Biology subjects

Friml, J.

Publications and source records attributed to Friml, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Clathrin-mediated endocytosis-independent function of VAN3 ARF GTPase Activating Protein at the plasma membrane

ARF small GTPases in plants serve important cellular functions in subcellular trafficking and developmental functions in auxin-mediated patterning of the plant body. The Arabidopsis thaliana ARF regulator ARF-GAP VAN3 has been implicated to act at the plasma membrane (PM) and linked functionally to the clathrin- and dynamin-mediated endocytosis. Here we re-evaluated the localization of VAN3 at the PM and its function in endocytosis. Using Total Internal Reflection Fluorescence microscopy we observed remarkably transient associations of VAN3 to the PM at discrete foci, however, devoid of clathrin, the dynamin isoform DRP1A, or the ARF regulator GNOM, which is also involved in a developmental patterning function mediated from the PM. Clathrin-coated pits are abundant and endocytosis appears to proceed normally in van3-1 knockout mutant. In turn, post-translational silencing of clathrin expression indicates that the localization of VAN3 at the PM depends on clathrin function, presumably on clathrin-mediated endocytosis.

plant biology↗

bioRxiv2021

The role of clathrin in exocytosis and the mutual regulation of endo- and exocytosis in plant cells

Within the plant endomembrane system, the vesicle coat protein clathrin localizes to the plasma membrane (PM) and the trans-Golgi Network/Early Endosome (TGN/EE). While the role of clathrin as a major component of endocytosis at the PM is well established, its function at TGN/EE, possibly in exocytosis or the vacuolar pathway, is a matter of debate. This shared function of clathrin also opens a question whether plant cells possess a homeostatic mechanisms that balance rates of opposite trafficking routes, such as endo- and exocytosis. Here we address these questions using lines inducibly silencing CLATHRIN HEAVY CHAIN (CHC). We find a relocation of exocytic soluble and integral membrane protein cargoes to the vacuole, supporting a function of clathrin in exocytosis. A comparison with lines overexpressing AUXILIN-LIKE1, where inhibition of CME precedes rerouting of secretory cargoes, does not support a homeostatic regulatory mechanism adjusting exocytosis to the rates of endocytosis. Complementary experiments reveal only minor and variably detectable reductions in the rates of CME in secretory mutants, also not indicative of a converse homeostatic mechanism adjusting rates of endocytosis to the rates of secretion.

plant biology↗

bioRxiv2021

Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

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 AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) 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 trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis 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.

cell biology↗

bioRxiv2021

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis

Clathrin-mediated endocytosis in plants is an essential process but the underlying mechanisms are poorly understood, not least because of the extreme intracellular turgor pressure acting against the formation of endocytic vesicles. In contrast to other models, plant endocytosis is independent of actin, indicating a mechanistically distinct solution. Here, by using biochemical and advanced microscopy approaches, we show that the plant-specific TPLATE complex acts outside of endocytic vesicles as a mediator of membrane bending. Cells with disrupted TPLATE fail to generate spherical vesicles, and in vitro biophysical assays identified protein domains with membrane bending capability. These results redefine the role of the TPLATE complex as a key component of the evolutionarily distinct mechanism mediating membrane bending against high turgor pressure to drive endocytosis in plant cells. One Sentence SummaryWhile plant CME is actin independent, we identify that the evolutionarily ancient octameric TPLATE complex mediates membrane bending against high turgor pressure in plant clathrin-mediated endocytosis.

cell biology↗

bioRxiv2021

Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis

The signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.

plant biology↗

bioRxiv2021

Auxin-triggered changes in the Arabidopsis root tip (phospho)proteome reveal novel root growth regulators

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different auxin concentrations on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results together with previously published studies suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip. ONE SENTENCE SUMMARYAn auxin-triggered Arabidopsis root tip (phospho)proteome reveals novel root growth regulators

plant biology↗

bioRxiv2021

The PELOTA-HBS1 Complex Orchestrates mRNA Translation Surveillance and PDK1-mediated Plant Growth and Development

The quality control system for messenger RNA is fundamental for cellular activities in eukaryotes. To elucidate the molecular mechanism of 3-Phosphoinositide-Dependent Protein Kinase1 (PDK1), an essential regulator throughout growth and development of eukaryotes, a forward genetic approach was employed to screen for suppressors of the loss-of-function T-DNA insertional pdk1.1 pdk1.2 double mutant in Arabidopsis. Notably, the severe growth attenuation of pdk1.1 pdk1.2 is rescued by sop21 (suppressor of pdk1.1 pdk1.2) that harbours a loss-of-function mutation in PELOTA1 (PEL1). PEL1 is a homologue of mammalian PELOTA and yeast DOM34, which form a heterodimeric complex with the GTPase HBS1, responsible for ribosome rescue to assure the quality and fidelity of mRNA molecules. Genetic analysis further reveals that the dysfunction of PEL1-HBS complex fails to degrade the T-DNA-disrupted, truncated but functional PDK1 transcripts, thus rescuing pdk1.1 pdk1.2. Our studies demonstrate the functionality and identify the essential functions of a homologous PELOTA-HBS1 complex in higher plant, and provide novel insights into the mRNA quality control mechanism.

