Deep learning -- promises for 3D nuclear imaging: a guide for biologists.

For the past century, the nucleus has been the focus of extensive investigations in cell biology. However, many questions remain about how its shape and size are regulated during development, in different tissues, or during disease and aging. To track these changes, microscopy has long been the tool of choice. Image analysis has revolutionized this field of research by providing computational tools that can be used to translate qualitative images into quantitative parameters. Many tools have been designed to delimit objects in 2D and, eventually, in 3D in order to define their shapes, their number or their position in nuclear space. Today, the field is driven by deep-learning methods, most of which take advantage of convolutional neural networks. These techniques are remarkably adapted to biomedical images when trained using large datasets and powerful computer graphics cards. To promote these innovative and promising methods to cell biologists, this Review summarizes the main concepts and terminologies of deep learning. Special emphasis is placed on the availability of these methods. We highlight why the quality and characteristics of training image datasets are important and where to find them, as well as how to create, store and share image datasets. Finally, we describe deep-learning methods well-suited for 3D analysis of nuclei and classify them according to their level of usability for biologists. Out of more than 150 published methods, we identify fewer than 12 that biologists can use, and we explain why this is the case. Based on this experience, we propose best practices to share deep-learning methods with biologists.

A nuclear localization signal targets tail-anchored membrane proteins to the inner nuclear envelope in plants.

Protein targeting to the inner nuclear membrane (INM) is one of the least understood protein targeting pathways. INM proteins are important for chromatin organization, nuclear morphology and movement, and meiosis, and have been implicated in human diseases. In opisthokonts, one mechanism for INM targeting is transport factor-mediated trafficking, in which nuclear localization signals (NLSs) function in nuclear import of transmembrane proteins. To explore whether this pathway exists in plants, we fused the SV40 NLS to a plant ER tail-anchored protein and showed that the GFP-tagged fusion protein was significantly enriched at the nuclear envelope (NE) of leaf epidermal cells. Airyscan subdiffraction limited confocal microscopy showed that this protein displays a localization consistent with an INM protein. Nine different monopartite and bipartite NLSs from plants and opisthokonts, fused to a chimeric tail-anchored membrane protein, were all sufficient for NE enrichment, and both monopartite and bipartite NLSs were sufficient for trafficking to the INM. Tolerance for different linker lengths and protein conformations suggests that INM trafficking rules might differ from those in opisthokonts. The INM proteins developed here can be used to target new functionalities to the plant nuclear periphery. This article has an associated First Person interview with the first author of the paper.

Aluminium-silicon interactions in higher plants: an update.

Aluminium (Al) and silicon (Si) are abundant in soils, but their availability for plant uptake is limited by low solubility. However, Al toxicity is a major problem in naturally occurring acid soils and in soils affected by acidic precipitation. When, in 1995, we reviewed this topic for the Journal of Experimental Botany, it was clear that under certain circumstances soluble Si could ameliorate the toxic effects of Al, an effect mirrored in organisms beyond the plant kingdom. In the 25 years since our review, it has become evident that the amelioration phenomenon occurs in the root apoplast, with the formation of hydroxyaluminosilicates being part of the mechanism. A much better knowledge of the molecular basis for Si and Al uptake by plants and of Al toxicity mechanisms has been developed. However, relating this work to amelioration by Si is at an early stage. It is now clear that co-deposition of Al and Si in phytoliths is a fairly common phenomenon in the plant kingdom, and this may be important in detoxification of Al. Relatively little work on Al-Si interactions in field situations has been done in the last 25 years, and this is a key area for future development.

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

The Role of Cognitive Control in the Self-Regulation and Reinforcement of Smoking Behavior.

