Subcellular Reorganization Research Articles

Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity

The plant homolog of vertebrate necroptosis inducer mixed-lineage kinase domain-like (MLKL) contributes to downstream steps in Toll-interleukin-1 receptor domain NLR (TNL)-receptor-triggered immunity. Here, we show that Arabidopsis MLKL1 (AtMLKL1) clusters into puncta at the plasma membrane upon TNL activation and that this sub-cellular reorganization is dependent on the TNL signal transducer, EDS1. We find that AtMLKLs confer TNL-triggered immunity in parallel with RPW8-type HeLo-domain-containing NLRs (RNLs) and that the AtMLKL N-terminal HeLo domain is indispensable for both immunity and clustering. We show that the AtMLKL HeLo domain mediates cytoplasmic Ca2+ ([Ca2+]cyt) influx in plant and human cells, and AtMLKLs are responsible for sustained [Ca2+]cyt influx during TNL-triggered, but not CNL-triggered, immunity. Our study reveals parallel immune signaling functions of plant MLKLs and RNLs as mediators of [Ca2+]cyt influx and a potentially common role of the HeLo domain fold in the Ca2+-signal relay of diverse organisms.

Near-Native Imaging of Label-Free Silver Nanoparticles-Triggered 3D Subcellular Ultrastructural Reorganization in Microalgae.

Understanding the spatial orientation of nanoparticles and the corresponding subcellular architecture events favors uncovering fundamental toxic mechanisms and predicting response pathways of organisms toward environmental stressors. Herein, we map the spatial location of label-free citrate-coated Ag nanoparticles (Cit-AgNPs) and the corresponding subcellular reorganization in microalgae by a noninvasive 3D imaging approach, cryo-soft X-ray tomography (cryo-SXT). Cryo-SXT near-natively displays the 3D maps of Cit-AgNPs presenting in rarely identified sites, namely, extracellular polymeric substances (EPS) and the cytoplasm. By comparative 3D morphological assay, we observe that Cit-AgNPs disrupt the cellular ultrastructural homeostasis, triggering a severe malformation of cytoplasmic organelles with energy-producing and stress-regulating functions. AgNPs exposure causes evident disruption of the chloroplast membrane, significant attenuation of the pyrenoid matrix and starch sheath, extreme swelling of starch granules and lipid droplets, and shrinkage of the nucleolus. In accompaniment, the number and volume occupancy of starch granules are significantly increased. Meanwhile, the spatial topology of starch granules extends from the chloroplast to the cytoplasm with a dispersed distribution. Linking the dynamics of the internal structure and the alteration of physiological properties, we derive a comprehensive cytotoxic and response pathway of microalgae exposed to AgNPs. This work provides a perspective for assessing the toxicity at subcellular scales to achieve label-free nanoparticle-caused ultrastructure remodeling of phytoplankton.

Direct Visualization and Restoration of Metallic Ion-Induced Subcellular Ultrastructural Remodeling.

Analysis of cellular ultrastructure dynamics and metal ions' fate can provide insights into the interaction between living organisms and metal ions. Here, we directly visualize the distribution of biogenic metallic aggregates, ion-induced subcellular reorganization, and the corresponding regulation effect in yeast by the near-native 3D imaging approach, cryo-soft X-ray tomography (cryo-SXT). By comparative 3D morphometric assessment, we observe the gold ions disrupting cellular organelle homeostasis, resulting in noticeable distortion and folding of vacuoles, apparent fragmentation of mitochondria, extreme swelling of lipid droplets, and formation of vesicles. The reconstructed 3D architecture of treated yeast demonstrates ∼65% of Au-rich sites in the periplasm, a comprehensive quantitative assessment unobtained by TEM. We also observe some AuNPs in rarely identified subcellular sites, namely, mitochondria and vesicles. Interestingly, the amount of gold deposition is positively correlated with the volume of lipid droplets. Shifting the external starting pH to near-neutral results in the reversion of changes in organelle architectures, boosting the amount of biogenic Au nanoparticles, and increasing cell viability. This study provides a strategy to analyze the metal ions-living organism interaction from subcellular architecture and spatial localization perspectives.

Spatial proteomics reveals subcellular reorganization in human keratinocytes exposed to UVA light

SummaryThe effects of UV light on the skin have been extensively investigated. However, systematic information about how the exposure to ultraviolet-A (UVA) light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass-spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1,600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins, and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

Revealing Lipid Body Formation and its Subcellular Reorganization in Oleaginous Microalgae Using Correlative Optical Microscopy and Infrared Nanospectroscopy.