plant biology↗

bioRxiv2020

Pharmacological and genetic manipulations of Ca2+ signaling have contrasting effects on auxin-regulated trafficking

A large part of a plants developmental plasticity relies on the activities of the phytohormone auxin and the regulation of its own distribution. This process involves a cohort of transcriptional and non-transcriptional effects of auxin on polar auxin transport, regulating the abundancy, biochemical activity and polar localization of the molecular components, predominantly PIN auxin exporters. While the transcriptional auxin signaling cascade has been well characterized, the mechanism and role of non-transcriptional auxin signaling remains largely elusive. Here, we addressed the potential involvement of auxin-induced Ca2+ signaling in auxins inhibitory effect on PIN endocytic trafficking. On the one hand, exogenous manipulations of Ca2+ availability and signaling effectively antagonized auxin effects suggesting that auxin-induced Ca2+ signaling is required for inhibition of internalization. On the other hand, we addressed the auxin-mediated inhibition of PIN internalization in the auxin signaling (tir1afb2,3) or Ca2+ channel (cngc14) mutants. These mutants were strongly defective in auxin-triggered Ca2+ signaling, but not in auxin-inhibited internalization. These data imply that, while Ca2+ signaling may be required for normal PIN trafficking, auxin-mediated increase in Ca2+ signaling is not a direct part of a downstream mechanism that mediates auxin effects on Brefeldin A-visualized PIN intercellular aggregation. These contrasting results obtained by comparing the mutant analysis versus the exogenous manipulations of Ca2+ availability and signaling illustrate the critical importance of genetics to unravel the role of Ca2+ in a process of interest.

cell biology↗

bioRxiv2020

Synaptotagmins Maintain Diacylglycerol Homeostasis at Endoplasmic Reticulum-Plasma Membrane Contact Sites during Abiotic Stress

Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

molecular biology↗

bioRxiv2020

Modulation of root growth by nutrient-defined fine-tuning of polar auxin transport

Nitrogen is an essential macronutrient and its availability in soil plays a critical role in plant growth, development and impacts agricultural productivity. Plants have evolved different strategies to sense and respond to heterogeneous nitrogen distribution. Modulating root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to varying nitrogen sources are poorly understood. Here, using a combination of physiological, live in vivo high- and super resolution imaging, we describe a novel adaptation strategy of root growth on available nitrogen source. We show that growth, i.e. tissue-specific cell division and elongation rates are fine-tuned by modulating auxin flux within and between tissues. Changes in auxin redistribution are achieved by nitrogen source dependent post-translational modification of PIN2, a major auxin efflux carrier, at an uncharacterized, evolutionary conserved phosphosite. Further, we generate a computer model based on our results which successfully recapitulate our experimental observations and creates new predictions that could broaden our understanding of root growth mechanisms in the dynamic environment.

plant biology↗

bioRxiv2020

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana

Advanced transcriptome sequencing has uncovered that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Here we reveal, in the plant model Arabidopsis thaliana, that the PIN7 gene, which encodes a polarly localized transporter for the phytohormone auxin, produces two evolutionary-conserved transcripts. These isoforms PIN7a and PIN7b, differing in a 4 amino acid motif, are present at nearly equal levels in most cells. Although both variants do not differ in the subcellular localization and transport auxin with similar capacity, they closely associate and mutually influence their stability within the plasma membrane. Phenotypic complementation tests reveal that the functional contribution of PIN7b per se is minor but it markedly attenuates the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. These results establish alternative splicing of the PIN family as a conserved, functionally-relevant mechanism, unveiling an additional regulatory level of auxin-mediated plant development.

plant biology↗

bioRxiv2020

High temporal resolution reveals simultaneous plasma membrane recruitment of the TPLATE complex subunits.

The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC contains six evolutionary conserved subunits and two plant specific subunits, AtEH1/Pan1 and AtEH2/Pan1, which are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and a lowered temperature. Our data show that lowering the temperature slows down endocytosis and thereby enhances the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE, and the AtEH/Pan1 proteins, exhibited simultaneous recruitment at the PM. These results, together with our co-localization analysis of different TPC subunits, allow us to conclude that in plant cells, TPC is not recruited to the PM sequentially but as an octameric complex. One sentence summaryLowering the temperature increases spatiotemporal resolution of protein recruitment at the plasma membrane.

cell biology↗

bioRxiv2019

Evolutionary unique mechanistic framework of clathrin-mediated endocytosis in plants

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME follows the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the surface but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

plant biology↗

bioRxiv2019

A lipid code-dependent phosphoswitch directs PIN-mediated auxin efflux in Arabidopsis development