Cognitive control (CC)-the ability to regulate attention and memory-plays an important role in a variety of health behaviors, including smoking behavior. In this theoretical review of the literature, we propose a CC and smoking behavior framework that includes (1) the positive influence of CC on the self-regulation of smoking, (2) nicotine-induced improvements in CC that may indirectly reinforce smoking (including withdrawal reversal effects), and (3) the long-term effects of smoking on the brain that may result in reduced CC. Integration of these literatures suggests that CC contributes to both self-regulation (ie, brake pedal) and nicotine-related reinforcement (ie, gas pedal) amid the catastrophic effects of long-term smoking, which may reduce self-regulatory control over smoking while also enhancing indirect reinforcement. Supportive evidence and limitations of this approach will be presented, as well as ideas for future research directions that may fully examine this multifaceted modeling of CC in relation to smoking behavior. IMPLICATIONS: There is substantial evidence that CC contributes to self-regulation (ie, brake pedal) and reinforcement (ie, gas pedal) of smoking behavior as well as evidence that long-term smoking may cause reduced CC. The proposed model delineates how these opposing influences of CC may mask the unique contribution of self-regulation and reinforcement in maintaining smoking behavior. Targeting CC for treating nicotine dependence will require more nuanced approaches that consider the independent and combined effects of self-regulation and reinforcement to improve smoking cessation success rates.

Nicotine Withdrawal Induces Neural Deficits in Reward Processing.

INTRODUCTION: Nicotine withdrawal reduces neurobiological responses to nonsmoking rewards. Insight into these reward deficits could inform the development of targeted interventions. This study examined the effect of withdrawal on neural and behavioral responses during a reward prediction task. METHODS: Smokers (N = 48) attended two laboratory sessions following overnight abstinence. Withdrawal was manipulated by having participants smoke three regular nicotine (0.6 mg yield; satiation) or very low nicotine (0.05 mg yield; withdrawal) cigarettes. Electrophysiological recordings of neural activity were obtained while participants completed a reward prediction task that involved viewing four combinations of predictive and reward-determining stimuli: (1) Unexpected Reward; (2) Predicted Reward; (3) Predicted Punishment; (4) Unexpected Punishment. The task evokes a medial frontal negativity that mimics the phasic pattern of dopaminergic firing in ventral tegmental regions associated with reward prediction errors. RESULTS: Nicotine withdrawal decreased the amplitude of the medial frontal negativity equally across all trial types (p < .001). Exploratory analyses indicated withdrawal increased time to initiate the next trial following unexpected punishment trials (p < .001) and response time on reward trials during withdrawal was positively related to nicotine dependence (p < .001). CONCLUSIONS: Nicotine withdrawal had equivocal impact across trial types, suggesting reward processing deficits are unlikely to stem from changes in phasic dopaminergic activity during prediction errors. Effects on tonic activity may be more pronounced. Pharmacological interventions directly targeting the dopamine system and behavioral interventions designed to increase reward motivation and responsiveness (eg, behavioral activation) may aid in mitigating withdrawal symptoms and potentially improving smoking cessation outcomes. IMPLICATIONS: Findings from this study indicate nicotine withdrawal impacts reward processing signals that are observable in smokers' neural activity. This may play a role in the subjective aversive experience of nicotine withdrawal and potentially contribute to smoking relapse. Interventions that address abnormal responding to both pleasant and unpleasant stimuli may be particularly effective for alleviating nicotine withdrawal.

Absence of SUN1 and SUN2 proteins in Arabidopsis thaliana leads to a delay in meiotic progression and defects in synapsis and recombination.

The movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC-84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force-generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified. Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1-1 Atsun2-2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock-like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.

A novel family of plant nuclear envelope-associated proteins.

This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

The plant LINC complex at the nuclear envelope.

Significant advances in understanding the plant nuclear envelope have been made over the past few years; indeed, knowledge of the protein network at the nuclear envelope is rapidly growing. One such network, the linker of nucleoskeleton and cytoskeleton (LINC) complex, is known in animals to connect chromatin to the cytoskeleton through the nuclear envelope. The LINC complex is made of Sad1/Unc84 (SUN) and Klarsicht/Anc1/Syne1 homology (KASH) proteins which have been recently characterized in plants. SUN proteins are located within the inner nuclear membrane, while the KASH proteins are included into the outer nuclear membrane. SUN and KASH domains interact and bridge the two nuclear membranes. In Arabidopsis, KASH proteins also interact with the tryptophan-proline-proline (WPP) domain-interacting tail-anchored protein 1 (WIT1), associated with the nuclear pore complex and with myosin XI-i which directly interacts with the actin cytoskeleton. Although evidence for a plant LINC complex connecting the nucleus to the cytoskeleton is growing, its interaction with chromatin is still unknown, but knowledge gained from animal models strongly suggests its existence in plants. Possible functions of the plant LINC complex in cell division, nuclear shape, and chromatin organization are discussed.

