Phenotypical microRNA screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Hsu, Alan Y.; Wang, Dongqi; Liu, S.; Lu, Jian-chen; Sobreira, R. S.; Bennin, David A.; Huttenlocher, Anna; Wan, Jun; Deng, Qing

doi:10.1101/574335

Cited by 15 publications

(20 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ice-cold stop solution (20% TCA, 40 mM NaF and 20 mM β-glycerophosphate) was immediately added to the cells at 1:1 volume and put on ice for 1 h. Lysates were pelleted (14,000 g for 10 min) and washed once with 0.75 ml of ice-cold 0.5% TCA and resuspended in 2× Laemmli sample buffer. Western blotting was performed as described previously ( Hsu et al, 2019 ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

Zhou

Hsu

Wang

et al. 2020

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

Neutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (Mfn2) regulates neutrophil homeostasis and chemotaxis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue, trapped in the vasculature in zebrafish embryos, and not capable of chemotaxis. Consistently, human neutrophil-like cells deficient with MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis on 2D surfaces. Deletion of Mfn2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mechanistically, MFN2-deficient neutrophil-like cells display disrupted mitochondria-ER interaction, heightened intracellular calcium levels, and elevated Rac activation after chemokine stimulation. Restoring mitochondria-ER tether rescues the abnormal calcium levels, Rac hyperactivation, and chemotaxis defect resulted from MFN2 depletion. Finally, inhibition of Rac activation restores chemotaxis in MFN2-deficient neutrophils. Altogether, we identified that MFN2 regulates neutrophil migration via maintaining mitochondria-ER interaction to suppress Rac activation and uncovered a previously unrecognized role of MFN2 in regulating cell migration and the actin cytoskeleton.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Differentiated HL-60 NETosis induction was performed as described previously ( Hsu et al, 2019 ). Briefly, dHL-60 cells were resuspended in HBSS in 20 mM HEPES with 0.5% FBS and allowed to attach to fibrinogen-coated slides for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

Zhou

Hsu

Wang

et al. 2020

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…not have a dominant function but only impacts neutrophil mobility in the rac2 knockout background. Rac2 CA alone cannot coordinate neutrophil migration.Our previous study revealed an unexpected and critical role of Cdk2 in neutrophil migration and chemotaxis27 . To ensure that our neutrophil-specific knockout system is feasible for disrupting other genes, we injected plasmids carrying cdk2 sgRNAs into Tg(lyzC:cas9, cry:GFP) pu26 embryos.…”

mentioning

confidence: 89%

A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

Wang¹,

Hsu²,

Walton

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Tissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

show abstract

“…Lysates were pelleted and washed once with 0.75 ml of ice cold 0.5% TCA and resuspended in 2× Laemmli sample buffer (Bio-Rad Laboratories). Western blot was performed as described (Hsu et al, 2019) with anti-phospho-PAK (Cell signaling #2605S), anti-PAK (Cell signaling #2604) along with…”

Section: Western Blotmentioning

confidence: 99%

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

Zhou

Hsu

Wang

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Neutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (Mfn2) regulates neutrophil homeostasis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue and trapped in the vasculature in zebrafish embryos. Human neutrophil-like cells deficient with MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis. Deletion of Mfn2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mfn2, but not Mfn1, -null mouse embryonic fibroblast cells have altered actin structure and are impaired in wound closure. MFN2-deficient neutrophil-like cells display heightened intracellular calcium levels and Rac activation after chemokine stimulation. Mechanistically, MFN2 maintains mitochondria-ER interaction. Restoring mitochondria-ER tether rescues the chemotaxis defect and Rac activation resulted from MFN2 depletion. Finally, inhibition of Rac restores chemotaxis in MFN2-deficient neutrophils. Altogether, we identified that MFN2 regulates neutrophil migration via suppressing Rac activation and uncovered a previously unrecognized role of MFN2 in regulating the actin cytoskeleton.

show abstract

Phenotypical microRNA screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Cited by 15 publications

References 70 publications

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

Contact Info

Product

Resources

About