The purpose of this work is to develop an integrated imaging approach to characterize without labeling at the sub-cellular level the formation of lipid body droplets (LBs) in microalgae undergoing nitrogen starvation. First conventional optical microscopy approaches, gas chromatography, and turbidimetry measurements allowed to monitor the biomass and the total lipid content in the oleaginous microalgae Parachlorella kesslerii during the starvation process. Then a local analysis of the LBs was proposed using an innovative infrared nanospectroscopy technique called atomic force microscopy-based infrared spectroscopy (AFM-IR). This label-free technique assessed the formation of LBs and allowed to look into the LB composition thanks to the acquisition of local infrared spectra. Last correlative measurements using fluorescence microscopy and AFM-IR were performed to investigate the subcellular reorganization of LB and the chloroplasts.

Are Flavonoids Effective Antioxidants in Plants? Twenty Years of Our Investigation.

Whether flavonoids play significant antioxidant roles in plants challenged by photooxidative stress of different origin has been largely debated over the last few decades. A critical review of the pertinent literature and our experimentation as well, based on a free-of-scale approach, support an important antioxidant function served by flavonoids in plants exposed to a wide range of environmental stressors, the significance of which increases with the severity of stress. On the other side, some questions need conclusive answers when the putative antioxidant functions of plant flavonoids are examined at the level of both the whole-cell and cellular organelles. This partly depends upon a conclusive, robust, and unbiased definition of “a plant antioxidant”, which is still missing, and the need of considering the subcellular re-organization that occurs in plant cells in response to severe stress conditions. This likely makes our deterministic-based approach unsuitable to unveil the relevance of flavonoids as antioxidants in extremely complex biological systems, such as a plant cell exposed to an ever-changing stressful environment. This still poses open questions about how to measure the occurred antioxidant action of flavonoids. Our reasoning also evidences the need of contemporarily evaluating the changes in key primary and secondary components of the antioxidant defense network imposed by stress events of increasing severity to properly estimate the relevance of the antioxidant functions of flavonoids in an in planta situation. In turn, this calls for an in-depth analysis of the sub-cellular distribution of primary and secondary antioxidants to solve this still intricate matter.

Reduction of endocytic activity accelerates cell elimination during tissue remodeling of the Drosophila epidermal epithelium.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Cells that are no longer needed are quickly removed without compromising the barrier function of the tissue. During metamorphosis, insects undergo developmentally programmed tissue remodeling. However, the mechanisms that regulate this rapid tissue remodeling are not precisely understood. Here, we show that the temporal dynamics of endocytosis modulate physiological cell properties to prime larval epidermal cells for cell elimination. Endocytic activity gradually reduces as tissue remodeling progresses. This reduced endocytic activity accelerates cell elimination through the regulation of Myosin II subcellular reorganization, junctional E-cadherin levels, and caspase activation. Whereas the increased Myosin II dynamics accelerates cell elimination, E-cadherin plays a protective role against cell elimination. Reduced E-cadherin is involved in the amplification of caspase activation by forming a positive-feedback loop with caspase. These findings reveal the role of endocytosis in preventing cell elimination and in the cell-property switching initiated by the temporal dynamics of endocytic activity to achieve rapid cell elimination during tissue remodeling.

A Proteomic Map to Navigate Subcellular Reorganization in Fatty Liver Disease

In a tour de force in this issue of Developmental Cell, Krahmer etal. leverage proteomic platforms to reveal subcellular proteome dynamics during development of nonalcoholic fatty liver disease. Their findings uncover a remarkable degree of subcellular reorganization, alterations in protein localization, and remodeling of organelle contact sites.

Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy.

Dysfunctional astrocytes are increasingly recognized as key players in the development and progression of mesial temporal lobe epilepsy (MTLE). One of the dramatic changes astrocytes undergo in MTLE with hippocampal sclerosis (HS) is loss of gap junction coupling. To further elucidate molecular mechanism(s) underlying this alteration, we assessed expression, cellular localization and phosphorylation status of astrocytic gap junction proteins in human and experimental MTLE-HS. In addition to conventional confocal analysis of immunohistochemical staining we employed expansion microscopy, which allowed visualization of blood-brain-barrier (BBB) associated cellular elements at a sub-µm scale. Western Blot analysis showed that plasma membrane expression of connexin43 (Cx43) and Cx30 were not significantly different in hippocampal specimens with and without sclerosis. However, we observed a pronounced subcellular redistribution of Cx43 toward perivascular endfeet in HS, an effect that was accompanied by increased plaque size. Furthermore, in HS Cx43 was characterized by enhanced C-terminal phosphorylation of sites affecting channel permeability. Prominent albumin immunoreactivity was found in the perivascular space of HS tissue, indicating that BBB damage and consequential albumin extravasation was involved in Cx43 dysregulation. Together, our results suggest that subcellular reorganization and/or abnormal posttranslational processing rather than transcriptional downregulation of astrocytic gap junction proteins account for the loss of coupling reported in human and experimental TLE. The observations of the present study provide new insights into pathological alterations of astrocytes in HS, which may aid in the identification of novel therapeutic targets and development of alternative anti-epileptogenic strategies.