Characterization of two distinct subfamilies of SUN-domain proteins in Arabidopsis and their interactions with the novel KASH-domain protein AtTIK.

SUN-domain proteins belong to a gene family including classical Cter-SUN and mid-SUN subfamilies differentiated by the position of the SUN domain within the protein. Although present in animal and plant species, mid-SUN proteins have so far remained poorly described. Here, we used a combination of genetics, yeast two-hybrid and in planta transient expression methods to better characterize the SUN family in Arabidopsis thaliana. First, we validated the mid-SUN protein subfamily as a monophyletic group conserved from yeast to plant. Arabidopsis Cter-SUN (AtSUN1 and AtSUN2) and mid-SUN (AtSUN3 and AtSUN4) proteins expressed as fluorescent protein fusions are membrane-associated and localize to the nuclear envelope (NE) and endoplasmic reticulum. However, only the Cter-SUN subfamily is enriched at the NE. We investigated interactions in and between members of the two subfamilies and identified the coiled-coil domain as necessary for mediating interactions. The functional significance of the mid-SUN subfamily was further confirmed in mutant plants as essential for early seed development and involved in nuclear morphology. Finally, we demonstrated that both subfamilies interact with the KASH domain of AtWIP1 and identified a new root-specific KASH-domain protein, AtTIK. AtTIK localizes to the NE and affects nuclear morphology. Our study indicates that Arabidopsis Cter-SUN and mid-SUN proteins are involved in a complex protein network at the nuclear membranes, reminiscent of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex found in other kingdoms.

In Depth Topological Analysis of Arabidopsis Mid-SUN Proteins and Their Interaction with the Membrane-Bound Transcription Factor MaMYB.

Mid-SUN proteins are a neglected family of conserved type III membrane proteins of ancient origin with representatives in plants, animals, and fungi. Previous higher plant studies have associated them with functions at the nuclear envelope and the endoplasmic reticulum (ER). In this study, high-resolution confocal light microscopy is used to explore the localisation of SUN3 and SUN4 in the perinuclear region, to explore topology, and to study the role of mid-SUNs on endoplasmic reticulum morphology. The role of SUN3 in the ER is reinforced by the identification of a protein interaction between SUN3 and the ER membrane-bound transcription factor maMYB. The results highlight the importance of mid-SUNs as functional components of the ER and outer nuclear membrane.

Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms.

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)-GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing.

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF -ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360-460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF -ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360-460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.

Characterization of SUN-domain proteins at the higher plant nuclear envelope.

Sad1/UNC-84 (SUN)-domain proteins are inner nuclear membrane (INM) proteins that are part of bridging complexes linking cytoskeletal elements with the nucleoskeleton, and have been shown to be conserved in non-plant systems. In this paper, we report the presence of members of this family in the plant kingdom, and investigate the two Arabidopsis SUN-domain proteins, AtSUN1 and AtSUN2. Our results indicate they contain the highly conserved C-terminal SUN domain, and share similar structural features with animal and fungal SUN-domain proteins including a functional coiled-coil domain and nuclear localization signal. Both are expressed in various tissues with AtSUN2 expression levels relatively low but upregulated in proliferating tissues. Further, we found AtSUN1 and AtSUN2 expressed as fluorescent protein fusions, to localize to and show low mobility in the nuclear envelope (NE), particularly in the INM. Deletion of various functional domains including the N terminus and coiled-coil domain affect the localization and increase the mobility of AtSUN1 and AtSUN2. Finally, we present evidence that AtSUN1 and AtSUN2 are present as homomers and heteromers in vivo, and that the coiled-coil domains are required for this. The study provides evidence suggesting the existence of cytoskeletal-nucleoskeletal bridging complexes at the plant NE.

The smoking N-back: a measure of biased cue processing at varying levels of cognitive load.

