Competition between myosin II and ßH-spectrin regulates cytoskeletal tension.

Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two α-spectrins and two ß-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. We have investigated the role and regulation of the Drosophila ß-heavy spectrin (ßH-spectrin, encoded by the karst gene) in wing imaginal discs. Our results establish that ßH-spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While we find that α-spectrin also regulates Hippo signaling through Jub, unexpectedly, we find that ßH-spectrin localizes and functions independently of α-spectrin. Instead, ßH-spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which ßH-spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of ßH-spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how ßH-spectrin participates in ratcheting mechanisms associated with cell shape change.

Analysis of the Drosophila Ajuba LIM protein defines functions for distinct LIM domains.

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.

TRIP6 is required for tension at adherens junctions.

Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.

E2 and Gamma distributions in polygonal networks.

From solar supergranulation to salt flat in Bolivia, from veins on leaves to cells on Drosophila wing discs, polygon-based networks exhibit great complexities, yet similarities and consistent patterns emerge. Based on analysis of 99 polygonal tessellations of a wide variety of physical origins, this work demonstrates the ubiquity of an exponential distribution in the squared norm of the deformation tensor, E2, which directly leads to the ubiquitous presence of Gamma distributions in polygon aspect ratio as recently demonstrated by Atia et al. [Nat. Phys. 14, 613 (2018)]. In turn an analytical approach is developed to illustrate its origin. E2 relates to most energy forms, and its Boltzmann-like feature allows the definition of a pseudo-temperature that promises utility in a thermodynamic ensemble framework.

Integration of Hippo-YAP Signaling with Metabolism.

The Hippo-Yes-associated protein (YAP) signaling network plays a central role as an integrator of signals that control cellular proliferation and differentiation. The past several years have provided an increasing appreciation and understanding of the diverse mechanisms through which metabolites and metabolic signals influence Hippo-YAP signaling, and how Hippo-YAP signaling, in turn, controls genes that direct cellular and organismal metabolism. These connections enable Hippo-YAP signaling to coordinate organ growth and homeostasis with nutrition and metabolism. In this review, we discuss the current understanding of some of the many interconnections between Hippo-YAP signaling and metabolism and how they are affected in disease conditions.

Rapping about Mechanotransduction.

Mechanical cues can regulate cell proliferation and differentiation through the Hippo-YAP signaling network. Reporting in Nature, Meng et al. (2018) show that the Ras-related GTPase RAP2 connects extracellular matrix stiffness to Hippo pathway regulation, adding to our understanding of how mechanical cues are converted into changes in YAP activity.

Tension-dependent regulation of mammalian Hippo signaling through LIMD1.

Hippo signaling is regulated by biochemical and biomechanical cues that influence the cytoskeleton, but the mechanisms that mediate this have remained unclear. We show that all three mammalian Ajuba family proteins - AJUBA, LIMD1 and WTIP - exhibit tension-dependent localization to adherens junctions, and that both LATS family proteins, LATS1 and LATS2, exhibit an overlapping tension-dependent junctional localization. This localization of Ajuba and LATS family proteins is also influenced by cell density, and by Rho activation. We establish that junctional localization of LATS kinases requires LIMD1, and that LIMD1 is also specifically required for the regulation of LATS kinases and YAP1 by Rho. Our results identify a biomechanical pathway that contributes to regulation of mammalian Hippo signaling, establish that this occurs through tension-dependent LIMD1-mediated recruitment and inhibition of LATS kinases in junctional complexes, and identify roles for this pathway in both Rho-mediated and density-dependent regulation of Hippo signaling.

Drosophila p115 is required for Cdk1 activation and G2/M cell cycle transition.

Golgi complex inheritance and its relationship with the cell cycle are central in cell biology. Golgi matrix proteins, known as golgins, are one of the components that underlie the shape and functionality of this organelle. In mammalian cells, golgins are phosphorylated during mitosis to allow fragmentation of the Golgi ribbon and they also participate in spindle dynamics; both processes are required for cell cycle progression. Little is known about the function of golgins during mitosis in metazoans in vivo. This is particularly significant in Drosophila, in which the Golgi architecture is distributed in numerous units scattered throughout the cytoplasm, in contrast with mammalian cells. We examined the function of the ER/cis-Golgi golgin p115 during the proliferative phase of the Drosophila wing imaginal disc. Knockdown of p115 decreased tissue size. This phenotype was not caused by programmed cell death or cell size reductions, but by a reduction in the final cell number due to an accumulation of cells at the G2/M transition. This phenomenon frequently allows mitotic bypass and re-replication of DNA. These outcomes are similar to those observed following the partial loss of function of positive regulators of Cdk1 in Drosophila. In agreement with this, Cdk1 activation was reduced upon p115 knockdown. Interestingly, these phenotypes were fully rescued by Cdk1 overexpression and partially rescued by Myt1 depletion, but not by String (also known as Cdc25) overexpression. Additionally, we confirmed the physical interaction between p115 and Cdk1, suggesting that the formation of a complex where both proteins are present is essential for the full activation of Cdk1 and thus the correct progression of mitosis in proliferating tissues.

