title: Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts.
abstract: To explain how micrometer-sized cellular structures generate and respond to forces, we need to characterize their micromechanical properties. Here we provide a protocol to build and use a dual force-calibrated microneedle-based setup to quantitatively analyze the micromechanics of a metaphase spindle assembled in Xenopus laevis egg extracts. This cell-free extract system allows for controlled biochemical perturbations of spindle components. We describe how the microneedles are prepared and how they can be used to apply and measure forces. A multimode imaging system allows the tracking of microtubules, chromosomes and needle tips. This setup can be used to analyze the viscoelastic properties of the spindle on timescales ranging from minutes to sub-seconds. A typical experiment, along with data analysis, is also detailed. We anticipate that our protocol can be readily extended to analyze the micromechanics of other cellular structures assembled in cell-free extracts. The entire procedure can take 3-4 d.
authors.affiliation: Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York, USA.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Shimamoto
authors.forename: Tarun M
authors.initials: TM
authors.lastname: Kapoor
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena; D002457:Cell Extracts; D002474:Cell-Free System; D003584:Cytological Techniques / Q000295:instrumentation; D008941:Spindle Apparatus / Q000502:physiology* / Q000648:ultrastructure; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts