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Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

ABSTRACT: The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems-growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

SUBMITTER: Ory EC

PROVIDER: S-EPMC5738915 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

Ory Eleanor C EC Bhandary Lekhana L Boggs Amanda E AE Chakrabarti Kristi R KR Parker Joshua J Losert Wolfgang W Martin Stuart S SS

Physical biology 20170420 2

The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems-growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; ...[more]

PMID: 28092269

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Project description:Abstract The ability of glioma cells to invade adjacent brain tissue remains a major obstacle to therapeutic disease management. Therefore, the development of novel treatment modalities that disrupt glioma cell motility could facilitate greater disease control. Tumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. This novel loco-regional treatment has demonstrated efficacy and safety and is FDA-approved in patients with glioblastoma and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials, including the phase 3 METIS trial (brain metastases from NSCLC; NCT02831959). Although established as an anti-mitotic treatment, the anti-metastatic potential of TTFields and its effects on cytoskeleton rapid dynamics during cellular motility warrant further investigation. Previous studies have demonstrated that TTFields inhibits metastatic properties of cancer cells. Identification of a unifying mechanism connecting the versatile TTFields-induced molecular responses is required to optimize the therapeutic potential of TTFields. In this study, confocal microscopy, computational tools, and biochemical analyses were utilized to show that TTFields disrupt glioma cellular polarity by interfering with microtubule assembly and directionality. Under TTFields application, changes in microtubule organization resulted in activation of GEF-H1, which led to an increase in active RhoA levels and consequent focal adhesion formation with actin cytoskeleton architectural changes. Furthermore, the optimal TTFields frequency for inhibition of invasion in glioma cells was 300 kHz, which differed from the optimal anti-mitotic frequency leading to glioma cell death of 200 kHz. The inhibitory effect of TTFields on migration was observed at fields intensities of 0.6 V/cm RMS (below the threshold of 1 V/cm RMS previously reported for cytotoxic effects). Together, these data identify discrete TTFields effects that disrupt processes crucial for glioma cell motility.

Dataset Information

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

Publications

Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

Similar Datasets

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