The Hippo pathway governs cell growth, proliferation, and differentiation and is frequently deregulated in cancer. Yes-associated protein (YAP) is the central transcriptional coactivator of the Hippo pathway and interacts with β-catenin to coordinate YAP-Wnt signaling crosstalk. Both pathways are modulated by diverse upstream signals including mechanical cues, cell density, and cell polarity, yet how such signals are integrated remains poorly understood. Here, we demonstrate that Homer scaffolding proteins coordinate YAP and Wnt signaling downstream of the Crumbs polarity complex. Homers interact directly via their EVH1 domains with the Crumbs component PATJ and the NDR kinase scaffold Furry-like (FRYL). Homers antagonize FRYL to promote YAP activation while cooperating with FRYL to enhance Wnt/β-catenin signaling, revealing pathway-selective regulation. PATJ, in contrast, recruits Homers to the cortex and restrains YAP activity. We further show that Homers form biomolecular condensates in nonpolarized epithelial and colorectal cancer cells, whose assembly and signaling properties are differentially modulated by PATJ and FRYL. Whereas FRYL promotes the formation of cytoplasmic droplets, PATJ drives the assembly of phase-separated compartments at or near the plasma membrane. Collectively, our findings establish Homer-driven phase separation as a tunable signaling mechanism to translate polarity cues into transcriptional output.

During de novo lumenogenesis, epithelial cells establish luminal identity by directing apical cargo to an apical membrane initiation site (AMIS). Although this process has been widely studied, the mechanisms governing AMIS formation and its progression into a luminal precursor remain poorly understood. Here we combine quantitative light and electron microscopy with proximity proteomics to demonstrate that apical cargo is delivered to the AMIS in large apical precursor organelles, termed vacuolar apical compartments (VACs). VACs possess a microvilli-rich cortex and undergo exocytic fusion at the AMIS to generate a nascent lumen. Lumen initiation is tightly coordinated with the assembly and rearrangement of apical cell junctions and requires the Crumbs complex protein PatJ. Together, our results show that PatJ is a key structural determinant of the apical-lateral interface and indicate that VACs act as specialized transport organelles that deliver a preassembled apical surface to the AMIS, enabling rapid and efficient lumen initiation.

Chromatin architecture is critical in determining nuclear mechanics. Most studies focus on the mechanical rigidity conferred by chromatin condensation from densely packed heterochromatin, but less is known on how transient chromatin decompaction impinge on nucleus stiffness. Here, we used an array of vertically aligned nanopillars to study nuclear deformability in situ after chromatin decompaction in cells. The nucleus significantly stiffened within 4 h of chromatin decompaction but softened at longer timescales. This acute stiffening of the nucleus was underpinned predominantly by an increase in nucleus volume and nuclear import, and partially by enhanced lamin protein recruitment to the periphery. The coupling between nucleus stiffening and acute chromatin decompaction was observed in low malignancy cancer cell lines (e.g. MCF7, PEO1, A549) but weakened in highly malignant counterparts (e.g. MDA-MB-231, HEYA8, HT1080) due to the capacity to efficiently compact heterochromatin into foci that sustains nucleus deformability required for confined migration. Our work signals how rapid chromatin remodeling is a physiologically relevant pathway to modulate nucleus mechanics and cell migration behavior.

Tight junction protein-1 and -2 (Tjp1/ZO-1 and Tjp2/ZO-2) function as scaffold proteins within the tight junction complexes of the blood-retinal barrier (BRB). Although the breakdown of the BRB is implicated in retinopathies, the contribution of ZO-1/2 in the pathogenesis of retinopathies is unknown. To understand their role, we generated RPE-specific conditional ZO-1/2 single KOs (T1KO/T2KO) and ZO-1/2 double knockout (DKO) mice. While T1KO and T2KO did not exhibit overt retinal phenotypes, DKO demonstrated a strong retinal phenotype from 1-month post-KO induction. This includes the loss of RPE integrity and retinal thinning. Furthermore, RPE in DKO re-entered the cell cycle with upregulated YAP. At 12 months, we observed severe structural and functional retinal deterioration. In response to laser-induced damage, RPE displayed persistent hyper-proliferation, delayed wound repair, and the up-regulation of YAP. These studies confirmed the critical role of ZO-1/2 in maintaining an intact BRB and revealed a role of ZO-1/2 in wound healing.

