Medicago LINC complexes function in nuclear morphology, nuclear movement, and root nodule symbiosis 1[OPEN]

Nuclear movement is involved in cellular and developmental processes across eukaryotic life, often driven by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, which bridge the nuclear envelope (NE) via the interaction of Klarsicht/ ANC-1/Syne-1 Homology (KASH) and Sad1/UNC-84 (SUN) proteins. Arabidopsis (Arabidopsis thaliana) LINC complexes are involved in nuclear movement and positioning in several cell types. Observations since the 1950s have described targeted nuclear movement and positioning during symbiosis initiation between legumes and rhizobia, but it has not been established whether these movements are functional or incidental. Here, we identify and characterize LINC complexes in the model legume Medicago truncatula. We show that LINC complex characteristics such as NE localization, dependence of KASH proteins on SUN protein binding for NE enrichment, and direct SUN-KASH binding are conserved between plant species. Using a SUN dominant-negative strategy, we demonstrate that LINC complexes are necessary for proper nuclear shaping and movement in Medicago root hairs, and are important for infection thread initiation and nodulation.National Science Foundation NSF-1440019, NSF-1613501Biotechnology and Biological Sciences Research Council BB/P007112/1European Molecular Biology Organization 699

Adenokarzinome mit Lokalisation am gastroösophagealen Übergang: Lymphatisches Metastasierungsmuster als Grundlage für die Definition des Planungszielvolumens bei neoadjuvanter Radiochemotherapie

Aim of the study Regional nodal metastasis after neoadjuvant chemoradiation (ypN) of adenocarcinoma of the esophagogastric junction (AEG) has a high impact on the odds of the survival of patients. It is the aim of this study to clarify the lymph node distribution of AEG according to location of the tumor mass and invasion of neighboring areas for the selection of radiotherapy planning target volume (PTV) margins. Methods Patterns of regional spread were analyzed in pathologic reports of 326 primary resected patients with AEG with ≥ 15 lymph nodes examined. Tumors were classified into three AEG types (I, II and III) based on endoscopic and pathologic reports. Fisher’s exact test was used to compare nodal disease and tumor characteristics. Results Nodes were positive in 81% of T2-4 tumors. AEG type, tumor size, lymphovascular invasion and grading significantly influenced nodal distribution. We assessed the following significant correlations: Marked esophageal invasion of AEG II/III significantly correlates with paraesophageal nodal disease and T3-4 AEG II/III has a significant rate of splenic hilum/artery nodes. For radiotherapy planning the following nodes should be included into the planning target volume (PTV): Middle and lower paraesophageal nodes should be treated in T2-4 AEG I and AEG II with ≥ 15 mm involvement above the Z-line, and T3-4 AEG II. The splenic hilum and artery nodes on the other hand can be spared in T2 AEG tumors, especially type I tumors. The influence of paraesophageal nodal treatment on the risk of postoperative pulmonary complications can be estimated from dose-volume-histograms. Conclusions Accurate pretherapeutic staging predicts the risk of subclinical nodal disease and should be used to select the appropriate radiotherapeutic planning target volume (PTV). Careful selection of the PTV can be used to maximize the therapeutic window in multimodal therapy for AEG.Ziel der Studie Regionale Lymphknotenmetastasen bei Adenokarzinomen am ösophagogastrealen Übergang (AEG) sind ein prognostischer Faktor nach einer neoadjuvanten Radiochemotherapie. Ziel dieser Arbeit ist es, die Verteilung von Lymphknotenmetastasen bei AEG-Tumoren unter Berücksichtigung der Tumorlokalisation und des Tumorwachstums in Nachbarregionen zu identifizieren, um adäquate Grenzen für das Bestrahlungs-Planungszielvolumen erstellen zu können. Methodik Bei 326 Patienten mit AEG Tumoren wurde das Metastasierungsmuster von regionalen Lymphknoten anhand von Pathologiebefunden analysiert. Bei den Betroffenen wurde eine radikale Tumorresektion ohne vorausgegangene Therapie durchgeführt. Hierbei wurden mindestens 15 Lymphknoten untersucht. Basierend auf Endoskopie- und Pathologiebefunden wurden die Tumoren in drei AEG-Typen (I-III) klassifiziert. Mit Hilfe des Fishers Exact Tests wurden die Lymphknotenmetastasen und die Tumorcharakteristika verglichen. Ergebnisse Bei T2- bis T4-Tumoren waren 81 % der Lymphknoten positiv. AEG-Typ, Tumorgröße, Lymphgefäßeinbrüche und Grading haben einen deutlichen Einfluss auf die Verteilung von Lymphknotenmetastasen. Wir stellten folgende signifikante Korrelationen fest: Eine Tumorinvasion proximal der Z-Linie in den distalen Ösophagus korrelierte bei AEG II/III-Tumoren mit paraösophagealen Lymphknotenmetastasen und das Vorliegen eines Tumorstadiums T3- und T4-Tumoren der AEG-Typen II/III korrelierte mit Lymphknotenmetastasen im Bereich der A. splenica und des Milzhilus. Für die Bestrahlungsplanung sollten bei allen AEG I-Tumoren, bei AEG II-Tumoren mit ≥ 15 mm Beteiligung oberhalb der Z-Linie im T2- bis T4-Stadium und bei AEG II-Tumoren im T3/T4-Stadium die mittleren und unteren paraösophagealen Lymphknoten mit in das PTV eingeschlossen werden. Andererseits können Lymphknoten im Bereich der A. splenica und des Milzhilus bei T2 AEG-Tumoren, insbesondere bei AEG-Typ I-Tumoren, bei der Therapie ausgespart werden. Das Risiko von postoperativen pulmonalen Komplikationen durch die Behandlung von paraösophagealen Lymphknoten kann anhand eines Dosis-Volumen-Histogramms abgeschätzt werden. Schlussfolgerung Durch ein sorgfältiges prätherapeutisches Staging des Primärtumors kann das Risiko für subklinische Lymphknotenmetastasen prognostiziert werden. Es sollte für die Wahl eines adäquaten radiotherapeutischen Planungszielvolumens herangezogen werden. Die sorgfältig bedachte Wahl des Planungszielvolumens trägt dazu bei, das therapeutische Fenster bei der multimodalen Therapie bei AEG-Tumoren zu maximieren

