Abstract
Simple SummaryThe microtubule (MT) cytoskeleton is a crucial factor for organized cell motility and migration of cancer as well as benign cells. Mitotic centromere-associated kinesin (MCAK/KIF2C) is a member of the kinesin-13 family, which is important for the regulation of MT dynamics. Its overexpression has been reported to be related to increased metastasis in various tumor entities. Our study further elucidate how MCAK’s is able to modulate cell migration and invasion. Interfering with the precise regulated expression of MCAK led to impaired FA protein composition and altered their phosphorylation status, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. MCAK regulates these processes by affecting the actin-MT cytoskeleton dynamics, providing molecular mechanisms by which a deregulation of MCAK could promote tumor metastasis.The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it’s up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.
Highlights
Migration is essential for a variety of physiological processes, including the development of organisms, repair of damaged tissue and immune response, and for multiple pathological events like tumor metastasis [1,2]
To elucidate precisely how mitotic centromere-associated kinesin (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 focal adhesion (FA) turnover associated with compromised MT- and actin-cytoskeleton dynamics
The relative gene expression of KIF2C was reduced by 73% in HeLa CRISPRi sgRNA against MCAK (sgMCAK) (HeLa CRISPRi) cells and by 90% in retinal pigment epithelium (RPE) CRISPRi sgMCAK (RPE CRISPRi) cells compared to both control sgcon cells (Figure 1A,B, 1st and 2nd bars)
Summary
Migration is essential for a variety of physiological processes, including the development of organisms, repair of damaged tissue and immune response, and for multiple pathological events like tumor metastasis [1,2]. The main proteins linking the integrin transmembrane receptors to the actin cytoskeleton are mediated by the interactions of focal adhesion kinase (FAK)/paxillin, talin/vinculin and α-actinin/zyxin/VASP (vasodilator stimulated phosphoprotein) to ensure a proper directional migration [12]. The formation of stable FAs is initiated by a signaling cascade triggered by the binding of extracellular matrix (ECM) components to integrins, leading to the activation of FAK. This enables FAK to phosphorylate paxillin at tyrosine 118 and tyrosine 31, promoting docking sites for several FA proteins including vinculin, α-actinin, and talin, leading to the stabilization of newly formed FAs [13,14]
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