Directional intercellular transport of the phytohormone auxin mediated by PIN FORMED (PIN) efflux carriers plays essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. Multilevel regulations of PIN activity under internal and external cues are complicated; however, the underlying molecular mechanism remains elusive. Here we demonstrate that 3-Phosphoinositide-Dependent Protein Kinase1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub integrating the upstream lipid signalling and the downstream substrate activity through phosphorylation. Genetic analysis uncovers that loss-of-function Arabidopsis mutant pdk1.1 pdk1.2 exhibits a plethora of abnormalities in organogenesis and growth, due to the defective PIN-dependent auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 Protein Kinase to facilitate its activity towards PIN proteins. Our studies establish a lipid-dependent phosphorylation cascade connecting membrane composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.

plant biology↗

bioRxiv2019

Cell-surface receptors enable perception of extracellular cytokinins

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience 1,2. Although cytokinin receptors are substantially localised to the endoplasmic reticulum 3-5, the cellular sites of cytokinin perception continue to be debated 1,6,7. Several cytokinin types display bioactivity 8,9 and recently a cell-specific cytokinin gradient was reported in roots 10. Yet, the importance of spatially heterogeneous cytokinin distribution and the specific cytokinin(s) that account for the different responses remain unclear. Here we show that cytokinin perception by plasma membrane receptors is an effective path for cytokinin response in root cells. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP;11) is closely matched to intracellular cytokinin content, yet a proportion of bioactive cytokinins are detected in the extracellular fluid. Using cytokinins covalently linked to beads that could not pass the plasma membrane, we demonstrate that strong TCSn activation still occurs and that this response is greatly diminished in cytokinin receptor mutants. Although intracellular receptors play significant roles, we argue for a revision of concepts of cytokinin perception to include the spatial dimensions. In particular, selective ligand-receptor affinities, cellular localisation and tissue distribution of bioactive cytokinins, their receptors, transporters and inactivation enzymes appear all to be components of the signalling regulatory mechanisms.

plant biology↗

bioRxiv2019

A putative ER-PM contact site complex relocalizes in response to Rare Earth Elements-induced endocytosis

In plant cells, environmental stressors induce changes in the cytosolic concentration of calcium ([Ca2+]cyt) that are transduced by Ca2+-sensing proteins. To confer specificity to the stress signaling response, [Ca2+]cyt sensing must be tightly regulated in space and time; the molecular mechanisms that restrict the localization and dynamics of Ca2+ sensors in plants, however, are largely unknown. In this report, we identify a putative Ca2+-sensitive complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+-dependent lipid binding protein (CLB1), which is enriched at ER-PM contact sites (EPCS) and relocalizes in response to Rare Earth Elements (REEs)-induced endocytosis. Our results show that endocytosed REEs influence cytosolic Ca2+ signaling, as indicated by the activation of the Ca2+/Calmodulin-based ratiometric sensor GCaMP3, and promote the cytoskeleton-dependent accumulation of ER-PM contact sites at the cell cortex. Based on these results, we propose that the EPCS-localized SYT1/SYT5/CLB1 complex is part of an evolutionarily conserved and spatially regulated Ca2+-responsive mechanism that control cER-PM communication during stress episodes.

plant biology↗

bioRxiv2019

Antagonistic and auxin-dependent phosphoregulation of columella PIN proteins controls lateral root gravitropic setpoint angle in Arabidopsis

Lateral roots of many species are maintained at non-vertical angles with respect to gravity. These gravitropic setpoint angles (GSAs) are intriguing because their maintenance requires that roots are able to effect gravitropic response both with and against the gravity vector. Here we have used the Arabidopsis lateral root in order to investigate the molecular basis of the maintenance of non-vertical GSAs. We show that gravitropism in the lateral root is angle-dependent and that both upward and downward graviresponse requires auxin transport and the generation of auxin asymmetries consistent with the Cholodny-Went model. We show that the symmetry in auxin distribution in lateral roots growing at GSA can be traced back to a net, balanced polarization of PIN3 and PIN7 auxin transporters in the columella cells. Further, upward and downward graviresponse in lateral roots correlates with corresponding changes in PIN3 and PIN7 polar localisation. Finally, we show that auxin, in addition to driving tropic growth in the lateral root, acts within the columella to regulate GSA via the PIN phosphatase subunit RCN1 in a PIN3-dependent and PIN7-independent manner. Together, these findings provide a molecular framework for understanding gravity-dependent nonvertical growth in Arabidopsis lateral roots.

plant biology↗

bioRxiv2018

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division. In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI (SOK) proteins that integrate apical-basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. SOK proteins structurally resemble the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation. Our work identifies a plant compass, interpreted by SOK proteins. Furthermore, despite fundamental differences, polarity in plants and animals converge upon the same protein domain.

plant biology↗