as a negative control for chaperone activity and shows TDP-43-induced growth impairment on galactose. The left spotting plate shows equal spotting and no growth impairment of yeast on glucose media, which does not induce TDP-43 or chaperone expression. The right plate shows growth phenotypes upon induction with galactose. Spotting data are representative images of four independent trials. The expression of TDP-43 and chaperones was detected via Western blotting; PGK1 is used as a loading control ( right side ).

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Expressing, Cotransfection, Fluorescence, Serial Dilution, Spotting Assay, Plasmid Preparation, Negative Control, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Phase separation of proSAAS into spheres results in core sequestration of TDP-43 216-414 aggregates

doi: 10.1101/2020.04.15.039578

Figure Lengend Snippet: Dynamic widefield microscopy of Cherry-cyto-proSAAS ( red ) and TDP-43 216-414 (green, with DIC) beginning 18 h post transfection at 2 min intervals for 5 h. See also Supplemental Movie 1. Panel A : Images shown at 24 min intervals (top) depict generation of multiple spheres, some containing TDP-43 216-414 aggregates which condense and fuse into larger spheres. A subset of images (box) is shown below at 2 min intervals, which captures two large spheres condensing into one. Panel B : Rarely, internal TDP43 216-414 was found to rapidly and wholly exit from the center of cyto-proSAAS spheres. Max projections are shown, but 3D analysis reveals TDP-43 216-424 to be entirely within the sphere at the beginning of the time course. Cyto-proSAAS, TDP-43 216-414 and Cherry-cyto-proSAAS were co-transfected in a ratio 1:1:0.2, as indicated in Methods. Scale bar, 10 μm. Magnified inserts correspond to 10 μm.

Article Snippet: The Addgene catalog numbers are as follows: GFP-tagged TDP-43 constructs TDP-43 216-414 , #28197; TDP-43 1-193 (#28202); TDP-43 1-273 (#28200); TDP-43 86-414 (#28195) and TDP-43 170-414 (#28196), described in ( ); HTT-Q74 (HTT exon 1 Q74, His- and HA-tagged, #40264, #40262); and GFP-HTT-Q74, in ( ); EGFP-Tau, #46904, and EGFP-TauE14, #46907 ( ); EGFP-α-Syn, #40822 and EGFP-α-Syn-A53T, #40823 ( ); and pEGFP-N1-FUS/TLS-FLAGC, #60362 ( ).

Techniques: Microscopy, Transfection