RESUMEN
Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.
RESUMEN
The molecular mechanisms guiding oriented cell divisions in the root vascular tissues of Arabidopsis thaliana are still poorly characterised. By overlapping bulk and single-cell transcriptomic datasets, we unveiled TETRASPANIN1 (TET1) as a putative regulator in this process. TET1 is expressed in root vascular cells, and loss-of-function mutants contain fewer vascular cell files. We further generated and characterised a CRISPR deletion mutant and showed, unlike previously described mutants, that the full knock out is additionally missing endodermal cells in a stochastic way. Finally, we show that HA-tagged versions of TET1 are functional in contrast to fluorescent TET1 translational fusions. Immunostaining using HA-TET1 lines complementing the mutant phenotype suggested a dual plasma membrane and intracellular localisation in the root vasculature and a polar membrane localisation in the young cortex, endodermal and initial cells. Taken together, we show that TET1 is involved in both vascular proliferation and ground tissue patterning. Our initial results pave the way for future work to decipher its precise mode of action.
Asunto(s)
Arabidopsis , Arabidopsis/genética , División Celular , Membrana Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Perfilación de la Expresión GénicaRESUMEN
Protein-protein interactions orchestrate plant development and serve as crucial elements for cellular and environmental communication. Understanding these interactions offers a gateway to unravel complex protein networks that will allow a better understanding of nature. Methods for the characterization of protein-protein interactions have been around for a long time, yet the complexity of some of these interactions fuels the development of new techniques that provide a better understanding of the underlying dynamics. In many cases, the application of these techniques is limited by the nature of the available sample. While some methods require an in vivo set up, others solely depend on protein sequences to study protein-protein interactions via an in silico set up. The vast amount of techniques available to date calls for a way to select the appropriate tools for the study of specific interactions. Here, we classify widely spread tools and new emerging techniques for the characterization of protein-protein interactions based on sample requirements while providing insights into the information that they can potentially deliver. We provide a comprehensive overview of commonly used techniques and elaborate on the most recent developments, showcasing their implementation in plant research.
RESUMEN
Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.
Asunto(s)
Clatrina , Endocitosis , Membrana Celular/metabolismo , Clatrina/metabolismoRESUMEN
The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Hipocótilo/genética , Hipocótilo/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Matriz Nuclear/metabolismo , Regulación de la Expresión Génica de las Plantas , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismoRESUMEN
Endocytosis is an essential eukaryotic process that maintains the homeostasis of the plasma membrane proteome by vesicle-mediated internalization. Its predominant mode of operation utilizes the polymerization of the scaffold protein clathrin forming a coat around the vesicle; therefore, it is termed clathrin-mediated endocytosis (CME). Throughout evolution, the machinery that mediates CME is marked by losses, multiplications, and innovations. CME employs a limited number of conserved structural domains and folds, whose assembly and connections are species dependent. In plants, many of the domains are grouped into an ancient multimeric complex, the TPLATE complex, which occupies a central position as an interaction hub for the endocytic machinery. In this review, we provide an overview of the current knowledge regarding the structural aspects of plant CME, and we draw comparisons to other model systems. To do so, we have taken advantage of recent developments with respect to artificial intelligence-based protein structure prediction.
Asunto(s)
Clatrina , Endocitosis , Plantas , Endocitosis/fisiología , Clatrina/metabolismo , Clatrina/química , Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Evolución Biológica , Membrana Celular/metabolismo , Evolución MolecularRESUMEN
Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here, we make use of the accessibility of the Arabidopsis (Arabidopsis thaliana) root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes as part of the terminal differentiation program that is orchestrated by the NO APICAL MERISTEM, ARABIDOPSIS THALIANA ACTIVATING FACTOR, CUP-SHAPED COTYLEDON (NAC) transcription factor SOMBRERO. Surprisingly, the breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. In analogy to animal apoptosis, the plasma membrane remains impermeable for proteins during and after PCD execution. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in terminally differentiated root cap cells, suggesting that these ion fluxes act as PCD-triggering signals. This detailed information on the cellular processes occurring during developmental PCD in plants is a pivotal prerequisite for future research into the molecular mechanisms of cell death execution.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Apoptosis/fisiología , Muerte CelularRESUMEN
Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Endocitosis , Regulación de la Expresión Génica de las Plantas , Meristema/genética , Plantas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
The plasma membrane (PM) houses a wide variety of proteins, facilitating interactions between the cell and its surroundings. Perception of external stimuli leads to selective internalization of membrane proteins via endocytosis. A multitude of endocytic signals affect protein internalization; however, their coordination and the exact mechanism of their recognition still remain elusive. In this review, we summarized the up-to-date knowledge of different internalization signals in PM cargo proteins and their involvement during protein trafficking.
