NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Genome-wide screening in human kidney organoids identifies developmental and disease-related aspects of nephrogenesis.

Human organoids allow the study of proliferation, lineage specification, and 3D tissue development. Here we present a genome-wide CRISPR screen in induced pluripotent stem cell (iPSC)-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling, and endpoint sorting of tubular and stromal cells generated a complex, high-quality dataset uncovering a broad spectrum of insightful biology from early development to "adult" epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two additional congenital anomalies of the kidney and urinary tract (CAKUT) genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can reveal ways of communication between heterogeneous cell populations in complex tissues. These data serve as a rich resource for the kidney research community and as a benchmark for future iPSC-derived organoid CRISPR screens.

The tumor suppressor WT1 drives progenitor cell progression and epithelialization to prevent Wilms tumorigenesis in human kidney organoids.

Wilms tumor is the most widespread kidney cancer in children and frequently associated with homozygous loss of the tumor suppressor WT1. Pediatric tumorigenesis is largely inaccessible in humans. Here, we develop a human kidney organoid model for Wilms tumor formation and show that deletion of WT1 during organoid development induces overgrowth of kidney progenitor cells at the expense of differentiating glomeruli and tubules. Functional and gene expression analyses demonstrate that absence of WT1 halts progenitor cell progression at a pre-epithelialized cell state and recapitulates the transcriptional changes detected in a subgroup of Wilms tumor patients with ectopic myogenesis. By "transplanting" WT1 mutant cells into wild-type kidney organoids, we find that their propagation requires an untransformed microenvironment. This work defines the role of WT1 in kidney progenitor cell progression and tumor suppression, and establishes human kidney organoids as a phenotypic model for pediatric tumorigenesis.

Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1.

The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.

Dissociation of membrane-chromatin contacts is required for proper chromosome segregation in mitosis.

The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells. Using a membrane-chromatin tether that cannot be dissociated when cells enter mitosis, we show that a failure in breaking membrane-chromatin interactions impairs mitotic chromatin organization, chromosome segregation and cytokinesis, and induces an aberrant NE morphology in postmitotic cells. In contrast, chromosome segregation and cell division proceed successfully when membrane attachment to chromatin is induced during metaphase, after chromosomes have been singularized and aligned at the metaphase plate. These results indicate that the separation of membranes and chromatin is critical during prometaphase to allow for proper chromosome compaction and segregation. We propose that one cause of these defects is the multivalency of membrane-chromatin interactions.

Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology.

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER.

Mechanisms and functions of nuclear envelope remodelling.

As a compartment border, the nuclear envelope (NE) needs to serve as both a protective membrane shell for the genome and a versatile communication interface between the nucleus and the cytoplasm. Despite its important structural role in sheltering the genome, the NE is a dynamic and highly adaptable boundary that changes composition during differentiation, deforms in response to mechanical challenges, can be repaired upon rupture and even rapidly disassembles and reforms during open mitosis. NE remodelling is fundamentally involved in cell growth, division and differentiation, and if perturbed can lead to devastating diseases such as muscular dystrophies or premature ageing.

Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis.

Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5'ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth.

A Wounded Nucleus Needs ESCRT Service.

Two recent publications in Science report on frequent rupturing events of the nuclear envelope induced by migration of cells through narrow openings. Nuclear envelope "wounds" are repaired by the ESCRT-III machinery to limit DNA damage, nuclear fragmentation, and cell death.

An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane.

Newly synthesized membrane proteins are inserted into the endoplasmic reticulum (ER) from where they are constantly sorted to various cellular compartments. To analyze and visualize sorting of membrane proteins to the inner nuclear membrane (INM), we developed a trap-release system that uncouples membrane integration into the ER from transport. This assay allows the simultaneous release of a large pool of an INM-destined membrane protein from the ER and microscopy-based monitoring of targeting to the INM. The use of semi-permeabilized HeLa cells further enables the identification and characterization of essential requirements of the targeting process. This protocol provides a detailed description of reporter construction, in vitro reconstitution, and visualization of trafficking.

Establishment of NE asymmetry­targeting of membrane proteins to the inner nuclear membrane.

The inner nuclear membrane (INM) represents a specialized subdomain of the endoplasmic reticulum (ER). The INM houses a unique set of integral membrane proteins that perform key functions in the organization of intranuclear architecture, control of gene expression and coupling of the nucleus to the cytoskeleton. However, the molecular mechanism of membrane protein sorting from the ER to the INM has remained enigmatic. Recently, novel approaches combining visual kinetic assays and computational modeling were used to define the requirements of trafficking to the INM in human cells. These studies reveal that nuclear retention, diffusional mobility in the ER as well as the number and architecture of NPCs are major determinants of INM targeting, collectively lending support to a diffusion-retention-based mechanism.

Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane.

Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2ß as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention-based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo.

Traversing the NPC along the pore membrane: targeting of membrane proteins to the INM.

The inner nuclear membrane (INM) accommodates a specific set of integral membrane proteins many of which interact with chromatin and/or in metazoan cells with the lamina network. The localization of these proteins characterizes this membrane area of the nuclear envelope (NE) despite the fact that the INM forms a membrane continuum with the outer nuclear membrane (ONM) and the remaining endoplasmic reticulum (ER). In fact, the INM can be regarded as a highly specialized membrane subdomain of the ER. How the specific protein composition of the INM is established and maintained and whether this is achieved via a single unifying mechanism is by and large unclear. Recent experiments shed light on some aspects of the process.

A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane.

Integral membrane proteins of the inner nuclear membrane (INM) are inserted into the endoplasmic reticulum membrane during their biogenesis and are then targeted to their final destination. We have used human SUN2 to delineate features that are required for INM targeting and have identified multiple elements that collectively contribute to the efficient localization of SUN2 to the nuclear envelope (NE). One such targeting element is a classical nuclear localization signal (cNLS) present in the N-terminal, nucleoplasmic domain of SUN2. A second motif proximal to the cNLS is a cluster of arginines that serves coatomer-mediated retrieval of SUN2 from the Golgi. Unexpectedly, also the C-terminal, lumenal SUN domain of SUN2 supports NE localization, showing that targeting elements are not limited to cytoplasmic or transmembrane domains of INM proteins. Together, SUN2 represents the first mammalian INM protein relying on a functional cNLS, a Golgi retrieval signal and a perinuclear domain to mediate targeting to the INM.

Control and signal processing by transcriptional interference.

A transcriptional activator can suppress gene expression by interfering with transcription initiated by another activator. Transcriptional interference has been increasingly recognized as a regulatory mechanism of gene expression. The signals received by the two antagonistically acting activators are combined by the polymerase trafficking along the DNA. We have designed a dual-control genetic system in yeast to explore this antagonism systematically. Antagonism by an upstream activator bears the hallmarks of competitive inhibition, whereas a downstream activator inhibits gene expression non-competitively. When gene expression is induced weakly, the antagonistic activator can have a positive effect and can even trigger paradoxical activation. Equilibrium and non-equilibrium models of transcription shed light on the mechanism by which interference converts signals, and reveals that self-antagonism of activators imitates the behavior of feed-forward loops. Indeed, a synthetic circuit generates a bell-shaped response, so that the induction of expression is limited to a narrow range of the input signal. The identification of conserved regulatory principles of interference will help to predict the transcriptional response of genes in their genomic context.