Crosstalk between ROP GTPase signaling and plant hormones.

Rho of Plants (ROPs) constitute a plant-specific subset of small guanine nucleotide-binding proteins within the Cdc42/Rho/Rac family. These versatile proteins regulate diverse cellular processes, including cell growth, cell division, cell morphogenesis, organ development, and stress responses. In recent years, the dynamic cellular and subcellular behaviors orchestrated by ROPs have unveiled a notable connection to hormone-mediated organ development and physiological responses, thereby expanding our knowledge of the functions and regulatory mechanisms of this signaling pathway. This article delineates advancements in understanding the interplay between plant hormones and the ROP signaling cascade, centering primarily on the connections with auxin and abscisic acid pathways, alongside preliminary discoveries in cytokinin, brassinosteroid, and salicylic acid responses. It endeavors to shed light on the intricate, coordinated mechanisms bridging cell-level and tissue-level signals that underlie plant cell behavior, organ development, and physiological processes, and highlight future research prospects and challenges in this rapidly developing field.

Effects of fluorescent tags and activity status on the membrane localization of ROP GTPases.

Plant-specific Rho-type GTPases (ROPs) are master regulators of cell polarity and development. Over the past 30 years, their localization and dynamics have been largely examined with fluorescent proteins fused at the amino terminus without investigating their impact on protein function. The moss Physcomitrium patens genome encodes four rop genes. In this study, we introduce a fluorescent tag at the endogenous amino terminus of ROP4 in wild-type and rop1,2,3 triple mutant via homologous recombination and demonstrate that the fluorescent tag severely impairs ROP4 function and inhibits its localization on the plasma membrane. This phenotype is exacerbated in mutants lacking ROP-related GTPase-activating proteins. By comparing the localization of nonfunctional and functional ROP4 fusion reporters, we provide insight into the mechanism that governs the membrane association of ROPs.

Two subtypes of GTPase-activating proteins coordinate tip growth and cell size regulation in Physcomitrium patens.

The establishment of cell polarity is a prerequisite for many developmental processes. However, how it is achieved during tip growth in plants remains elusive. Here, we show that the RHO OF PLANTs (ROPs), ROP GUANINE NUCLEOTIDE EXCHANGE FACTORs (RopGEFs), and ROP GTPASE-ACTIVATING PROTEINs (RopGAPs) assemble into membrane domains in tip-growing cells of the moss Physcomitrium patens. The confinement of membrane domains requires redundant global inactivation of ROPs by PpRopGAPs and the PLECKSTRIN HOMOLOGY (PH) domain-containing RenGAP PpREN. Unexpectedly, PpRopGAPs and PpREN exert opposing effects on domain size and cell width upon overexpression. Biochemical and functional analyses indicate that PpRopGAPs are recruited to the membrane by active ROPs to restrict domain size through clustering, whereas PpREN rapidly inactivates ROPs and inhibits PpRopGAP-induced clustering. We propose that the activity- and clustering-based domain organization by RopGAPs and RenGAPs is a general mechanism for coordinating polarized cell growth and cell size regulation in plants.

The putative myristoylome of Physcomitrium patens reveals conserved features of myristoylation in basal land plants.

KEYMESSAGE: The putative myristoylome of moss P. patens opens an avenue for studying myristoylation substrates in non-canonical model plants. A myristoylation signal was shown sufficient for membrane targeting and useful for membrane dynamics visualization during cell growth. N-myristoylation (MYR) is one form of lipid modification catalyzed by N-myristoyltransferase that enables protein-membrane association. MYR is highly conserved in all eukaryotes. However, the study of MYR is limited to a few models such as yeasts, humans, and Arabidopsis. Here, using prediction tools, we report the characterization of the putative myristoylome of the moss Physcomitrium patens. We show that basal land plants display a similar signature of MYR to Arabidopsis and may have organism-specific substrates. Phylogenetically, MYR signals have mostly co-evolved with protein function but also exhibit variability in an organism-specific manner. We also demonstrate that the MYR motif of a moss brassinosteroid-signaling kinase is an efficient plasma membrane targeting signal and labels lipid-rich domains in tip-growing cells. Our results provide insights into the myristoylome in a basal land plant and lay the foundation for future studies on MYR and its roles in plant evolution.

ROP GTPase-dependent polarity establishment during tip growth in plants.

