Crowded Nuclei Research Articles

Attention guided multi-scale cluster refinement with extended field of view for amodal nuclei segmentation

Nuclei segmentation plays a crucial role in disease understanding and diagnosis. In whole slide images, cell nuclei often appear overlapping and densely packed with ambiguous boundaries due to the underlying 3D structure of histopathology samples. Instance segmentation via deep neural networks with object clustering is able to detect individual segments in crowded nuclei but suffers from a limited field of view, and does not support amodal segmentation. In this work, we introduce a dense feature pyramid network with a feature mixing module to increase the field of view of the segmentation model while keeping pixel-level details. We also improve the model output quality by adding a multi-scale self-attention guided refinement module that sequentially adjusts predictions as resolution increases. Finally, we enable clusters to share pixels by separating the instance clustering objective function from other pixel-related tasks, and introduce supervision to occluded areas to guide the learning process. For evaluation of amodal nuclear segmentation, we also update prior metrics used in common modal segmentation to allow the evaluation of overlapping masks and mitigate over-penalization issues via a novel unique matching algorithm. Our experiments demonstrate consistent performance across multiple datasets with significantly improved segmentation quality.

Evolutionarily conserved protein motifs drive interactions between the plant nucleoskeleton and nuclear pores.

The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear shape and gene expression. To understand how the Arabidopsis (Arabidopsis thaliana) nucleoskeleton physically connects to the nuclear periphery in plants, we investigated the Arabidopsis nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. We identified 3 conserved peptide motifs within the N-terminal region of KAKU4, which are required for intermolecular interactions of KAKU4 with itself, interaction with the nucleoskeleton protein CROWDED NUCLEI (CRWN), localization at the nuclear periphery, and nuclear elongation in differentiated tissues. Unexpectedly, we find these motifs to be present also in NUP82 and NUP136, 2 plant-specific nucleoporins from the nuclear pore basket. We further show that NUP82, NUP136, and KAKU4 have a common evolutionary history predating nonvascular land plants with KAKU4 mainly localizing outside the nuclear pore suggesting its divergence from an ancient nucleoporin into a new nucleoskeleton component. Finally, we demonstrate that both NUP82 and NUP136, through their shared N-terminal motifs, interact with CRWN and KAKU4 proteins revealing the existence of a physical continuum between the nuclear pore and the nucleoskeleton in plants.

Arabidopsis lamin-like proteins CRWN1 and CRWN2 interact with SUPPRESSOR OF NPR1-1 INDUCIBLE 1 and RAD51D to prevent DNA damage.

Plants cope with various recurring stress conditions that often induce DNA damage, ultimately affecting plant genome integrity, growth, and productivity. The CROWDED NUCLEI (CRWN) family comprises lamin-like proteins with multiple functions, such as regulating gene expression, genome organization, and DNA damage repair in Arabidopsis (Arabidopsis thaliana). However, the mechanisms and consequences of CRWNs in DNA damage repair are largely unknown. Here, we reveal that CRWNs maintain genome stability by forming repairing nuclear bodies at DNA double-strand breaks. We demonstrate that CRWN1 and CRWN2 physically associate with the DNA damage repair proteins RAD51D and SUPPRESSOR OF NPR1-1 Inducible 1 (SNI1) and act in the same genetic pathway to mediate this process. Moreover, CRWN1 and CRWN2 partially localize at γ-H2AX foci upon DNA damage. Notably, CRWN1 and CRWN2 undergo liquid-liquid phase separation to form highly dynamic droplet-like structures with RAD51D and SNI1 to promote the DNA damage response (DDR). Collectively, our data shed light on the function of plant lamin-like proteins in the DDR and maintenance of genome stability.

Two-step regulation of centromere distribution by condensin II and the nuclear envelope proteins.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. After entering interphase, the positions of the scattered centromeres are then stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWN proteins have little effect on chromatin organization involved in the control of gene expression, indicating a robustness of chromatin organization regardless of the type of centromere distribution.

