Validation of an Algorithm to Quantify Changes in Actin Cytoskeletal Organization

Vindin, Howard; Bischof, Leanne; Gunning, Peter W.; Stehn, Justine R.

doi:10.1177/1087057113503494

Cited by 19 publications

(13 citation statements)

References 38 publications

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“…After treatment with LatA, we phalloidin stained U2OS cells and observe decreased F-actin complexfluorescence ( Fig. 3a) in agreement with previous findings 53 . With SIFTER, we observe a statistically significant 2.7-fold decrease in the median F-actin complex-levels upon LatA treatment (Mann-Whitney P-value < 0.0001, Fig.…”

Section: Quantifying Distributions Of Total Actin and Fratio Across Csupporting

confidence: 92%

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Vlassakis

Hansen

Higuchi‐Sanabria

et al. 2020

Preprint

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Multimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for robust endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Validating with actin-destabilizing Latrunculin A (LatA), we quantify 2.7-fold lower median F-actin complex-levels in LatA-treated single cells. Further clustering analysis of U2OS cells treated with LatA detects a subpopulation (~11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin, and find heat shock dysregulates F and monomeric G-actin correlation. The assay overcomes selectivity limitations of existing methods to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

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Section: Quantifying Distributions Of Total Actin and Fratio Across Csupporting

confidence: 92%

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Vlassakis

Hansen

Higuchi‐Sanabria

et al. 2020

Preprint

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“…In contrast, Tpm2.1 overexpressing clones showed numerous well‐defined actin filament cables that traversed the length of the cell (Figure D–F), a phenotype not seen in the other tropomyosin overexpressing clones (Bach, Schevzov, Bryce, Gunning, & O'neill, ; Bryce et al, ; Creed et al, ; Curthoys et al, ). To quantify actin filament bundles in a high throughput and nonbiased manner, a previously validated linear feature algorithm was used to detect actin filament bundles (Vindin, Bischof, Gunning, & Stehn, ). Digital output from this algorithm showed the accuracy of actin filament detection in both control (Figure C) and Tpm2.1 overexpressing clones (Figure F).…”

Section: Resultsmentioning

confidence: 99%

“…Quantification of the number of actin filament bundles per cell was conducted using a novel linear feature algorithm developed in collaboration with the CSIRO (Vindin et al, ). Briefly, cells were stained with Phalloidin‐488 and DAPI and images were uploaded into the software application.…”

Section: Methodsmentioning

confidence: 99%

Tumor suppressor tropomyosin Tpm2.1 regulates sensitivity to apoptosis beyond anoikis characterized by changes in the levels of intrinsic apoptosis proteins

2017

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The actin cytoskeleton is a polymer system that acts both as a sensor and mediator of apoptosis. Tropomyosins (Tpm) are a family of actin binding proteins that form co-polymers with actin and diversify actin filament function. Previous studies have shown that elevated expression of the tropomyosin isoform Tpm2.1 sensitized cells to apoptosis induced by cell detachment (anoikis) via an unknown mechanism. It is not yet known whether Tpm2.1 or other tropomyosin isoforms regulate sensitivity to apoptosis beyond anoikis. In this study, rat neuroepithelial cells overexpressing specific tropomyosin isoforms (Tpm1.7, Tpm2.1, Tpm3.1, and Tpm4.2) were screened for sensitivity to different classes of apoptotic stimuli, including both cytoskeletal and non-cytoskeletal targeting compounds. Results showed that elevated expression of tropomyosins in general inhibited apoptosis sensitivity to different stimuli. However, Tpm2.1 overexpression consistently enhanced sensitivity to anoikis as well as apoptosis induced by the actin targeting drug jasplakinolide (JASP). In contrast the cancer-associated isoform Tpm3.1 inhibited the induction of apoptosis by a range of agents. Treatment of Tpm2.1 overexpressing cells with JASP was accompanied by enhanced sensitivity to mitochondrial depolarization, a hallmark of intrinsic apoptosis. Moreover, Tpm2.1 overexpressing cells showed elevated levels of the apoptosis proteins Bak (proapoptotic), Mcl-1 (prosurvival), Bcl-2 (prosurvival) and phosphorylated p53 (Ser392). Finally, JASP treatment of Tpm2.1 cells caused significantly reduced Mcl-1, Bcl-2 and p53 (Ser392) levels relative to control cells. We therefore propose that Tpm2.1 regulates sensitivity to apoptosis beyond the scope of anoikis by modulating the expression of key intrinsic apoptosis proteins which primes the cell for death.

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“…Cells were seeded at 4 × 10 3 cells/well, 8 wells per clone, in a 96-well plate (Cell Carrier-96 Black Optically Clear Bottom plates, PerkinElmer) and cultured for 24 hrs. The fixation and immunostaining of cells was performed as previously described [ 40 ] with minor modifications. In brief, cells were fixed in 4% (w/v) paraformaldehyde/phosphate-buffered saline (PBS) for 30 min and washed twice with PBS.…”

Section: Methodsmentioning

confidence: 99%

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

et al. 2015

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The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

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Validation of an Algorithm to Quantify Changes in Actin Cytoskeletal Organization

Cited by 19 publications

References 38 publications

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Tumor suppressor tropomyosin Tpm2.1 regulates sensitivity to apoptosis beyond anoikis characterized by changes in the levels of intrinsic apoptosis proteins

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

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