Polar cell growth in plants requires a cell peripheral region that undergoes membrane extension and cell wall remodeling. Since the 1990s, RHO-RELATED GTPASES FROM PLANTS (ROPs) have been identified as master regulators that determine the site of cell growth. ROPs function to regulate actin and microtubule cytoskeletons, calcium gradients, and exocytosis, thus directing the delivery of materials for membrane and cell wall extension. In recent years, our understanding of the regulatory mechanisms underlying polar localization and the activation of ROPs has greatly advanced. Evidence points to the crucial roles of membrane lipids, receptor-like kinases, and cell wall components. In this review, we provide updates on the mechanisms underlying polarity control in tip-growing cells, with a focus on ROP effectors and membrane-associated signals. By integrating knowledge from pollen tubes, root hairs, and findings in bryophyte protonema cells and rhizoids, we hope to offer important insights into a common conceptual framework on polarity establishment governed by intercellular and extracellular signals.

Exogenous 6-benzylaminopurine inhibits tip growth and cytokinesis via regulating actin dynamics in the moss Physcomitrium patens.

MAIN CONCLUSION: Exogenous BAP but not 2iP disrupts actin structures and induces tip-growth retardation and cytokinesis failure in the moss Physcomitrium patens. Synthetic cytokinins have been widely used to address hormonal responses during plant development. However, exogenous cytokinins can cause a variety of cellular effects. A detailed characterization of such effects has not been well studied. Here, using Physcomitrium patens as a model, we show that the aromatic cytokinin 6-benzylaminopurine (BAP) inhibits tip growth at concentrations above 0.2 µM. At higher concentrations (0.6-1 µM), BAP can additionally block mitotic entry and induce cytokinesis defects and cell death. These effects are associated with altered actin dynamics and structures. By contrast, 2-isopentenyladenine (2iP) does not cause marked defects at various concentrations up to 10 µM, while t-zeatin (tZ) can moderately inhibit moss growth. Our results provide mechanistic insight into the inhibitory effects of BAP on cell growth and cell division and call for attention to the use of synthetic cytokinins for bioassays.

Division site determination during asymmetric cell division in plants.

During development, both animals and plants exploit asymmetric cell division (ACD) to increase tissue complexity, a process that usually generates cells dissimilar in size, morphology, and fate. Plants lack the key regulators that control ACD in animals. Instead, plants have evolved two unique cytoskeletal structures to tackle this problem: the preprophase band (PPB) and phragmoplast. The assembly of the PPB and phragmoplast and their contributions to division plane orientation have been extensively studied. However, how the division plane is positioned off the cell center during asymmetric division is poorly understood. Over the past 20 years, emerging evidence points to a critical role for polarly localized membrane proteins in this process. Although many of these proteins are species- or cell type specific, and the molecular mechanism underlying division asymmetry is not fully understood, common features such as morphological changes in cells, cytoskeletal dynamics, and nuclear positioning have been observed. In this review, we provide updates on polarity establishment and nuclear positioning during ACD in plants. Together with previous findings about symmetrically dividing cells and the emerging roles of developmental cues, we aim to offer evolutionary insight into a common framework for asymmetric division-site determination and highlight directions for future work.

RNA editing restricts hyperactive ciliary kinases.

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.

Rho of Plants GTPases and Cytoskeletal Elements Control Nuclear Positioning and Asymmetric Cell Division during Physcomitrella patens Branching.

Branching morphogenesis is a widely used mechanism for development [1, 2]. In plants, it is initiated by the emergence of a new growth axis, which is of particular importance for plants to explore space and access resources [1]. Branches can emerge either from a single cell or from a group of cells [3-5]. In both cases, the mother cells that initiate branching must undergo dynamic morphological changes and/or adopt oriented asymmetric cell divisions (ACDs) to establish the new growth direction. However, the underlying mechanisms are not fully understood. Here, using the bryophyte moss Physcomitrella patens as a model, we show that side-branch formation in P. patens protonemata requires coordinated polarized cell expansion, directional nuclear migration, and orientated ACD. By combining pharmacological experiments, long-term time-lapse imaging, and genetic analyses, we demonstrate that Rho of plants (ROP) GTPases and actin are essential for cell polarization and local cell expansion (bulging). The growing bulge acts as a prerequisite signal to guide long-distance microtubule (MT)-dependent nuclear migration, which determines the asymmetric positioning of the division plane. MTs play an essential role in nuclear migration but are less involved in bulge formation. Hence, cell polarity and cytoskeletal elements act cooperatively to modulate cell morphology and nuclear positioning during branch initiation. We propose that polarity-triggered nuclear positioning and ACD comprise a fundamental mechanism for increasing multicellularity and tissue complexity during plant morphogenesis.