植物細胞の核のカタチを制御する分子機構

Most animal cells harbor spherical and oval nuclei, whereas the nuclei in terrestrial plants do not only show spherical and oval shapes but also show various morphologies such as spindle-shaped and rod-shaped. The morphology of animal nuclei is regulated by lamin proteins, which form the nuclear lamina. In contrast, lamin orthologs are not conserved in plants; therefore, nuclear lamina proteins that regulate nuclear morphology in plants have not been identified. In my research, I detected CROWDED NUCLEI (CRWN) proteins in crude nuclear lamina fraction prepared from Arabidopsis thaliana. Phenotype analysis of crwn disruptants and the subcellular localization analysis of CRWNs demonstrated that CRWNs are plant-specific nuclear lamina proteins and play essential roles in regulating nuclear morphology. In addition, I found that CRWNs govern the transcriptional control of many environmental stress-responsive genes. Particularly, I revealed that CRWNs alter the position of the CA gene cluster in the nucleus affecting CA gene expression, contributing to tolerance to excess copper.

Characterization of a Plant Nuclear Matrix Constituent Protein in Liverwort.

The nuclear lamina (NL) is a complex network of nuclear lamins and lamina-associated nuclear membrane proteins, which scaffold the nucleus to maintain structural integrity. In animals, type V intermediate filaments are the main constituents of NL. Plant genomes do not encode any homologs of these intermediate filaments, yet plant nuclei contain lamina-like structures that are present in their nuclei. In Arabidopsis thaliana, CROWDED NUCLEI (CRWN), which are required for maintaining structural integrity of the nucleus and specific perinuclear chromatin anchoring, are strong candidates for plant lamin proteins. Recent studies revealed additional roles of Arabidopsis Nuclear Matrix Constituent Proteins (NMCPs) in modulating plants’ response to pathogen and abiotic stresses. However, detailed analyses of Arabidopsis NMCP activities are challenging due to the presence of multiple homologs and their functional redundancy. In this study, we investigated the sole NMCP gene in the liverwort Marchantia polymorpha (MpNMCP). We found that MpNMCP proteins preferentially were localized to the nuclear periphery. Using CRISPR/Cas9 techniques, we generated an MpNMCP loss-of-function mutant, which displayed reduced growth rate and curly thallus lobes. At an organelle level, MpNMCP mutants did not show any alteration in nuclear morphology. Transcriptome analyses indicated that MpNMCP was involved in regulating biotic and abiotic stress responses. Additionally, a highly repetitive genomic region on the male sex chromosome, which was preferentially tethered at the nuclear periphery in wild-type thalli, decondensed in the MpNMCP mutants and located in the nuclear interior. This perinuclear chromatin anchoring, however, was not directly controlled by MpNMCP. Altogether, our results unveiled that NMCP in plants have conserved functions in modulating stress responses.

Effect of vitamin B17 on experimentally induced colon cancer in adult male albino rat.

Colon cancer is considered to be the third most common cancer worldwide. At diagnosis of colon cancer, 3.7-11% developed bone metastasis. Diet based strategies are important for prevention and treatment of colon cancer. This study investigated the effect of vitamin B17 on a DMH induced rat model of colon cancer. Eighty young adult male albino rats were divided into five groups: group I (control group), group II (vitamin B17), group III (colon cancer), group IV (protected) and group V (treated). Distal colon sections were prepared for light and scanning electron microscopic examination. Lumbar vertebrae specimens were prepared for light microscopic study. Morphometric and statistical analysis were done. In comparison with the control, both colon cancer and treated groups showed invasion of the colonic tissue by pleomorphic branching colonic glands of variable shapes and sizes lined with dysplastic elongated hyperchromatic nuclei with frequent mitotic figures or stratified multi-layered crowded nuclei with an extremely significant (p < 0.0001) reduction of goblet cell number when compared to the control together with major pathological bone changes were observed in colon cancer and the treated groups. While the protected group showed impressive improvement of all previously mentioned diameters.

Subnuclear gene positioning through lamina association affects copper tolerance

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated (CA) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions.

Nuclear lamina CRWN proteins regulate chromatin organization, gene expression, and nuclear body formation in plants.