Two fission yeast high mobility group box proteins in the maintenance of genomic integrity following doxorubicin insult

Drug resistance is a challenge in chemotherapy, and, to date, there has been little resolution as to how it is induced. We previously isolated a host of doxorubicin resistance (DXR) genes in fission yeast and here we investigate the regulation of this resistance through two high mobility group (HMG) motif-containing DXR proteins, Nht1 and Hap2. The concurrent deletion of nht1 and hap2 did not confer cumulative sensitivity to doxorubicin, indicating that these factors cooperate closely in similar epistatic groups. We show that doxorubicin treatment resulted in the subcellular reorganization of Rhp54, a homologous recombination-dependent DNA damage repair protein. The disruption of either nht1 or hap2 attenuated Rhp54-foci formation, suggesting that these factors modulate the repair of doxorubicin-induced DNA lesions via the recruitment of homologous recombination machinery. Epistatic analyses further confirmed that Nht1 and Hap2 act in similar functional groups with complexes related to DSB repair but act synergistically with factors that regulate transcription and chromosome segregation. Overall, this work shows the molecular crosstalk coordinated by HMG proteins in conferring doxorubicin resistance in fission yeast.

An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions

We report a protein-fragment complementation assay (PCA) based on the engineered Deinococcus radiodurans infrared fluorescent protein IFP1.4. Unlike previous fluorescent protein PCAs, the IFP PCA is reversible, allowing analysis of spatiotemporal dynamics of hormone-induced signaling complexes in living yeast and mammalian cells at nanometer resolution. The inherently low background of infrared fluorescence permitted detection of subcellular reorganization of a signaling complex expressed at low abundance.

Involvement of Adhesive Puncta Adherentia Type Junctions in the Formation of Synaptic Connections between Dentate Fascia Transplants and the Recipient Brain

The dentate fascia of 20-day Wistar rat fetuses were transplanted heterotopically into the somatosensory area of the neocortex of adult rats. Granule cells in grafts projected their axons (mossy fibers) into recipient brains and established synaptic contacts with inappropriate targets. Electron microscopy was used to study the characteristics of the organization of ectopic mossy fiber synapses. These studies showed that ectopic synapses reproduced the determinant structural features of giant hippocampal synapses and induced subcellular reorganization of postsynaptic dendrites in the neocortex. Morphometric analysis showed significant increases in the number and extents of adhesive junctions, puncta adherentia, in ectopic synapses as compared with controls. These data indicate the involvement of adhesive mechanisms in the structural-chemical adaptation of target neurons to foreign axons growing from transplants.

3D high content imaging: high level phenotypic quantification new opportunity for drug discovery

Advances in automated imaging microscopy allow fast acquisitions of multidimensional biological samples. Those microscopes open new possibilities for analyzing subcellular structures and spatial cellular arrangements. We present a 3D image analysis framework perfectly well suited for medium-throughput screening. Upon adaptive and regularized segmentation, followed by precise 3D reconstruction, we achieve automatic quantification of numerous relevant 3D descriptors related to the shape, texture, and fluorescence intensity of multiple stained subcellular structures. A global analysis of the 3D reconstructed scene shows additional possibilities to quantify the relative position of organelles. Implementing this methodology, we analyzed the subcellular reorganization of the nucleus, the Golgi apparatus and the centrioles occurring during the cell cycle. In addition, we quantified the effect of a genetic mutation associated with the early onset primary dystonia on the redistribution of torsinA from the bulk endoplasmic reticulum to the perinuclear space of the nuclear envelope. We show that the method enables the classification of various translocation levels of torsinA and opens the possibility for compound-based screening campaigns. Finally we present real applications of 3D cellular phenotype quantification in the screening context.

Contextual Automated 3D Analysis of Subcellular Organelles Adapted to High-Content Screening

Advances in automated imaging microscopy allow fast acquisitions of multidimensional biological samples. Those microscopes open new possibilities for analyzing subcellular structures and spatial cellular arrangements. In this article, the authors describe a 3D image analysis framework adapted to medium-throughput screening. Upon adaptive and regularized segmentation, followed by precise 3D reconstruction, they achieve automatic quantification of numerous relevant 3D descriptors related to the shape, texture, and fluorescence intensity of multiple stained subcellular structures. A global analysis of the 3D reconstructed scene shows additional possibilities to quantify the relative position of organelles. Implementing this methodology, the authors analyzed the subcellular reorganization of the nucleus, the Golgi apparatus, and the centrioles occurring during the cell cycle. In addition, they quantified the effect of a genetic mutation associated with the early onset primary dystonia on the redistribution of torsinA from the bulk endoplasmic reticulum to the perinuclear space of the nuclear envelope. They show that their method enables the classification of various translocation levels of torsinA and opens the possibility for compound-based screening campaigns restoring the normal torsinA phenotype.

