Forming the queue: A role for WPR proteins in establishing cell polarity

To produce distinct cell lineages from one mitotic event, cells undergo asymmetric cell division. A prerequisite for asymmetric cell division is that the dividing cell is polarized. This is particularly true in the cell divisions required to make stomata (Guo et al., 2021). In the grasses, stomatal complexes are made of four cells: two guard cells and two subsidiary cells. The asymmetric cell division of subsidiary mother cells (SMCs) generates the subsidiary cells. Understanding the molecular underpinnings for SMC polarization has been the subject of years of work (reviewed in Nunes et al., 2020). Although many mechanistic questions remain to be answered, several proteins have been shown to participate in SMC polarization. Two such proteins, PANGLOSS2 (PAN2) and PAN1, are catalytically dead kinases that interact with Rho of Plants (ROP) (Zhang et al., 2012). They could be scaffold proteins, but what the PANs interact with, and to what extent, is an open question. Recent work from Qiong Nan and colleagues (Nan et al., 2022) in maize (Zea mays) identified four proteins as important elements of SMC polarization that interact with the PANs. This research describes new proteins in the SMC polarization pathway and uncovers a novel filamentous actin (F-actin)-binding domain (See Figure).

Figure.

ZmWPRB2 (“GFP-B2”) is a novel F-actin-binding protein. Heterologous coexpression of GFP-WPRB2 (A) with the live-cell F-actin marker Lifeact–RFP (B) in Nicotiana benthamiana leaf epidermis, merged image (C), reveals a previously uncharacterized F-actin-binding protein. Adapted from Nan et al. (2022), Figure 6, L–N.

Several protein polarization events occur prior to SMC division. BRICK1 (BRK1), a promoter of branched F-actin networks, is the first to polarize within the SMC. After BRK1, the kinase-dead leucine-rich repeat receptor-like protein PAN2, followed by PAN1, polarize to the same region. Finally, members of the ROP GTPases, which interact with PAN1, polarize. Only then will F-actin patches form (Facette et al., 2015). To find novel interactors of the PANs, Nan et al. performed co-immunoprecipitation coupled with mass spectrometry. Four proteins identified in PAN2 pull-downs contained a Domain of Unknown Function 827 (DUF827). The family of DUF827-containing proteins includes WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT 1 (WEB1) and PLASTID MOVEMENT IMPAIRED (PMI) 2-like proteins. The authors cluster the candidates into a WEB1/PMI2-Related (WPR) family. Therefore, the candidate proteins from the pull-down are WPRA1, WPRA2, WPRB1, and WPRB2.

To investigate WPR–PAN interactions, the authors perform targeted co-immunoprecipitation and yeast two-hybridization assays. Both PANs co-precipitated and interact directly in yeast two hybrid with both WPRBs. Conversely, the WPRAs associate with the PANs indirectly. The authors propose a model by which WPRBs drive heterodimerization with WPRAs and PANs. In support of this, WPRAs co-precipitate with WPRBs, suggesting frequent heterodimerization. The authors discover the WPRs polarize in SMCs after BRK1, but simultaneously with the PANs. Additionally, CFP-WPRA2 and RFP-WPRB2 are always polarly co-localized.

To elucidate the function of WPRs, the authors made mutants with CRISPR/Cas9. Interestingly, they were unable to obtain wpra1;wpra2 homozygous lines. This may be an indication that these proteins are essential in pollen. Homozygous wprb1;wprb2 lines, however, had misshapen subsidiary cells reminiscent of brk1, pan1, and pan2 mutants. These results validate that WPRBs are indeed novel regulators of proper subsidiary cell formation. The authors then sought to find a molecular function of the WPRs. Because founding members of the WPR group have roles in actin-based organelle movement, the authors hypothesized that the WPRs may function through F-actin. By over-expressing full-length and truncated forms of the WPRBs, the authors discovered a novel F-actin-binding domain in the N-terminus (see Figure). WPRB–actin interactions were confirmed with co-sedimentation assays. This work opens the door for novel investigations into polarized F-actin formation during asymmetrical SMC division.

Generating diverse cell identities within an organ, such as a leaf, often requires asymmetrical cell division. The work presented in Nan et al. elucidates not only novel regulators in SMC polarization but also lays the foundation for further work into a new F-actin regulatory gene family as well. Additionally, the stage is now set to investigate the role of WPR homologs in other cell polarization events—from branch cell emergence in bryophytes through mother guard cell formation in angiosperms.