The metazoan cell nucleus is physically supported by nuclear lamina structures, which are mainly composed of filamentous protein lamins. CROWDED NUCLEI (CRWNs)/nuclear matrix constituent proteins are considered potential candidates of lamin analogs in plants. CRWNs specifically localize to the nuclear periphery, maintain nuclear morphology, and interact with several inner nuclear membrane proteins. CRWNs also regulate several nuclear events. In this review, I summarize the functions of CRWNs in regulating chromatin organization, gene expression, and nuclear body formation, and provide insights into novel plant nuclear lamina functions and structures.

Plant lamin-like proteins mediate chromatin tethering at the nuclear periphery

BackgroundThe nuclear envelope not only serves as a physical barrier separating nuclear content from the cytoplasm but also plays critical roles in modulating the three-dimensional organization of genomic DNA. For both plants and animals, the nuclear periphery is a functional compartment enriched with heterochromatin. To date, how plants manage to selectively tether chromatin at the nuclear periphery is unclear.ResultsBy conducting dual-color fluorescence in situ hybridization experiments on 2C nuclei, we show that in Arabidopsis thaliana, specific chromatin positioning at the nuclear periphery requires plant lamin-like proteins CROWDED NUCLEI 1 (CRWN1), CRWN4, and DNA methylation in CHG and CHH contexts. With chromosome painting and Hi-C analyses, we show global attenuation of spatial chromatin compartmentalization and chromatin positioning patterns at the nuclear periphery in both the crwn1 and crwn4 mutants. Furthermore, ChIP-seq analysis indicates that CRWN1 directly interacts with chromatin domains localized at the nuclear periphery, which mainly contains non-accessible chromatin.ConclusionsIn summary, we conclude that CRWN1 is a key component of the lamina-chromatin network in plants. It is functionally equivalent to animal lamins, playing critical roles in modulating patterns of chromatin positioning at the nuclear periphery.

SAT-586 Primary Fallopian Tube Carcinoma Metastasizing to the Thyroid Gland: A Rare Cause of Hypothyroidism

Background: Primary fallopian tube carcinoma (PFTC) has been considered to account for only about 0.1-0.5% of all gynecologic cancers. It is notorious for metastasis to the ovaries, omentum, and uterus but has never been reported to present with hypothyroidism as a metastatic thyroid nodule. Case: A 53-year-old female presented with fatigue, generalized weakness and constipation for the last 4 months. Her past medical history was significant for stage IV poorly differentiated PFTC status post total abdominal hysterectomy, omentectomy and tumor debulking due to the local spread of the PFTC. She also underwent multiple cycles of chemotherapy with paclitaxel, cisplatin, doxorubicin and bevacizumab and was declared in remission by her oncologist. On examination, her vital signs were stable. Thyroid examination showed palpable nodules in the right and left lobes. Her laboratory investigation revealed normal liver and renal function tests, but the blood chemistry was significant for raised TSH levels of 68.8 mcIU/ml and decreased T3 and T4 levels of 45 ng/dL and 0.54 ng/dL respectively. Thyroid Peroxidase Antibody level was less than 3 IU/ml (reference range <6 IU/ML). Thyroid ultrasound showed a 2.3 x 0.7 x 2 cm solid nodule in the medial lower pole of the right lobe with and a 1.9 x 0.8 x 1.1 cm solid nodule in the medial lower pole of left lobe with increased vascularity. The fine needle aspiration cytology revealed groups of highly atypical cells showing enlarged, irregular, crowded nuclei which were intermixed with scattered bland follicular cells and macrophages. These atypical cells were identical to the serous carcinoma cells seen on previous PFTC specimens, confirming the hematogenous spread of PFTC to the thyroid gland. This diagnosis was further substantiated by immunohistochemical staining, which showed two different populations of cells, thyroid cells stained positive for TTF-1 (Thyroid transcription factor-1) while other cells were positive for WT-1 immunohistochemical stain strongly suggestive of metastatic serous carcinoma. Her serum cancer antigen 125 (CA 125) was also found to be elevated at 395 U/ml (normal range 0-35 U/ML) suggesting recurrence of PFTC. Niraparib chemotherapy was initiated. She was also started on 75 mcg of Levothyroxine per day. TSH levels after two months were 4.2 mcIU/ml. Conclusion: PFTC can be diagnosed early in its course using transvaginal Doppler ultrasound to detect areas of neovascularization within the fallopian tube. PFTC has histological and clinical resemblance with ovarian and uterine primaries and hence can be challenging to diagnose. It is incredibly uncommon to suspect thyroid metastasis from the residuals of PFTC as a possible etiology of hypothyroidism. Thus, physicians should keep a very high index of suspicion in appropriate clinical settings. Prognosis remains poor as most patients present with metastatic disease.