Carbon Nanotubes Reorganize Actin Structures in Cells and ex Vivo

The ability of globular actin to form filaments and higher-order network structures of the cytoskeleton is essential for cells to maintain their shape and perform essential functions such as force generation, motility, and division. Alterations of actin structures can dramatically change a cell's ability to function. We found that purified and dispersed single wall carbon nanotubes (SWCNTs) can induce actin bundling in cells and in purified model actin systems. SWCNTs do not induce acute cell death, but cell proliferation is greatly reduced in SWCNT-treated cells with an increase in actin-related division defects. Actin, normally present in basal stress fibers in control cells, is located in heterogeneous structures throughout the SWCNT-treated cell. These SWCNT-induced changes in actin structures are seen functionally in multinucleated cells and with reduced force generation. Ex vivo, purified actin filaments cross-linked with alpha-actinin and formed isotropic networks, whereas SWCNTs caused purified actin filaments to assemble into bundles. While purified, isolated SWCNTs do not appear acutely toxic, this subcellular reorganization may cause chronic changes to cellular functions.

Triggering cell detachment from patterned electrode arrays by programmed subcellular release

Programmed subcellular release is an in vitro technique for the quantitative study of cell detachment. The dynamics of cell contraction are measured by releasing cells from surfaces to which they are attached with spatial and temporal control. Release of subcellular regions of cells is achieved by plating cells on an electrode array created by standard microfabrication methods. The electrodes are then biochemically functionalized with an arginine-glycine-aspartic acid (RGD)-terminated thiol. Application of a voltage pulse results in electrochemical desorption of the RGD-terminated thiols, triggering cell detachment. This method allows for the study of the full cascade of events from detachment to subsequent subcellular reorganization. Fabrication of the electrode arrays may take 1-2 d. Preparation for experiments, including surface functionalization and cell plating, can be completed in 10 h. A series of cell release experiments on one device may last several hours.

Differential association of Orc1 and Sir2 proteins to telomeric domains in Plasmodium falciparum

Telomeres have the capacity to recruit proteins that facilitate the spreading of heterochromatin into subtelomeric DNA regions. In the human protozoan pathogen Plasmodium falciparum, the telomere-associated protein Sir2 has been shown to control the silencing of members of virulence genes at some, but not all, chromosome-end loci, indicating that additional proteins are involved in telomere position effect. Here, we identified, in P. falciparum, a novel telomere-associated protein that displays homology with the origin-of-recognition-complex 1 protein Orc1. Antibodies raised against this P. falciparum protein localized to telomeric clusters in the nuclear periphery and the nucleolus. It was found that, prior to DNA replication, P. falciparum Orc1 and Sir2 undergo drastic subcellular reorganization, such as dissociation from the telomere cluster and spreading into the nucleus and parasite cytoplasm. Relocation of Orc1 and Sir2 was also linked to the partial dissociation of telomere clusters. Super gel-shift and chromatin-immunoprecipitation experiments showed the physical association of Orc1 with telomere repeats but revealed a differential association with adjacent non-coding repeat DNA elements. Our data suggest that Plasmodium telomeres might fold back and that Orc1 cooperates with Sir2 in telomeric silencing.

Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

BackgroundPlant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle.ResultsWithin 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed.ConclusionOur results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the pathogen as it attempts to invade the epidermal cell surface.

Directional sensing in eukaryotic chemotaxis: a balanced inactivation model.

Many eukaryotic cells, including Dictyostelium discoideum amoebae, fibroblasts, and neutrophils, are able to respond to chemoattractant gradients with high sensitivity. Recent studies have demonstrated that, after the introduction of a chemoattractant gradient, several chemotaxis pathway components exhibit a subcellular reorganization that cannot be described as a simple amplification of the external gradient. Instead, this reorganization has the characteristics of a switch, leading to a well defined front and back. Here, we propose a directional sensing mechanism in which two second messengers are produced at equal rates. The diffusion of one of them, coupled with an inactivation scheme, ensures a switch-like response to external gradients for a large range of gradient steepness and average concentration. Furthermore, our model is able to reverse the subcellular organization rapidly, and its response to multiple simultaneous chemoattractant sources is in good agreement with recent experimental results. Finally, we propose that the dynamics of a heterotrimeric G protein might allow for a specific biochemical realization of our model.