Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants.

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.

Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach.

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana , which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues. Graphical abstract.

Phosphatidylinositol-4-phosphate joins the dance of plant autophagosome formation.

In plants, macroautophagy/autophagy is a key mechanism that contributes to their ability to cope with a wide range of environmental constraints such as drought, nutrient starvation or pathogen resistance. Nevertheless, the molecular mechanisms of plant autophagy, and notably that of autophagosome formation, remain poorly understood. As the starting point of our recent paper, we considered the potential functional contribution of lipids in the numerous membrane-remodeling steps involved in this process. By combining biochemistry, genetics, cell biology and high-resolution 3D imaging, we unraveled the function of the lipid phosphatidylinositol-4-phosphate (PtdIns4P) in autophagy in Arabidopsis thaliana, thus providing novel insights into the assembly of autophagosomes in plant cells.

Novel phonotactic learning by children and infants: Generalizing syllable-position but not co-occurrence regularities.

Restrictions in the sequencing of sounds (phonotactic constraints) can be represented at the level of sound co-occurrences (e.g., in baF.Pev, F and P co-occur) and at the level of the syllable (e.g., F is syllable-coda/end, P is syllable-onset/start). Can children (5-year-olds) and infants (11-month-olds) represent constraints as sound co-occurrences and/or relative to syllable positions? Participants listened to artificial languages displaying both word-medial consonant restrictions in co-occurrence pairs (e.g., FP or DZ but not FZ) and in the position of consonants within syllables (e.g., P/Z onsets and D/F codas) in words like baF.Pev and tiD.Zek. Children responded similarly to novel words with the same (e.g., FP) versus different (e.g., FZ) co-occurrence pairs, but they were more misled (i.e., responded "heard it before") by novel words with consonants in the same (e.g., onset-P) versus different (e.g., coda-P) syllable positions (Experiment 1). With the same training stimuli, infants had similar orientation times for novel words with the same versus different co-occurrence pairs, but they had longer orientation times for novel words with consonants in the same versus different syllable positions (Experiment 2). Thus, across different methods and ages, syllable-position information was more readily available for generalization than consonant co-occurrence information. The results suggest that when multiple regularities are present simultaneously, some phonotactic constraints (e.g., consonants in particular syllable positions) may be spontaneously represented and generalized by children and infants, whereas others (e.g., consonant co-occurrences) might not be available. The results contribute toward understanding how children and infants represent sound sequences.

SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome-lysosome fusion.

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.

Phosphatidylinositol-4-phosphate controls autophagosome formation in Arabidopsis thaliana.

Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.

Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer.

Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.

A Shift in the Direction of the Production Effect in Children Aged 2-6 Years.

Research has found mixed evidence for the production effect in childhood. Some studies have found a positive effect of production on word recognition and recall, while others have found the reverse. This paper takes a developmental approach to investigate the production effect. Children aged 2-6 years (n = 150) from a predominantly white population in Ottawa, Canada were trained on familiar words which were either seen, heard or produced, followed by a recall task. Results showed a developmental shift: younger participants showed a reverse production effect, recalling more words that were heard during training, while older children showed the typical production effect, recalling more produced words. The effect of production on recall is not unidirectional and varies by age.

How Lipids Contribute to Autophagosome Biogenesis, a Critical Process in Plant Responses to Stresses.

Throughout their life cycle, plants face a tremendous number of environmental and developmental stresses. To respond to these different constraints, they have developed a set of refined intracellular systems including autophagy. This pathway, highly conserved among eukaryotes, is induced by a wide range of biotic and abiotic stresses upon which it mediates the degradation and recycling of cytoplasmic material. Central to autophagy is the formation of highly specialized double membrane vesicles called autophagosomes which select, engulf, and traffic cargo to the lytic vacuole for degradation. The biogenesis of these structures requires a series of membrane remodeling events during which both the quantity and quality of lipids are critical to sustain autophagy activity. This review highlights our knowledge, and raises current questions, regarding the mechanism of autophagy, and its induction and regulation upon environmental stresses with a particular focus on the fundamental contribution of lipids. How autophagy regulates metabolism and the recycling of resources, including lipids, to promote plant acclimation and resistance to stresses is further discussed.

The Effects of Confinement on Neuropsychiatric Symptoms in Alzheimer's Disease During the COVID-19 Crisis.