INTRODUCTION: Recent cognitive models of drug addiction have emphasized attentional bias to drug-related cues. This bias manifests as increased accessibility to affect-laden drug-related content relative to less emotionally evocative stimuli and ideation. We examined whether biased processing of smoking-related content would differentially affect performance on a cognitive task as a function of smoking status and task complexity. METHODS: Twenty-one smokers and 15 nonsmokers completed increasingly difficult 1-, 2-, and 3-back versions of the Smoking N-back task. RESULTS: There were no reaction time effects that included smoking status nor was there an effect for accuracy on the 1-back task. However, smokers showed poorer accuracy on matched trials relative to nonmatched trials for smoking words on the 2- and 3-back tasks, which involve more effortful cognitive processing. Among nonsmokers, this effect was present within the 3-back condition only. CONCLUSIONS: These findings suggest that cognitive bias to drug-related cues may be modulated by task complexity. Future research on cognitive bias should better account for this factor. Additional research will be needed to validate these findings by controlling for various potential confounds (e.g., nicotine withdrawal, task fatigue) as well as determine the clinical relevance of cognitive bias across varying levels of task complexity.

The plant nuclear envelope in focus.

Recent progress in understanding the plant NE (nuclear envelope) has resulted from significant advances in identifying and characterizing the protein constituents of the membranes and nuclear pores. Here, we review recent findings on the membrane integral and membrane-associated proteins of the key domains of the NE, the pore domain and inner and outer NEs, together with information on protein targeting and NE function.

Organelle biogenesis and positioning in plants.

The biogenesis and positioning of organelles involves complex interacting processes and precise control. Progress in our understanding is being made rapidly as advances in analysing the nuclear and organellar genome and proteome combine with developments in live-cell microscopy and manipulation at the subcellular level. This paper introduces the collected papers resulting from Organelle Biogenesis and Positioning in Plants, the 2009 Biochemical Society Annual Symposium. Including papers on the nuclear envelope and all major organelles, it considers current knowledge and progress towards unifying themes that will elucidate the mechanisms by which cells generate the correct complement of organelles and adapt and change it in response to environmental and developmental signals.

Nuclear envelope proteins and their role in nuclear positioning and replication.

Controlled movement of the nucleus is important in a wide variety of plant cellular events. Positioning involving intact nuclei occurs in cell division, development, tip growing systems such as the root hair and in response to stimuli, including light, touch and infection. Positioning is also essential in the division and replication of nuclear components, ranging from chromosome attachment to the breakdown and reformation of the nuclear envelope. Although description and understanding of the processes involved have advanced rapidly in recent years, significant gaps remain in our knowledge, especially concerning nuclear proteins involved in anchoring and interacting with cytoskeletal and nucleoskeletal elements involved in movement. In the present review, processes involving the movement and positioning of nuclei and nuclear components are described together with novel proteins implicated in nucleoskeletal and cytoskeletal interactions.

Advancing knowledge of the plant nuclear periphery and its application for crop science.

In this review, we explore recent advances in knowledge of the structure and dynamics of the plant nuclear envelope. As a paradigm, we focused our attention on the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, a structurally conserved bridging complex comprising SUN domain proteins in the inner nuclear membrane and KASH domain proteins in the outer nuclear membrane. Studies have revealed that this bridging complex has multiple functions with structural roles in positioning the nucleus within the cell, conveying signals across the membrane and organizing chromatin in the 3D nuclear space with impact on gene transcription. We also provide an up-to-date survey in nuclear dynamics research achieved so far in the model plant Arabidopsis thaliana that highlights its potential impact on several key plant functions such as growth, seed maturation and germination, reproduction and response to biotic and abiotic stress. Finally, we bring evidences that most of the constituents of the LINC Complex and associated components are, with some specificities, conserved in monocot and dicot crop species and are displaying very similar functions to those described for Arabidopsis. This leads us to suggest that a better knowledge of this system and a better account of its potential applications will in the future enhance the resilience and productivity of crop plants.

Nicotine self-medication of cognitive-attentional processing.

This article selectively reviews research concerning nicotine's effects on cognition, including the neurobiological mechanism for these effects, task and experimental features that may be important for elucidating these effects, and why these effects may have amplified motivational significance among smokers with cognitive deficit. Nicotine has effects on various cognitive processes, though most studies in humans have focused on the amelioration of cognitive deficits experienced during drug withdrawal. The direct cognitive-enhancing effect of nicotine remains a controversial topic. The relationship between attentional and non-attentional cognitive effects of nicotine is discussed in the context of cognitive self-medication. Further research should include theory-driven examination of cognitive effects of nicotine, and develop targeted smoking cessation programs based on an improved understanding of the role of cognitive self-medication in high-risk individuals.