1189449_Rozario,A_2021.pdf (5.73 MB)
Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy
journal contributionposted on 2022-05-06, 01:24 authored by AM Rozario, S Duwé, C Elliott, RB Hargreaves, GW Moseley, P Dedecker, Donna WhelanDonna Whelan, TDM Bell
Background: The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Results: We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30–80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics. Conclusions: Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.
Support from the Australian Research Council through its Discovery program (DP170104477) is gratefully acknowledged. Dr. Whelan is the recipient of an Australian Research Council Australian Discovery Early Career Research Award (DE200100584) funded by the Australian Government.
Article NumberARTN 260
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Science & TechnologyLife Sciences & BiomedicineBiologyLife Sciences & Biomedicine - Other TopicsSuper-resolutionMicrotubulesFilament curvatureLive cell imagingdSTORMSOFIAntimitoticColcemidCOLCHICINETUBULINRESOLUTIONAGENTSPOLYMERIZATIONORGANELLEBINDINGTARGETCytoskeletonDemecolcineMicroscopy, FluorescenceDevelopmental Biology