BACKGROUND: Neuropsychiatric symptoms, such as depression, anxiety, apathy, agitation, and hallucinations, are frequent in Alzheimer's disease (AD) and their prevalence tends to increase with external stressors. OBJECTIVE: We offer the first investigation of the effects of confinement during the COVID-19 crisis on neuropsychiatric symptoms in patients with AD. METHODS: We contacted caregivers of 38 patients with AD who were confined to their homes for nearly two months and asked them to report whether patients experienced any change in neuropsychiatric symptoms during, compared to before, the confinement and rate its severity and impact on themselves using the Neuropsychiatric Inventory-Questionnaire. RESULTS: Among the 38 patients, only 10 demonstrated neuropsychiatric changes during the confinement. Cognitive function of these 10 patients, assessed with the Mini-Mental State Examination, was worse than that of patients who did not demonstrate neuropsychiatric changes. Interestingly, among the 10 patients with neuropsychiatric changes, the duration of confinement significantly correlated with the severity of symptoms as well as with their caregivers' distress. DISCUSSION: The confinement seems to impact neuropsychiatric symptomatology in AD patients with low baseline cognitive function.

Impact of Confinement on the Burden of Caregivers of Patients with the Behavioral Variant of Frontotemporal Dementia and Alzheimer Disease during the COVID-19 Crisis in France.

INTRODUCTION: The clinical presentation of the behavioral variant of frontotemporal dementia (bvFTD) differs from that of Alzheimer disease (AD), with major impairments in behavioral functions in bvFTD and cognitive impairment in AD. Both behavioral disturbances in bvFTD and cognitive impairment in AD contribute to caregiver burden. OBJECTIVE: To investigate the impact of home confinement during the COVID-19 crisis on the burden of caregivers of bvFTD or AD patients. METHODS: During the COVID-19 lockdown in France, neurologists and neuropsychologists from the Memory Center of Nantes Hospital conducted teleconsultations for 38 AD patients and 38 bvFTD patients as well as for their caregivers. During these consultations, caregivers were invited to rate the change in their burden during home confinement. They were also invited to rate behavioral or emotional changes in the patients during, compared with before, the confinement. RESULTS: Twenty-two bvFTD caregivers and 14 AD caregivers experienced an increase in burden. For bvFTD caregivers, this increased burden occurred regardless of behavioral changes, while AD caregivers experienced an increased burden related to changes in patients' neuropsychiatric symptoms. Among the whole cohort, 2 factors were associated with increased caregiver burden: behavioral change and bvFTD. CONCLUSION: The results demonstrate that during home confinement in the COVID-19 crisis, neuropsychiatric symptoms were the core factor that impacted caregiver burden in different ways depending on the disease.

Bidirectional roles of Dhh1 in regulating autophagy.

Macroautophagy/autophagy activity is carefully modulated to allow cells to adapt to changing environmental conditions and maintain energy homeostasis. This control notably occurs in part through the regulation of autophagy-related (ATG) gene expression. Others and we have jointly shown that under nutrient-rich conditions Dhh1 mediates the degradation of certain ATG mRNAs, most significantly that of ATG8, through a Dcp2-dependent decapping pathway to maintain gene expression and autophagy activity at a basal level. More recently, we illustrated that under nitrogen-starvation conditions Dhh1 switches its role to become a positive regulator of autophagy, and promotes the translation of ATG1 and ATG13 mRNAs to meet the increased demand for autophagy activity. This regulation helps selected ATG mRNAs to escape the general repression in translation that occurs when nutrients are limited and TOR is inhibited. Our studies also suggest that Dhh1's nutrient-dependent bidirectional regulation of auto-phagy is conserved in more complex eukaryotes. Abbreviations: ATG: autophagy related; EIF4EBP: EIF4E binding protein; UTR: untranslated region.

Arabidopsis CER1-LIKE1 Functions in a Cuticular Very-Long-Chain Alkane-Forming Complex.