Multi-layer boosting sparse convolutional model for generalized nuclear segmentation from histopathology images

It is a challenging problem to achieve generalized nuclear segmentation in digital histopathology images. Existing techniques, using either handcrafted features in learning-based models or traditional image analysis-based approaches, do not effectively tackle the challenging cases, such as crowded nuclei, chromatin-sparse, and heavy background clutter. In contrast, deep networks have achieved state-of-the-art performance in modeling various nuclear appearances. However, their success is limited due to the size of the considered networks. We solve these problems by reformulating nuclear segmentation in terms of a cascade 2-class classification problem and propose a multi-layer boosting sparse convolutional (ML-BSC) model. In the proposed ML-BSC model, discriminative probabilistic binary decision trees (PBDTs) are designed as weak learners in each layer to cope with challenging cases. A sparsity-constrained cascade structure enables the ML-BSC model to improve representation learning. Comparing to the existing techniques, our method can accurately separate individual nuclei in complex histopathology images, and it is more robust against chromatin-sparse and heavy background clutter. An evaluation carried out using three disparate datasets demonstrates the superiority of our method over the state-of-the-art supervised approaches in terms of segmentation accuracy.

Loss of CRWN Nuclear Proteins Induces Cell Death and Salicylic Acid Defense Signaling.

Defects in the nuclear lamina of animal cell nuclei have dramatic effects on nuclear structure and gene expression as well as diverse physiological manifestations. We report that deficiencies in CROWDED NUCLEI (CRWN), which are candidate nuclear lamina proteins in Arabidopsis (Arabidopsis thaliana), trigger widespread changes in transcript levels and whole-plant phenotypes, including dwarfing and spontaneous cell death lesions. These phenotypes are caused in part by ectopic induction of plant defense responses via the salicylic acid pathway. Loss of CRWN proteins induces the expression of the salicylic acid biosynthetic gene ISOCHORISMATE SYNTHASE1, which leads to spontaneous defense responses in crwn1 crwn2 and crwn1 crwn4 mutants, which are deficient in two of the four CRWN paralogs. The symptoms of ectopic defense response, including pathogenesis marker gene expression and cell death, increase in older crwn double mutants. These age-dependent effects are postulated to reflect an increase in nuclear dysfunction or damage over time, a phenomenon reminiscent of aging effects seen in animal nuclei and in some human laminopathy patients.

Roles of CRWN-family proteins in protecting genomic DNA against oxidative damage

CROWDED NUCLEI (CRWN) family in Arabidopsis consists of four members, CRWN1 to CRWN4. It has been previously reported that the CRWN proteins are involved in the control of nuclear morphology and degradation of ABI5. In this study, however, we discover that CRWN-family proteins are not only involved in attenuating responsiveness to abscisic acid (ABA), but also implicated in inhibiting reactive oxygen species (ROS) production and DNA damage induced by genotoxic agent methyl methanesulfonate (MMS). Our results demonstrate that three crwn double mutants, i.e. crwn1 crwn3, crwn2 crwn3, and crwn2 crwn4, show slightly earlier leaf senescence, enhanced leaf cell death, and obvious overaccumulation of ROS under regular growth conditions. When treated with 0.15 μM ABA or 0.01% MMS, two double mutants, crwn1 crwn3 and crwn2 crwn3, exhibit significant decreased germination rates as well as leaf opening and greening rates. Moreover, subsequent investigations indicate that the MMS treatment strongly inhibits the growth of crwn mutant seedlings, while this inhibition is substantially relieved by imidazole (IMZ); by contrast, DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) has no effect on relief of the growth inhibition. Further studies reveal that under 0.01% MMS treatment conditions, crwn mutants, especially the three double mutants, accumulate more ROS compared to Col-0, and their genomic DNA suffers from more severe DNA damage relative to Col-0, which is indicated by significantly higher 8-oxo-7-hydrodeoxyguanosine (8-oxo dG) content as observed in the crwn mutants. Altogether, these data clearly demonstrate that the CRWN-family proteins play important roles in diminishing ROS accumulation and protecting genomic DNA against excessive oxidative damage caused by MMS.

