A Septin Cytoskeleton-Targeting Small Molecule, Forchlorfenuron, Inhibits Epithelial Migration via Septin-Independent Perturbation of Cellular Signaling.

Lei Sun,Alex Feygin,Nayden G Naydenov,Susana Lechuga,Andrei I Ivanov,Xuelei Cao

doi:10.3390/cells9010084

Abstract

Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, filaments, and rings. The septin cytoskeleton has a number of cellular functions, including regulation of cytokinesis, cell migration, vesicle trafficking, and receptor signaling. A plant cytokinin, forchlorfenuron (FCF), interacts with septin subunits, resulting in the altered organization of the septin cytoskeleton. Although FCF has been extensively used to examine the roles of septins in various cellular processes, its specificity, and possible off-target effects in vertebrate systems, has not been investigated. In the present study, we demonstrate that FCF inhibits spontaneous, as well as hepatocyte growth factor-induced, migration of HT-29 and DU145 human epithelial cells. Additionally, FCF increases paracellular permeability of HT-29 cell monolayers. These inhibitory effects of FCF persist in epithelial cells where the septin cytoskeleton has been disassembled by either CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of septin 7, insinuating off-target effects of FCF. Biochemical analysis reveals that FCF-dependent inhibition of the motility of control and septin-depleted cells is accompanied by decreased expression of the c-Jun transcription factor and inhibited ERK activity. The described off-target effects of FCF strongly suggests that caution is warranted while using this compound to examine the biological functions of septins in cellular systems and model organisms.

Highlights

The cytoskeleton is a major determinant of the architecture and function of eukaryotic cells
Well-differentiated HT-29 cf8 human colonic epithelial cells and DU145 human prostate epithelial cells were used in this study; their spontaneous and hepatocyte growth factor (HGF)-induced migration was investigated using a classical scratch wound healing assay
In HT-29 cell monolayers, FCF significantly attenuated spontaneous cell migration (Figure 1). This compound completely blocked the increase in cell migration caused by HGF (Figure 1)

Summary

Introduction

The cytoskeleton is a major determinant of the architecture and function of eukaryotic cells. It is composed of various filaments formed via self-assembly and the polymerization of specialized structural proteins [1,2,3]. There are four types of cytoskeletal structures in eukaryotic cells: actin filaments, microtubules, intermediate filaments, and septin complexes. These cytoskeletal elements play crucial roles in regulating homeostatic and specialized functions of different cells. Such functions include regulation of cell shape and size, cell division, migration, vesicle trafficking, cell-cell interactions, receptor signaling, etc. Abnormal architecture and dynamics of different cytoskeletal structures are linked to the development of various diseases, most notably cancer and inflammation [4,5,6,7]

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Results

Conclusion