Anchorage of Plant RanGAP to the Nuclear Envelope Involves Novel Nuclear-Pore-Associated Proteins

SummaryThe Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, and nuclear envelope (NE) formation [1–4]. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1 [5–8]. Mammalian RanGAP1 targeting to the NE and kinetochores requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2 [9–14]. In contrast, Arabidopsis RanGAP1 is associated with the NE and cell plate, mediated by an N-terminal, plant-specific WPP domain [15–18]. In the absence of RanBP2 in plants, the mechanism for spatially sequestering plant RanGAP is unknown. Here, Arabidopsis WPP-domain interacting proteins (WIPs) that interact with RanGAP1 in vivo and colocalize with RanGAP1 at the NE and cell plate were identified. Immunogold labeling indicates that WIP1 is associated with the outer NE. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root-tip cells, whereas NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIPs are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms

GAP activity, but not subcellular targeting, is required for Arabidopsis RanGAP cellular and developmental functions

The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope

Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control

BACKGROUND: Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database. RESULTS: Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms. CONCLUSION: MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell

Nuclei in motion: movement and positioning of plant nuclei in development, signaling, symbiosis, and disease

While textbook figures imply nuclei as resting spheres at the center of idealized cells, this picture fits few real situations. Plant nuclei come in many shapes and sizes, and can be actively transported within the cell. In several contexts, this nuclear movement is tightly coupled to a developmental program, the response to an abiotic signal, or a cellular reprogramming during either mutualistic or parasitic plant-microbe interactions. While many such phenomena have been observed and carefully described, the underlying molecular mechanism and the functional significance of the nuclear movement are typically unknown. Here, we survey recent as well as older literature to provide a concise starting point for applying contemporary molecular, genetic and biochemical approaches to this fascinating, yet poorly understood phenomenon