Lamin-like Proteins Negatively Regulate Plant Immunity through NAC WITH TRANSMEMBRANE MOTIF1-LIKE9 and NONEXPRESSOR OF PR GENES1 in Arabidopsis thaliana

Nuclear lamins are involved in multiple biological processes in metazoan cells. The proteins of the CROWDED NUCLEI (CRWN) family are considered lamin-like candidates in Arabidopsis, although the functions of these proteins are largely unknown. In this article we show that crwn1 crwn2 double mutant displays an enhanced resistance against virulent bacterial pathogens, and both virulent bacteria and salicylic acid (SA) induce transcription of CRWN1 gene as well as proteasome-mediated degradation of CRWN1 protein. We also show that CRWN1 interacts with NAC WITH TRANSMEMBRANE MOTIF1-LIKE9 (NTL9), a NAC transcription factor involved in plant immunity. The interaction between CRWN1 and NTL9 enhances the binding of NTL9 to the promoter of the PATHOGENESIS-RELATED1 (PR1) gene, and inhibits PR1 expression. Further genetic experiments indicated that the defense-related phenotypes of crwn1 crwn2 double mutant are dependent on NONEXPRESSOR OF PR GENES1 (NPR1), a transcriptional cofactor of PR1. These findings revealed a regulatory network composed of lamin-like protein CRWN1, NTL9, and NPR1 for the regulation of PR1 expression.

A Dataset and a Technique for Generalized Nuclear Segmentation for Computational Pathology.

Nuclear segmentation in digital microscopic tissue images can enable extraction of high-quality features for nuclear morphometrics and other analysis in computational pathology. Conventional image processing techniques, such as Otsu thresholding and watershed segmentation, do not work effectively on challenging cases, such as chromatin-sparse and crowded nuclei. In contrast, machine learning-based segmentation can generalize across various nuclear appearances. However, training machine learning algorithms requires data sets of images, in which a vast number of nuclei have been annotated. Publicly accessible and annotated data sets, along with widely agreed upon metrics to compare techniques, have catalyzed tremendous innovation and progress on other image classification problems, particularly in object recognition. Inspired by their success, we introduce a large publicly accessible data set of hematoxylin and eosin (H&E)-stained tissue images with more than 21000 painstakingly annotated nuclear boundaries, whose quality was validated by a medical doctor. Because our data set is taken from multiple hospitals and includes a diversity of nuclear appearances from several patients, disease states, and organs, techniques trained on it are likely to generalize well and work right out-of-the-box on other H&E-stained images. We also propose a new metric to evaluate nuclear segmentation results that penalizes object- and pixel-level errors in a unified manner, unlike previous metrics that penalize only one type of error. We also propose a segmentation technique based on deep learning that lays a special emphasis on identifying the nuclear boundaries, including those between the touching or overlapping nuclei, and works well on a diverse set of test images.

