Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 μm wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.
The software is available on GitHub, the data are available on Dryad.
Viscoelastic properties of suspended cells measured with shear flow deformation cytometryDryad Digital Repository, doi:10.5061/dryad.5hqbzkh8p.
- Elham Mirzahossein
- Mar Eroles
- Mar Eroles
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Alphee Michelot, Institut de Biologie du Développement, France
© 2022, Gerum et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.
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