Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2

Abstract

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing SMIFH2, a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule Associated protein of 215 kD (XMAP215) homologue MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Four-dimensional quantitative analysis of cell plate development using lattice light sheet microscopy identifies robust transition points between growth phases

“…Directly detecting callose via fluorescent staining corresponds to these phases and validates the prediction of the biophysical model. Callose, due to its amorphous structure,can lead to alterations in the physical and mechanical properties at the deposition site (Piršelová and Matušíková, 2012; Zhang et al, 2021; Usak et al, 2023). The polysaccharide has the ability to enhance rigidity while maintaining flexibility and reduce permeability to various compounds across a range of functions (Yim and Bradford, 1998; Parre and Geitmann, 2005; Vaten et al, 2011).…”

Plant cytokinesis and the construction of new cell wall

“…Chemical biology using ES7 and SMIFH2 informed the role of callose and formins, respectively, in cytokinesis [77,78,108] and can be instrumental in dissecting different aspects of cytokinesis at a spatiotemporal level while overcoming lethality.…”

Reforming the Barrier: The Role of Formins in Wound Repair

“…SMIFH2 has been widely used in most recent studies to evaluate the role of formin in different biological processes. It has been shown to inhibit formin-dependent actin polymerization, reduce cell proliferation, and disrupt cytokinesis and microtubule dynamics [ 118 ]. The blocking effect of SMIFH2 on formins has been shown to perturb the actin cytoskeleton through the remodeling of actin filaments and microtubules [ 119 ].…”