Exploring the evolution of the proteins of the plant nuclear envelope

ABSTRACTIn this study, we explore the plasticity during evolution of proteins of the higher plant nuclear envelope (NE) from the most ancestral plant species to advanced angiosperms. The higher plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins. Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesized to be associated with it including Crowded Nuclei (CRWN; also termed LINC and NMCP), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. SUN domain proteins appear throughout with a key role for mid-SUN proteins suggested. Evolution of KASH domain proteins has resulted in increasing complexity, with some appearing in all species considered, while other KASH proteins are progressively gained during evolution. Failure to identify CRWN homologs in unicellular organisms included in the study and their presence in plants leads us to speculate that convergent evolution may have occurred in the formation of the lamina with each kingdom having new proteins such as the Lamin B receptor (LBR) and Lamin-Emerin-Man1 (LEM) domain proteins (animals) or NEAPs and KAKU4 (plants). Our data support a model in which increasing complexity at the nuclear envelope occurred through the plant lineage and suggest a key role for mid-SUN proteins as an early and essential component of the nuclear envelope.

Histochemical alterations in colorectal carcinoma and adenoma in Egyptian patients

Objective: To evaluate the histochemical alterations in DNA and total carbohydrates, in colorectal cancer cells. Methods: This study was carried out on 48 colorectal carcinoma and 10 adenoma specimens. Hematoxylin and Eosin staining was carried out for histopathological examination to confirm the diagnosis and to evaluate the histopathological characteristics of tumor. Histologic grade and pathologic stage was assessed according to TNM staging system. Staging was also assessed according to original Dukes’ staging system. DNA was demonstrated by Feulgen method and carbohydrates were demonstrated by periodic acid Schiff’s reaction. Results: Adenoma cases showed that the cells lining the glands of the polyp have more crowded, irregular and darker nuclei (hyperchromatic), anisonucleosis, abnormal mitotic figures with prominent nucleoli and variability in the size and shape of nuclei. Colorectal carcinoma cases showed a condensation and reduction in the size of a cell nucleus associated with hyperchromatosis, pyknotic nuclei, abnormal mitotic figures, anisonucleosis, irregular nuclear membrane and inequality in the size of the nuclei (Pleomorphosis). There was a statistical significant differences between adenoma and carcinoma regarding number of mitotic cells (P = 0.03) that was in favour of malignant group. Adenoma and colorectal carcinoma cases showed periodic acid Schiff’s reactivity with different degree. Conclusions: These histochemical alterations can be so characteristic of a given tumor type and stage that they are used in cancer diagnosis and might also be related to the altered functional properties of cancer cells. Article history:

The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination.

Plant nuclear shape is independently determined by the SUN-WIP-WIT2-myosin XI-i complex and CRWN1

Nuclei undergo dynamic shape changes during plant development, but the mechanism is unclear. In Arabidopsis, Sad1/UNC-84 (SUN) proteins, WPP domain-interacting proteins (WIPs), WPP domain-interacting tail-anchored proteins (WITs), myosin XI-i, and CROWDED NUCLEI 1 (CRWN1) have been shown to be essential for nuclear elongation in various epidermal cell types. It has been proposed that WITs serve as adaptors linking myosin XI-i to the SUN-WIP complex at the nuclear envelope (NE). Recently, an interaction between Arabidopsis SUN1 and SUN2 proteins and CRWN1, a plant analog of lamins, has been reported. Therefore, the CRWN1-SUN-WIP-WIT-myosin XI-i interaction may form a linker of the nucleoskeleton to the cytoskeleton complex. In this study, we investigate this proposed mechanism in detail for nuclei of Arabidopsis root hairs and trichomes. We show that WIT2, but not WIT1, plays an essential role in nuclear shape determination by recruiting myosin XI-i to the SUN-WIP NE bridges. Compared with SUN2, SUN1 plays a predominant role in nuclear shape. The NE localization of SUN1, SUN2, WIP1, and a truncated WIT2 does not depend on CRWN1. While crwn1 mutant nuclei are smooth, the nuclei of sun or wit mutants are invaginated, similar to the reported myosin XI-i mutant phenotype. Together, this indicates that the roles of the respective WIT and SUN paralogs have diverged in trichomes and root hairs, and that the SUN-WIP-WIT2-myosin XI-i complex and CRWN1 independently determine elongated nuclear shape. This supports a model of nuclei being shaped both by cytoplasmic forces transferred to the NE and by nucleoplasmic filaments formed under the NE.