RNAseq data.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.</div

Figs S1-S15.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.</div

TDP-43 screen hits (less aggregation).

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.</div

APEX2 proximity labeling reveals SRRD in close proximity to intermediate filaments and regulators of IF oligomerization.

1) HEK293Ts stably expressing SRRD-HA stained for HA and either CANX or mitochondria. B) Schematic of APEX2 proximity labeling experiment where APEX2 is fused to SRRD or to an NES control. C) Volcano plot of APEX2 proximity labeling mass spectrometry output, where fold change (x-axis) is plotted by significance (y-axis). Colored dots correspond to STRING clusters in 2E. (*) correspond to indicate functional annotations of interest highlighted in STRING cluster in 2E. D) Filtered GSEA (cellular compartment) of SRRD-APEX2 dataset. E) Clustering of top protein-protein interactions (STRING database) of top 88 proteins ranked by fold change and p-value. Clusters generated with MCL clustering and excludes proteins with no known connections and clusters with insignificant p-values. Clusters colored based on STRING annotated GO terms and proteins with functional annotations of interested are highlighted as follows: Protein-disulfide isomerases circled in pink, proteins involved in calcium binding circled in orange.</p

Antibodies used.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.</div

Plasmids submitted to AddGene.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.</div

SRRD regulates efficient assembly of aggresomes.

A) WT, SRRD clonal KO, and SRRD clonal KO + SRRD-mRuby3 293Ts treated for 16hrs with 5μM MG132 or DMSO control and stained for VIM and HDAC6. B) Quantification of the percentage of cells harboring aggresomes (perinuclear, HDAC6+, VIM cage+) after MG132 treatment. Dots indicate replicate wells treated, stained, and imaged in parallel. n = 4 replicates, one way anova with Tukey HSD test. Adjusted p-values WT:SRRD KO = 0; KO:rescue = 2.71e-09. C) WT, SRRD clonal KO, and SRRD clonal KO + SRRD-mRuby3 293Ts transfected with mClover3-TDP-43 ΔNLS and stained for VIM. D) Line intensity plots of TDP-43 ΔNLS (green) and VIM (blue) signals corresponding to white lines drawn in 3C. E) Line intensity drawings aggregating TDP-43 ΔNLS intensity data from WT, SRRD clonal KO, and SRRD clonal KO + SRRD-mRuby3 293Ts. Solid line indicates average value at each point, and shaded areas represent the standard deviation. F) Quantification of the percentage of TDP-43 ΔNLS aggregates that have at least a partial VIM cage surrounding it in WT, SRRD clonal KO, and SRRD clonal KO + SRRD-mRuby3 293Ts transfected with mClover3-TDP-43 ΔNLS and stained for VIM. G) WT, SRRD clonal KO, and SRRD clonal KO + SRRD-mRuby3 293Ts treated for 16hrs with 5μM MG132 and stained for SQSTM1 and HDAC6. H) Line intensity plots of SQSTM1 corresponding to white lines drawn in 3G. I) Line intensity drawings aggregating SQSTM1 data from WT, SRRD clonal KO, and SRRD clonal KO + SRRD-mRuby3 293Ts. Solid line indicates average value at each point, and shaded areas represent the standard deviation.</p

SRRD localizes to aggresomes following cellular proteotoxic stress.

A) Schematic of APEX2 proximity ligation assay with SRRD-V5-APEX2 clonal lines. B) Volcano plot of APEX2 proximity labeling mass spectrometry output, where fold change (x-axis) is plotted by significance (y-axis). Arrows indicate enrichment in +/- MG232 conditions, and each dot represents a protein. Colored dots correspond to STRING clusters in 2I. C) Clustering of top protein-protein interactions (STRING database) of top 100 proteins ranked by fold change and p-value associated with +MG132 (blue) and top associated proteins associated with -MG132 (red) conditions. Clusters generated with MCL clustering and excludes proteins with no known connections and clusters with insignificant p-values. Clusters colored based on STRING annotated GO terms. D) Enrichment analysis of ranked proteins after differential expression analysis of APEX +/- MG132 proximity labeling experiment using CORUM protein complex database. E) 293Ts expressing SRRD-mRuby3 treated with either 5μM MG132 or DMSO control for 16hrs, fixed and stained for VIM and HDAC6. F) 293Ts stably expressing SRRD-mRuby3 transfected with mClover3 control or mClover3-TDP-43 ΔNLS. G) Colocalization of mRuby3 and mClover3 in indicated protein pairs, measured in FIJI using Pearson’s correlation coefficient. Each dot represents correlation coefficient calculated for a single cell, boxes indicate median, upper, and lower quartiles. T-test p-values: SRRD-mClover:SRRD-TDP-43 = 1.055e-08; mRuby3-TDP-43:SRRD-TDP-43 = 7.305e-10.</p

APEX data with MG132.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.</div

Spacer sequences for sgRNAs used in validation studies.

Spacer sequences for sgRNAs used in validation studies.</p