DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells

Harterink, Martin; Silva, Marta Santos Esteves da; Will, Lena; Turan, Julia; Ibrahim, Adiljan; Lang, Alexander E.; Battum, Eljo Y. van; Pasterkamp, R. Jeroen; Kapitein, Lukas C.; Kudryashov, Dmitri S.; Barres, Ben A.; Hoogenraad, Casper C.; Zuchero, J. Bradley

doi:10.1038/nmeth.4257

Cited by 56 publications

(51 citation statements)

References 39 publications

(51 reference statements)

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“…In adipocytes, actin severing protein regulates insulin-dependent GLUT4 translocation through dynamic remodeling of actin. It has been reported that ectopic overexpression of gelsolin but not cofilin 1 markedly accelerates F-actin dissociation (37). To explore whether modulation of actin dynamics affects insulin-dependent glucose uptake, we overexpressed gelsolin in differentiated 3T3-L1 adipocytes.…”

Section: Resultsmentioning

confidence: 99%

During Adipocyte Remodeling, Lipid Droplet Configurations Regulate Insulin Sensitivity through F-Actin and G-Actin Reorganization

Kim

Park

et al. 2019

Molecular and Cellular Biology

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Adipocytes have unique morphological traits in insulin sensitivity control. However, how the appearance of adipocytes can determine insulin sensitivity has not been understood. Here, we demonstrate that actin cytoskeleton reorganization upon lipid droplet (LD) configurations in adipocytes plays important roles in insulin-dependent glucose uptake by regulating GLUT4 trafficking. Compared to white adipocytes, brown/beige adipocytes with multilocular LDs exhibited well-developed filamentous actin (F-actin) structure and potentiated GLUT4 translocation to the plasma membrane in the presence of insulin. In contrast, LD enlargement and unilocularization in adipocytes downregulated cortical F-actin formation, eventually leading to decreased F-actin-to-globular actin (G-actin) ratio and suppression of insulin-dependent GLUT4 trafficking. Pharmacological inhibition of actin polymerization accompanied with impaired F/G-actin dynamics reduced glucose uptake in adipose tissue and conferred systemic insulin resistance in mice. Thus, our study reveals that adipocyte remodeling with different LD configurations could be an important factor to determine insulin sensitivity by modulating F/G-actin dynamics.

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Section: Resultsmentioning

confidence: 99%

During Adipocyte Remodeling, Lipid Droplet Configurations Regulate Insulin Sensitivity through F-Actin and G-Actin Reorganization

Kim

Park

et al. 2019

Molecular and Cellular Biology

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“…Additionally, we tested whether PCM‐1 down‐regulation or F‐actin disruption similarly affect neuronal differentiation in the developing cortex. We electroporated in utero control shRNA or PCM‐1 shRNA, together with Venus, and DeAct plasmid—which impairs F‐actin dynamics —at E15 and analyzed the neuronal morphology at E18 in situ . Importantly, we found that down‐regulating PCM‐1 in the developing cortex and disrupting F‐actin in newly born neurons promotes neurite elongation in a similar manner (Appendix Fig S11E–H).…”

Section: Resultsmentioning

confidence: 99%

“…A pSilencer vector containing a random sequence hairpin insert was used as a control for the shRNAs . M. Harterink (from the laboratory of C. Hoogenraad, Utrecht University) kindly provided the DeAct‐SpvB plasmid (that was cloned into the pGW2‐GFP vector) . The Centrin2‐KR fusion protein was generated by cloning the Centrin2 cDNA in‐frame with KillerRed, using the BamHI and XhoI cloning sites of the pKillerRed‐N vector (Evrogen) .…”

Section: Methodsmentioning

confidence: 99%

Radial somatic F‐actin organization affects growth cone dynamics during early neuronal development

et al. 2019

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The centrosome is thought to be the major neuronal microtubule‐organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin‐organizing center, raising the possibility that neuronal development may, in addition, require a centrosome‐based actin radial organization. Here, we report, using super‐resolution microscopy and live‐cell imaging of cultured rodent neurons, F‐actin organization around the centrosome with dynamic F‐actin aster‐like structures with F‐actin fibers extending and retracting actively. Photoactivation/photoconversion experiments and molecular manipulations of F‐actin stability reveal a robust flux of somatic F‐actin toward the cell periphery. Finally, we show that somatic F‐actin intermingles with centrosomal PCM‐1 (pericentriolar material 1 protein) satellites. Knockdown of PCM‐1 and disruption of centrosomal activity not only affect F‐actin dynamics near the centrosome but also in distal growth cones. Collectively, the data show a radial F‐actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, hence sustaining initial neuronal development.

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“…While these must be custom-developed for each protein of interest, some could prove particularly useful. For example, conditional expression of a peptide that disrupts actin polymerization (DeAct) has been used to interfere with the morphogenesis of a specific neuron (Harterink et al 2017).…”

Section: Other Methods For Conditionally Inhibiting Proteinsmentioning

confidence: 99%

The Caenorhabditis elegans Transgenic Toolbox

2019

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The power of any genetic model organism is, in part, derived from the ease with which gene expression can be manipulated. The short generation time and invariant developmental lineage have made Caenorhabditis elegans very useful for understanding, e.g., developmental programs, basic cell biology, neurobiology, and aging. Over the last decade, the C. elegans transgenic toolbox has expanded considerably with the addition of a variety of methods to control expression and modify genes with unprecedented resolution. Here we provide a comprehensive overview of transgenic methods in C. elegans, with an emphasis on recent advances in transposon-mediated transgenesis, CRISPR/Cas9 gene editing, conditional gene and protein inactivation, and bipartite systems for temporal and spatial control of expression.

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DeActs: genetically encoded tools for perturbing the actin cytoskeleton in single cells

Cited by 56 publications

References 39 publications

During Adipocyte Remodeling, Lipid Droplet Configurations Regulate Insulin Sensitivity through F-Actin and G-Actin Reorganization

During Adipocyte Remodeling, Lipid Droplet Configurations Regulate Insulin Sensitivity through F-Actin and G-Actin Reorganization

Radial somatic F‐actin organization affects growth cone dynamics during early neuronal development

The Caenorhabditis elegans Transgenic Toolbox

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