Person:

Mitchison, Timothy

Background: Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. Results: We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. Conclusions: The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

Background: 2,3-Butanedione monoxime (BDM) has been widely used as a non-muscle myosin inhibitor to investigate the role of non-muscle myosinII in the process of actin retrograde flow and other actin cytoskeletal processes. Recent reports show that BDM does not inhibit any non-muscle myosins so far tested, including nm-myosinII, prompting the question, how were these process affected in BDM studies? Results: We have found that treatment of mammalian cells with BDM for only 1 min blocks actin incorporation at the leading edge in a permeabilized cell system. We show that inhibition of actin incorporation occurs through de-localization of leading edge proteins involved in actin polymerization – the Arp2/3 complex, WAVE, and VASP – that de-localize concomitantly with the leading edge actin network. Conclusion: De-localization of actin leading edge components by BDM treatment is a newly described effect of this compound. It may explain many of the results previously ascribed to inhibition of non-muscle myosinII by BDM, particularly in studies of leading edge dynamics. Though this effect of BDM is intriguing, future studies probing actin dynamics at the leading edge should use more potent and specific inhibitors.