Spatial regulation of MCAK promotes cell polarization and focal adhesion turnover to drive robust cell migration

Abstract

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK, in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared to the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning and focal adhesion dynamics that all help facilitate robust directional migration. [Media: see text] [Media: see text]

Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover

“…The knockdown of MCAK led to an impaired recruitment of FAK and paxillin, two positive key regulators for cell migration [64,65], leading to the reduced phosphorylation of these important FA structure proteins. These results are in accordance with a recently published paper by Zong et al, showing that MCAK knockdown interfered with directional movement, centrosome positioning and increased lifetime of FA's in RPE cells [21]. Moreover, both HeLa and RPE knockdown cells displayed a decreased FA size, which is also a vital factor related to cell migration [41].…”

“…The deregulation of its activity leads to severe mitotic defects and chromosomal instability [15,16,18]. In line with the close relationship between the MT and actin cytoskeleton, we and others have shown that functional MCAK has an impact on cancer cell migration [19][20][21]. The inhibitory phosphorylation of Aurora B at serine 192 of MCAK affects the directed migration as well as the invasion capacity of colorectal and cervix carcinoma cell lines [19].…”

“…MCAK has been shown to be associated with the migration capacity of endothelial, epithelial and cervical carcinoma cells [19][20][21] by regulating the polarization of MT growth and the FA turnover depending on RAC-1 [20,21]. In the present study, we utilized the CRISPR/dCas9 system [22] to further elucidate MCAK's molecular role in the process of cell motility and migration of cancer, as well as benign cells.…”

“…These results further support the notion that interfering with the expression of MCAK leads to disrupted FA dynamics, leading to the compromised migration and motility of CRISPRi/a cells. The MT plus-tip dynamics is an important parameter for cell motility, since it contributes to a front-rear cell polarity and coordinates FA turnover, promoting a robust directional migration [21,40]. To substantiate the regulatory role of MCAK on the MT plus-tip protein dynamics, a well-established EB3 (end-binding protein 3) comet detection assay [53] was performed with RPE and HeLa CRISPRi/a cells.…”

“…The inhibitory phosphorylation of Aurora B at serine 192 of MCAK affects the directed migration as well as the invasion capacity of colorectal and cervix carcinoma cell lines [19]. Moreover, it has been revealed that a signaling cascade of RAC1, Aurora B and MCAK facilitates polarization of endothelial cells [20], and that the activity of MCAK is crucial for positioning the centrosome towards the leading edge of cells, and the turnover of FAs [21]. To elucidate precisely how MCAK's activity regulates the motility of cancer cells, using MCAK CRISPRi/a knockdown and overexpression cell lines, we show that interfering with the expression of MCAK causes a decreased cell motility and migration, which is attributed to an impaired FA turnover associated with compromised MT-and actin-cytoskeleton dynamics.…”