Telomeres, the ends of linear chromosomes, are composed of repetitive DNA sequences, histones and a protein complex called shelterin. How DNA is packaged at telomeres is an outstanding question in the field with significant implications for human health and disease. Here, we studied the architecture of telomeres and their spatial association with other chromatin domains in different cell types using correlative light and electron microscopy. To this end, the shelterin protein TRF1 or TRF2 was fused in tandem to eGFP and the peroxidase APEX2, which provided a selective and electron-dense label to interrogate telomere organization by transmission electron microscopy, electron tomography and scanning electron microscopy. Together, our work reveals, for the first time, ultrastructural insight into telomere architecture. We show that telomeres are composed of a dense and highly compacted mesh of chromatin fibres. In addition, we identify marked differences in telomere size, shape and chromatin compaction between cancer and non-cancer cells and show that telomeres are in direct contact with other heterochromatin regions. Our work resolves the internal architecture of telomeres with unprecedented resolution and advances our understanding of how telomeres are organized in situ.

This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less-compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co-aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments.

Recent methodological advancements in microscopy and DNA sequencing-based methods provide unprecedented new insights into the spatio-temporal relationships between chromatin and nuclear machineries. We discuss a model of the underlying functional nuclear organization derived mostly from electron and super-resolved fluorescence microscopy studies. It is based on two spatially co-aligned, active and inactive nuclear compartments (ANC and INC). The INC comprises the compact, transcriptionally inactive core of chromatin domain clusters (CDCs). The ANC is formed by the transcriptionally active periphery of CDCs, called the perichromatin region (PR), and the interchromatin compartment (IC). The IC is connected to nuclear pores and serves nuclear import and export functions. The ANC is the major site of RNA synthesis. It is highly enriched in epigenetic marks for transcriptionally competent chromatin and RNA Polymerase II. Marks for silent chromatin are enriched in the INC. Multi-scale cross-correlation spectroscopy suggests that nuclear architecture resembles a random obstacle network for diffusing proteins. An increased dwell time of proteins and protein complexes within the ANC may help to limit genome scanning by factors or factor complexes to DNA exposed within the ANC.

BACKGROUND:Previous studies of higher order chromatin organization in nuclei of mammalian species revealed both structural consistency and species-specific differences between cell lines and during early embryonic development. Here, we extended our studies to nuclear landscapes in the human myelopoietic lineage representing a somatic cell differentiation system. Our longterm goal is a search for structural features of nuclei, which are restricted to certain cell types/species, as compared to features, which are evolutionary highly conserved, arguing for their basic functional roles in nuclear organization.RESULTS:Common human hematopoietic progenitors, myeloid precursor cells, differentiated monocytes and granulocytes analyzed by super-resolution fluorescence microscopy and electron microscopy revealed profound differences with respect to global chromatin arrangements, the nuclear space occupied by the interchromatin compartment and the distribution of nuclear pores. In contrast, we noted a consistent organization in all cell types with regard to two co-aligned networks, an active (ANC) and an inactive (INC) nuclear compartment delineated by functionally relevant hallmarks. The ANC is enriched in active RNA polymerase II, splicing speckles and histone signatures for transcriptionally competent chromatin (H3K4me3), whereas the INC carries marks for repressed chromatin (H3K9me3).CONCLUSIONS:Our findings substantiate the conservation of the recently published ANC-INC network model of mammalian nuclear organization during human myelopoiesis irrespective of profound changes of the global nuclear architecture observed during this differentiation process. According to this model, two spatially co-aligned and functionally interacting active and inactive nuclear compartments (ANC and INC) pervade the nuclear space.