Microtubule nucleation and organization without centrosomes.

Centrosomes play various critical roles in animal cells such as microtubule nucleation and stabilization, mitotic spindle morphogenesis, and spindle orientation. Land plants have lost centrosomes and yet must execute many of these functions. Recent studies have revealed the crucial roles played by morphologically distinct cytoplasmic microtubule-organizing centers (MTOCs) in initiating spindle bipolarity and maintaining spindle orientation robustness. These MTOCs resemble centrosomes in many aspects, implying an evolutionary divergence of MT-organizing structures in plants. However, their functions rely on conserved nucleation and amplification mechanisms, indicating a similarity in MT network establishment between animals and plants. Moreover, recent characterization of a plant-specific MT minus-end tracking protein suggests that plants have developed functionally equivalent modules to stabilize and organize MTs at minus ends. These findings support the theory that plants overcome centrosome loss by utilizing modified but substantially conserved mechanisms to organize MT networks.

The kinases male germ cell-associated kinase and cell cycle-related kinase regulate kinesin-2 motility in Caenorhabditis elegans neuronal cilia.

Kinesin-2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin-2 motors are regulated in vivo. Here, we performed forward genetic screens to isolate suppressors that rescue the ciliary defects of OSM-3-kinesin (homolog of mammalian homodimeric kinesin-2 KIF17) mutants in Caenorhabditis elegans. We identified the C. elegans dyf-5 and dyf-18, which encode the homologs of mammalian male germ cell-associated kinase and cell cycle-related kinase, respectively. Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin-2 (kinesin-II) at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are important for stabilizing the interaction between IFT particles and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

Centriole translocation and degeneration during ciliogenesis in Caenorhabditis elegans neurons.

Neuronal cilia that are formed at the dendritic endings of sensory neurons are essential for sensory perception. However, it remains unclear how the centriole-derived basal body is positioned to form a template for cilium formation. Using fluorescence time-lapse microscopy, we show that the centriole translocates from the cell body to the dendrite tip in the Caenorhabditis elegans sensory neurons. The centriolar protein SAS-5 interacts with the dynein light-chain LC8 and conditional mutations of cytoplasmic dynein-1 block centriole translocation and ciliogenesis. The components of the central tube are essential for the biogenesis of centrioles, which later drive ciliogenesis in the dendrite; however, the centriole loses these components at the late stage of centriole translocation and subsequently recruits transition zone and intraflagellar transport proteins. Together, our results provide a comprehensive model of ciliogenesis in sensory neurons and reveal the importance of the dynein-dependent centriole translocation in this process.

Dynein-Driven Retrograde Intraflagellar Transport Is Triphasic in C. elegans Sensory Cilia.

Cytoplasmic dynein-2 powers retrograde intraflagellar transport that is essential for cilium formation and maintenance. Inactivation of dynein-2 by mutations in DYNC2H1 causes skeletal dysplasias, and it remains unclear how the dynein-2 heavy chain moves in cilia. Here, using the genome-editing technique to produce fluorescent dynein-2 heavy chain in Caenorhabditis elegans, we show by high-resolution live microscopy that dynein-2 moves in a surprising way along distinct ciliary domains. Dynein-2 shows triphasic movement in the retrograde direction: dynein-2 accelerates in the ciliary distal region and then moves at maximum velocity and finally decelerates adjacent to the base, which may represent a physical obstacle due to transition zone barriers. By knocking the conserved ciliopathy-related mutations into the C. elegans dynein-2 heavy chain, we find that these mutations reduce its transport speed and frequency. Disruption of the dynein-2 tail domain, light intermediate chain, or intraflagellar transport (IFT)-B complex abolishes dynein-2's ciliary localization, revealing their important roles in ciliary entry of dynein-2. Furthermore, our affinity purification and genetic analyses show that IFT-A subunits IFT-139 and IFT-43 function redundantly to promote dynein-2 motility. These results reveal the molecular regulation of dynein-2 movement in sensory cilia.