Asunto(s)
Endocitosis , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Membrana Celular/metabolismo , Plantas/metabolismoRESUMEN
The formation of biomolecular condensates through phase separation is an important strategy to compartmentalize cellular functions. While it is now well established that condensates exist throughout eukaryotic cells, how condensates assemble and function on lipid membranes is only beginning to be understood. In this perspective, we highlight work from plant, animal, and yeast model systems showing that condensates assemble on many endomembrane surfaces to carry out diverse functions. In vesicle trafficking, condensation has reported roles in the formation of endocytic vesicles and autophagosomes and in the inactivation of secretory COPII vesicles. We briefly discuss how membranes and membrane lipids regulate the formation and function of membrane-associated condensates. This includes how membranes act as surfaces for condensate assembly, with lipids mediating the nucleation of condensates during endocytosis and other processes. Additionally, membrane-condensate interactions give rise to the biophysical property of "wetting", which has functional importance in shaping autophagosomal and vacuolar membranes. We also speculate on the existence of membrane-associated condensates during cell polarity in plants and discuss how condensation may help to establish functional plasma membrane domains. Lastly, we provide advice on relevant in vitro and in vivo approaches and techniques to study membrane-associated phase separation.
Asunto(s)
Proteínas , Vacuolas , Animales , Proteínas/metabolismo , Membrana Celular/metabolismo , Autofagosomas , BiologíaRESUMEN
Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fosforilación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Procesamiento Proteico-Postraduccional , Plantas Modificadas Genéticamente/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. In Arabidopsis thaliana, primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex, formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and division orientation by modulating the cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetics approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor, MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression, as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity, to ensure optimal cell proliferation rates during root vascular development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Meristema , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Raíces de Plantas/metabolismo , Retroalimentación , Transactivadores/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/metabolismo , División Celular , Citocininas/metabolismoRESUMEN
The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Ligandos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismoRESUMEN
Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Red trans-Golgi/metabolismo , Aparato de Golgi/metabolismo , Clatrina/metabolismoRESUMEN
Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.
Asunto(s)
Clatrina , Endocitosis , Clatrina/genética , Clatrina/metabolismo , Membrana Celular/metabolismo , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.
Asunto(s)
Proteínas de Arabidopsis , Fosfatos de Azúcar , Fosfatos de Azúcar/metabolismo , Trehalosa/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Plantas/metabolismo , Transducción de Señal , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
Asunto(s)
Arabidopsis , Vesículas Cubiertas por Clatrina , Arabidopsis/genética , Arabidopsis/metabolismo , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Proteoma/metabolismo , Proteómica , Factor de Transcripción AP-1/análisis , Factor de Transcripción AP-1/metabolismoRESUMEN
Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins, and auxin analogs. In this context, synthetic auxin analogs, such as 1-naphthalene acetic acid (1-NAA), are often favored over the endogenous auxin, indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven instrumental in revealing the various faces of auxin, they display in some cases bioactivities distinct from IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in brefeldin A-sensitive endosomal aggregations (BFA bodies), and correlation with the ability to elicit Ca2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin analog-induced Ca2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca2+ response, and their differential ability to elicit Ca2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca2+ signaling does not inhibit BFA body formation in Arabidopsis roots.