Formins bridle and unleash their way through cytokinesis: a phragmoplast-centered view

Cytokinesis is the last step of the cell cycle and ends with the division of the cytoplasm into distinct daughter cells. In animals, cytokinesis is an outside-in process with the invagination of the membrane driven through a contractile ring facilitating the split into two daughter cells. Plant cells, surrounded by a semi-rigid cell wall, use an inside-out approach instead through precise positioning of a newly synthesized cell plate at the site of division to partition the cells. The plant-specific cytokinetic structure, called the phragmoplast, forms during the transition from anaphase to telophase and facilitates cell plate assembly (Alberts et al., 2002).

Phragmoplasts are composed of actin filaments, microtubules, related membrane compartments, and cytosolic proteins. Cell plate biosynthesis begins from a phragmoplast initial centered between the daughter nuclei that scaffold the start of the cell plate assembly. The phragmoplast then expands outward through a series of microtubule polymerization at the forefront, or “leading zone,” loss at the back, or the “lagging zone,” and establishment of a central “transition zone” (Smertenko et al., 2018). A cell plate assembly matrix (CPAM) assembles the cell plate at the center of the phragmoplast and contains the necessary protein complexes and cargo to build the plate (Smertenko et al., 2017). The phragmoplast expands centrifugally until the cell plate hits the parental walls, after which the phragmoplast disassembles. Disruptions to microtubule dynamics affect phragmoplast expansion and cell plate assembly. However, the specific factors and proteins that control these dynamics remain largely unknown.

The study by Zhang et al. (2021) focuses on formins, an evolutionarily conserved family of eukaryotic proteins that have been implicated for their role in cytoskeletal dynamics in animals and fungi as well as plants (Breitsprecher and Goode, 2013; Van Gisbergen and Bezanilla, 2013). Formin proteins are classically involved in nucleation and elongation of actin filaments and stabilization of microtubules, with key functions in cell division and tissue morphogenesis (Pruyne et al., 2002; Pring et al., 2003; Chesarone et al., 2010). In plants, a homology search of the Arabidopsis (Arabidopsis thaliana) genome revealed 21 formins: 11 Class I formins with the conserved Formin Homology 1 and 2 (FH1 and FH2) domains and the N-terminal transmembrane domain, and 10 Class II formins without the N-terminal domain (Deeks et al., 2002; Grunt et al., 2008; Van Gisbergen and Bezanilla, 2013). A few formins localize on phragmoplast microtubules and the cell plate in Arabidopsis and Physcomitrium (Ingouff et al., 2005; Oulehlová et al., 2019; Van Gisbergen et al., 2020). Unraveling the factors that control cytoskeletal dynamics in planta is a challenge due to the presence of the cell wall and impermeability to conventional dyes that could provide a fast visual output. In the case of formins, redundancy from the large gene family presents an additional challenge.

In this issue of Plant Physiology, Zhang et al. (2021) employed a Small Molecular Inhibitor of Formin Homology 2 (SMIFH2) domain and the Nicotiana tabacum (tobacco) BY-2 cell culture system to unravel the function of formins during cytokinesis. SMIFH2 inhibits the ability of formin to promote actin polymerization and bind to microtubules. Thus, SMIFH2 is a neat system used frequently to understand the role of formins in animal cells and serves as a means to overcome the issue of functional redundancy.

Treatment of tobacco BY-2 cell cultures with SMIFH2 inhibits formin-dependent actin polymerization and suppresses cell proliferation. To define the role of formins in plant cytokinesis, the cells were blocked at different phases of the cell cycle and treated with SMIFH2. These studies revealed reduced microtubule polymerization during interphase and a delay in exit from metaphase and telophase, both of which are microtubule-dependent processes. The authors then examined lines expressing stable C-terminal GFP fusions of three Class I formins AtFH1, AtFH5, and AtFH8, after SMIFH2 treatment. Perturbed localization and turnover of formins at the cell plate demonstrate the need for formins during cytokinesis.

The authors next explored the impact of formins in microtubule polymerization and dynamics in the phragmoplast. They imaged BY-2 cells expressing tobacco α-tubulin upon SMIFH2 treatment and tracked END BINDING 1 (EB1) protein to study the growth and shape of microtubules. EB1 is a microtubule plus-end tracking protein that forms a comet-like structure in response to altered nucleotide state of tubulin at the growing end of the microtubule (Reid et al., 2019; Roth et al., 2019). Through these analyses they show formins are important for microtubule polymerization in the phragmoplast, but not in its expansion. Shorter and slower polymerization on EB1 comets in SMIFH2-treated cells show formins stabilize the tubulin flares in the growing end, and this regulation by formin seems to be evolutionarily conserved.

The authors also investigated the effect of SMIFH2 treatment on Arabidopsis mutants deficient in EB1. They found a skewed trajectory of root growth in the Ateb1 triple mutant resulting from disoriented cross-walls and cell plate orientation defects. This suggests formins work with EB1 to control microtubule stability and dynamics and thereby direction of root growth. Closer examination of the cell plate defects through transmission electron microscopy shows irregularity in cell plate shape and thickness. The authors speculate this could be a consequence of the defects in membrane recycling and suggestive of a role for formins in cell plate remodeling.

Through this work, Zhang et al. (2021) reveal the function of formins in the different processes surrounding phragmoplast machinery and subsequent cell plate formation (Figure 1). Precise positioning of the cell plate is critical for organized cell division and tissue morphogenesis. In plants, this is an area currently under extensive study in light of advances in imaging techniques using different cellular models in Arabidopsis, such as trichomes, shoot meristems, or root hairs, to name a few. It would be challenging but fascinating to study actin and microtubule dynamics in parallel to understand if the many formins have specialized individual functions but a collective effect on cytokinesis. A genetic engineering approach targeting several formin genes in Arabidopsis through CRISPR-cas9 systems could be a potential approach.

Figure 1.

Model depicting formin localization and functions centered on the phragmoplast during cytokinesis (Adapted from Figure 9 of Zhang et al., 2021). A) Formins function in F-actin nucleation by the cell plate; B) Microtubule elongation promoted through stabilization of tubulin protofilament at the growing plus-tip; C) Formins enable docking of the microtubule tip to the cell plate assembly matrix through interaction with END BINDING 1 (EB1) proteins; D) Formins localized within the CPAM and cell plate stabilizes microtubule plus-ends. E) A potential role in cell plate remodeling through an unknown mechanism. CPAM – Cell plate assembly matrix, DRP – Dynamin-related protein.

It would also be interesting to explore the regulatory cues that drive formin-dependent cytoskeletal dynamics. Could there be mechanical cues? What are the underlying molecular mechanisms regulating formin-mediated functions in the phragmoplast? The effect on cell plate remodeling hints at a role of formins in funneling cell wall components to the cell plate. Are they important for vesicular transport and/or delivery of wall components to the phragmoplast? Do they work independently or through coordination with other cytoskeletal proteins, and if so, which ones? Many exciting avenues remain to be explored!

Conflict of Interest statement. The authors declare that they have no competing interests.