Somatic CRISPR-Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia.

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2-driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT-dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.

Conditional gene knockout animals are valuable tools for studying the mechanisms underlying cell and developmental biology. We developed a conditional knockout strategy by spatiotemporally manipulating the expression of an RNA-guided DNA endonuclease, CRISPR-Cas9, in Caenorhabditis elegans somatic cell lineages. We showed that this somatic CRISPR-Cas9 technology provides a quick and efficient approach to generate conditional knockouts in various cell types at different developmental stages. Furthermore, we demonstrated that this method outperforms our recently developed somatic TALEN technique and enables the one-step generation of multiple conditional knockouts. By combining these techniques with live-cell imaging, we showed that an essential embryonic gene, Coronin, which is associated with human neurobehavioral dysfunction, regulates actin organization and cell morphology during C. elegans postembryonic neuroblast migration and neuritogenesis. We propose that the somatic CRISPR-Cas9 platform is uniquely suited for conditional gene editing-based biomedical research.

The application of transcription activator-like effector nucleases for genome editing in C. elegans.

The nematode Caenorhabditis elegans has been a powerful model system for biomedical research in the past decades, however, the efficient genetic tools are still demanding for gene knockout, knock-in or conditional gene mutations. Transcription activator-like effector nucleases (TALENs) that comprise a sequence-specific DNA-binding domain fused to a FokI nuclease domain facilitate the targeted genome editing in various cell types or organisms. Here we summarize the recent progresses and protocols using TALENs in C. elegans that generate gene mutations and knock-ins in the germ line and the conditional gene knockout in somatic tissues.

A proneural gene controls C. elegans neuroblast asymmetric division and migration.

Proneural genes control the generation of neuroblasts from the neuroepithelium, but their functions in neuroblast asymmetric division and migration remain elusive. Here, we identified Caenorhabditiselegans mutants of a proneural transcription factor (TF) lin-32, in which Q neuroblasts are produced. We showed that LIN-32 functions in parallel with a storkhead TF, HAM-1, to regulate Q neuroblast asymmetric division, and that Q neuroblast migration is inhibited in lin-32 alleles. Consistently, lin-32 is expressed throughout Q neuroblast lineage, suggesting that LIN-32 may promote different target gene expression. Our studies thus uncovered previously unknown functions of a proneural gene in neuroblast development.

Conditional targeted genome editing using somatically expressed TALENs in C. elegans.

We have developed a method for the generation of conditional knockouts in Caenorhabditis elegans by expressing transcription activator-like effector nucleases (TALENs) in somatic cells. Using germline transformation with plasmids encoding TALENs under the control of an inducible or tissue-specific promoter, we observed effective gene modifications and resulting phenotypes in specific developmental stages and tissues. We further used this method to bypass the embryonic requirement of cor-1, which encodes the homolog of human severe combined immunodeficiency (SCID) protein coronin, and we determined its essential role in cell migration in larval Q-cell lineages. Our results show that TALENs expressed in the somatic cells of model organisms provide a versatile tool for functional genomics.

Developmental stage-dependent transcriptional regulatory pathways control neuroblast lineage progression.

Neuroblasts generate neurons with different functions by asymmetric cell division, cell cycle exit and differentiation. The underlying transcriptional regulatory pathways remain elusive. Here, we performed genetic screens in C. elegans and identified three evolutionarily conserved transcription factors (TFs) essential for Q neuroblast lineage progression. Through live cell imaging and genetic analysis, we showed that the storkhead TF HAM-1 regulates spindle positioning and myosin polarization during asymmetric cell division and that the PAR-1-like kinase PIG-1 is a transcriptional regulatory target of HAM-1. The TEAD TF EGL-44, in a physical association with the zinc-finger TF EGL-46, instructs cell cycle exit after the terminal division. Finally, the Sox domain TF EGL-13 is necessary and sufficient to establish the correct neuronal fate. Genetic analysis further demonstrated that HAM-1, EGL-44/EGL-46 and EGL-13 form three transcriptional regulatory pathways. We have thus identified TFs that function at distinct developmental stages to ensure appropriate neuroblast lineage progression and suggest that their vertebrate homologs might similarly regulate neural development.