Assessment of protein inclusions in cultured cells using automated image analysis

McAlary, Luke; Shephard, V K; Sher, Mine; Rice, Lauren J.; Yerbury, Justin J.; Cashman, Neil R.; Plotkin, Steven S.

doi:10.1016/j.xpro.2022.101748

Cited by 5 publications

(8 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells expressing SOD1-EGFP constructs were imaged at time points 24, 48 and 72 h post transfection on the incucyte live cell imager (Sartorius) with a 10 x objective and excitation wavelength 470 nm. Images were illumination corrected using CellProfiler 28 software version 4.1 (Broad Institute, MIT Harvard) then analysed using CellPose 29 cell segmentation algorithm to count GFP positive cells across timepoints similar to our previous work 18 .…”

Section: Methodsmentioning

confidence: 99%

“…All of the variants are known FALS variants except for SOD1 I149D which is a synthetic variant designed to maximally destabilise the I149 site. Fluorescence microscopy and automated image analysis were used to determine the percentage of cells that contained inclusions and enumerate the number of GFP-positive cells across time to determine relative cell numbers 18 .…”

Section: Sod1 I149s Forms Inclusions and Is Toxic In A Motor Neuron-l...mentioning

confidence: 99%

See 1 more Smart Citation

Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1

Shephard,

Brown,

Thompson

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Variants of the oxygen free radical scavenging enzyme superoxide dismutase-1 (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These variants occur in roughly 20% of familial ALS cases, and 1% of sporadic ALS cases. Here, we identified a novel SOD1 variant in a patient in their 50s who presented with movement deficiencies and neuropsychiatric features. The variant was heterozygous and resulted in the isoleucine at position 149 being substituted with a serine (I149S). In silico analysis predicted the variant to be destabilising to the SOD1 protein structure. Expression of the SOD1I149S variant with a C-terminal EGFP tag in neuronal-like NSC-34 cells resulted in extensive inclusion formation and reduced cell viability. Immunoblotting revealed that the intramolecular disulphide between Cys57 and Cys146 was fully reduced for SOD1I149S. Furthermore, SOD1I149S was highly susceptible to proteolytic digestion, suggesting a large degree of instability to the protein fold. Finally, fluorescence correlation spectroscopy and native-PAGE of cell lysates showed that SOD1I149S was monomeric in solution in comparison to the dimeric SOD1WT. This experimental data was obtained within 3 months and resulted in the rapid re-classification of the variant from a variant of unknown significance to a clinically actionable likely pathogenic variant.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Sod1 I149s Forms Inclusions and Is Toxic In A Motor Neuron-l...mentioning

confidence: 99%

Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1

Shephard,

Brown,

Thompson

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“… For more information on how to use CellProfiler, please refer to publicly-available online tutorials ( https://cellprofiler.org/tutorials ) and step-by-step protocols. 3 , 4 , 5 …”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Protocol for neurogenin-2-mediated induction of human stem cell-derived neural progenitor cells

Guss,

Sathe,

Dai

et al. 2024

STAR Protocols

View full text Add to dashboard Cite

“…We followed previously published methods of image processing to quantify inclusions in our cells (39, 40) with some changes. Briefly, we performed illumination variation correction on all images using the background method.…”

Section: Methodsmentioning

confidence: 99%

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells

McAlary,

Nan,

Shyu

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified throughout the primary sequence, indicating that common structural features responsible for aggregation and toxicity may be present. Here, we used in silico tools to predict amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding 7 regions spread throughout the protein structure. We found that the introduction of proline residues into β-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in living cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in β-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that β-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

show abstract

Assessment of protein inclusions in cultured cells using automated image analysis

Cited by 5 publications

References 12 publications

Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1

Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1

Protocol for neurogenin-2-mediated induction of human stem cell-derived neural progenitor cells

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells

Contact Info

Product

Resources

About