Arthropodenfauna im Trichtermaterial humussammelnder Pflanzen in einem Tieflandregenwald in Costa Rica. Nahrstoffmangel in Boden tropischer Walder stellt einen wichtigen limitierenden Faktor fur das Pflanzenwachstum dar. Als alternative Ernahrungsstrategie entwickelten einige Pflanzen das so genannte Humussammeln bei dem herabfallendes, organisches Material wie Aste, Blatter, Fruchte usw. in der trichterformigen Krone gesammelt wird. Durch die rasche Zersetzung werden Nahrstoffe freigesetzt, welche von der Pflanze durch oberirdische Organe wie Adventivwurzeln oder Blattbasen aufgenommen werden konnen. Das angesammelte Material wird von einer Reihe wirbelloser Tiere bewohnt und teilweise sogar von Eidechsen und Vogeln zur Eiablage genutzt. Zwischen Juli und Oktober 2008 wurde an den zwei Standorten Hugelrucken und steiler Schluchtwald das Material zweier humussammelnder Pflanzen sowie Laubstreuproben des anliegenden Bodens gesammelt. Die Extrahierung der insgesamt 28.000 Arthropoden aus 25 Ordnungen erfolgte durch Berlese-Tullgren-Apparaturen und zeigte dass Acari, Collembola und Hymenoptera die mit Abstand am haufigsten vertretenen Tiergruppen sind. Letztere sind meist Formicidae, welche Ansammlungen von bis zu 300 Individuen pro Spezies bilden und oftmals mit anderen Vertretern der Familie im Material eines Trichters zusammenleben. Im Allgemeinen favorisieren Arthropoden das Trichtermaterial humussammelnder Pflanzen gegenuber der Laubstreu des anliegenden Bodens wobei das Material in Pflanzen entlang des Schluchtwaldes klar bevorzugt wird. Die Vielzahl an Nischen und Zersetzungsstadien machen dieses Habitat offenbar zum Attraktivsten und Vorteilhaftesten der untersuchten Bereiche.

The term correlative microscopy denotes the sequential visualization of one and the same cell using various microscopic techniques. Correlative microscopy provides a unique platform to combine the particular strength of each microscopic approach and compensate for its specific limitations. As an example, we report results of a correlative microscopic study exploring features of the nuclear landscape in HeLa cells. We present a detailed protocol to first investigate distinct structural features of a living cell in space and time (4D) using spinning disk laser scanning microscopy (SDLSM). Then, after fixation and staining of selected structures (e.g., by means of immunodetection), details of these structures are explored at increasingly higher resolution using three-dimensional (3D) confocal laser scanning microscopy (CLSM); super-resolution fluorescence microscopy, such as three-dimensional structured illumination microscopy (3D-SIM); and transmission electron microscopy (TEM). We discuss problems involved in the comparison of images of a given cell nucleus recorded with different microscopic approaches, which requires not only a compensation for different resolutions but also for various distortions.

Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a “shattered” chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.

Two chitinases (EC 3.2.1.14) and two beta-1,3-glucanases (EC 3.2.1.39) were purified from the culture medium of spruce (Picea abies [L.] Karst.) cells to study their role in modifying elicitors, cell walls, growth, and hyphal morphology of ectomycorrhizal fungi. The 36-kD class I chitinase (isoelectric point [pI] 8.0) and the 28-kD chitinase (pi 8.7) decreased the activity of elicitor preparations from Hebeloma crustuliniforme (Bull. ex Fries.) Quel., Amanita muscaria (L.) Pers., and Suillus variegatus (Sw.: Fr.) O.K., as demonstrated by using the elicitor-induced extracellular alkalinization in spruce cells as a test system. In addition, chitinases released monomeric products from the walls of these ectomycorrhizal fungi. The beta-1,3-glucanases (35 kD, pi 3.7 and 3.9), in contrast, had little influence on the activity of the fungal elicitors and released only from walls of A. muscaria some polymeric products. Furthermore, chitinases alone and in combination with beta-1,3-glucanases had no effect on the growth and morphology of the hyphae. Thus, it is suggested that apoplastic chitinases in the root cortex destroy elicitors from the ectomycorrhizal fungi without damaging the fungus. By this mechanism the host plant could attenuate the elicitor signal and adjust its own defense reactions to a level allowing symbiotic interaction.