Plant aerial organs are coated with cuticular waxes, a hydrophobic layer that primarily serves as a waterproofing barrier. Cuticular wax is a mixture of aliphatic very-long-chain molecules, ranging from 22 to 48 carbons, produced in the endoplasmic reticulum of epidermal cells. Among all wax components, alkanes represent up to 80% of total wax in Arabidopsis (Arabidopsis thaliana) leaves. Odd-numbered alkanes and their derivatives are produced through the alkane-forming pathway. Although the chemical reactions of this pathway have been well described, the enzymatic mechanisms catalyzing these reactions remain unclear. We previously showed that a complex made of Arabidopsis ECERIFERUM1 (CER1) and CER3 catalyzes the conversion of acyl-Coenzyme A's to alkanes with strict substrate specificity for compounds containing more than 29 carbons. To learn more about alkane biosynthesis in Arabidopsis, we characterized the biochemical specificity and physiological functions of a CER1 homolog, CER1-LIKE1. In a yeast strain engineered to produce very-long-chain fatty acids, CER1-LIKE1 interacted with CER3 and cytochrome B5 to form a functional complex leading to the production of alkanes that are of different chain lengths compared to that produced by CER1-containing complexes. Gene expression analysis showed that both CER1 and CER1-LIKE1 are differentially expressed in an organ- and tissue-specific manner. Moreover, the inactivation or overexpression of CER1-LIKE1 in Arabidopsis transgenic lines specifically impacted alkane biosynthesis and wax crystallization. Collectively, our study reports on the identification of a further plant alkane synthesis enzymatic component and supports a model in which several alkane-forming complexes with distinct chain-length specificities coexist in plants.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo.

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

The exoribonuclease Xrn1 is a post-transcriptional negative regulator of autophagy.

Macroautophagy/autophagy is a conserved catabolic process that promotes survival during stress. Autophagic dysfunction is associated with pathologies such as cancer and neurodegenerative diseases. Thus, autophagy must be strictly modulated at multiple levels (transcriptional, post-transcriptional, translational and post-translational) to prevent deregulation. Relatively little is known about the post-transcriptional control of autophagy. Here we report that the exoribonuclease Xrn1/XRN1 functions as a negative autophagy factor in the yeast Saccharomyces cerevisiae and in mammalian cells. In yeast, chromosomal deletion of XRN1 enhances autophagy and the frequency of autophagosome formation. Loss of Xrn1 results in the upregulation of autophagy-related (ATG) transcripts under nutrient-replete conditions, and this effect is dependent on the ribonuclease activity of Xrn1. Xrn1 expression is regulated by the yeast transcription factor Ash1 in rich conditions. In mammalian cells, siRNA depletion of XRN1 enhances autophagy and the replication of 2 picornaviruses. This work provides insight into the role of the RNA decay factor Xrn1/XRN1 as a post-transcriptional regulator of autophagy.

Lipids in membrane dynamics during autophagy in plants.

Autophagy is a critical pathway for plant adaptation to stress. Macroautophagy relies on the biogenesis of a specialized membrane named the phagophore that maturates into a double membrane vesicle. Proteins and lipids act synergistically to promote membrane structure and functions, yet research on autophagy has mostly focused on autophagy-related proteins while knowledge of supporting lipids in the formation of autophagic membranes remains scarce. This review expands on studies in plants with examples from other organisms to present and discuss our current understanding of lipids in membrane dynamics associated with the autophagy pathway in plants.

Novel phonotactic learning: Tracking syllable-position and co-occurrence constraints.

Are syllable-level and co-occurrence representations simultaneously available when one learns novel phonotactics? After training on word-medial consonant restrictions (e.g., word-medial onsets P/Z, codas D/F, and cross-syllable consonant clusters FP/DZ in items like baF.Pev, tiD.Zek), adults falsely recognized novel items containing restricted consonants with the same co-occurrences (e.g., FP) more often than those with different co-occurrences (e.g., FZ) when syllable-position information was kept constant (e.g., vuF.Pet vs. vuF.Zet; Experiment 1). Thus, adults tracked co-occurrence information. Additionally, even when co-occurrence information was different from training, participants more often falsely recognized novel items that contained restricted consonants in the same (e.g., onset-Z) rather than different syllable positions (e.g., coda-Z), whether the restricted consonants were in the same (word-medial, e.g., vuF.Zet vs. vuZ.Fet, Experiment 2) or different word positions (word-edge, e.g., Zut.veF vs. Fut.veZ, Experiment 3). Thus, adults also tracked syllable-level information. These findings show that adults spontaneously represent sound sequences at multiple levels.

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.