Differential growth triggers mechanical feedback that elevates Hippo signaling.

Mechanical stress can influence cell proliferation in vitro, but whether it makes a significant contribution to growth control in vivo, and how it is modulated and experienced by cells within developing tissues, has remained unclear. Here we report that differential growth reduces cytoskeletal tension along cell junctions within faster-growing cells. We propose a theoretical model to explain the observed reduction of tension within faster-growing clones, supporting it by computer simulations based on a generalized vertex model. This reduced tension modulates a biomechanical Hippo pathway, decreasing recruitment of Ajuba LIM protein and the Hippo pathway kinase Warts, and decreasing the activity of the growth-promoting transcription factor Yorkie. These observations provide a specific mechanism for a mechanical feedback that contributes to evenly distributed growth, and we show that genetically suppressing mechanical feedback alters patterns of cell proliferation in the developing Drosophila wing. By providing experimental support for the induction of mechanical stress by differential growth, and a molecular mechanism linking this stress to the regulation of growth in developing organs, our results confirm and extend the mechanical feedback hypothesis.

The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth.

The TOR signaling pathway is crucial in the translation of nutritional inputs into the protein synthesis machinery regulation, allowing animal growth. We recently identified the Bud32 (yeast)/PRPK (human) ortholog in Drosophila, Prpk (p53-related protein kinase), and found that it is required for TOR kinase activity. Bud32/PRPK is an ancient and atypical kinase conserved in evolution from Archeae to humans, being essential for Archeae. It has been linked with p53 stabilization in human cell culture and its absence in yeast causes a slow-growth phenotype. This protein has been associated to KEOPS (kinase, putative endopeptidase and other proteins of small size) complex together with Kae1p (ATPase), Cgi-121 and Pcc1p. This complex has been implicated in telomere maintenance, transcriptional regulation, bud site selection and chemical modification of tRNAs (tRNAs). Bud32p and Kae1p have been related with N6-threonylcarbamoyladenosine (t (6)A) synthesis, a particular chemical modification that occurs at position 37 of tRNAs that pair A-starting codons, required for proper translation in most species. Lack of this modification causes mistranslations and open reading frame shifts in yeast. The core constituents of the KEOPS complex are present in Drosophila, but their physical interaction has not been reported yet. Here, we present a review of the findings regarding the function of this complex in different organisms and new evidence that extends our recent observations of Prpk function in animal growth showing that depletion of Kae1 or Prpk, in accordance with their role in translation in yeast, is able to induce the unfolded protein response (UPR) in Drosophila. We suggest that EKC/KEOPS complex could be integrating t (6)A-modified tRNA availability with translational rates, which are ultimately reflected in animal growth.

Drosophila p53-related protein kinase is required for PI3K/TOR pathway-dependent growth.

Cell growth and proliferation are pivotal for final organ and body size definition. p53-related protein kinase (Bud32/PRPK) has been identified as a protein involved in proliferation through its effects on transcription in yeast and p53 stabilization in human cell culture. However, the physiological function of Bud32/PRPK in metazoans is not well understood. In this work, we have analyzed the role of PRPK in Drosophila development. Drosophila PRPK is expressed in every tissue analyzed and is required to support proliferation and cell growth. The Prpk knockdown animals show phenotypes similar to those found in mutants for positive regulators of the PI3K/TOR pathway. This pathway has been shown to be fundamental for animal growth, transducing the hormonal and nutritional status into the protein translation machinery. Functional interactions have established that Prpk operates as a transducer of the PI3K/TOR pathway, being essential for TOR kinase activation and for the regulation of its targets (S6K and 4E-BP, autophagy and bulk endocytosis). This suggests that Prpk is crucial for stimulating the basal protein biosynthetic machinery in response to insulin signaling and to changes in nutrient availability.

BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response.

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.

The import of S-adenosylmethionine into the Golgi apparatus is required for the methylation of homogalacturonan.

S-adenosylmethionine (SAM) is the substrate used in the methylation of homogalacturonan (HGA) in the Golgi apparatus. SAM is synthesized in the cytosol, but it is not currently known how it is then transported into the Golgi. In this study, we find that HGA methyltransferase is present in Golgi-enriched fractions and that its catalytic domain faces the lumen of this organelle. This suggests that SAM must be imported into the Golgi. We performed uptake experiments using [methyl-(14)C]SAM and found that SAM is incorporated into the Golgi vesicles, resulting in the methylation of polymers that are sensitive to pectinase and pectin methylesterase but not to proteases. To avoid detecting the transfer reaction, we also used [carboxyl-(14)C]SAM, the uptake of which into Golgi vesicles was found to be sensitive to temperature, detergents, and osmotic changes, and to be saturable with a K(m) of 33 microm. Double-label uptake experiments using [methyl-(3)H]SAM and [carboxyl-(14)C]SAM also revealed a time-dependent increase in the (3)H to (14)C ratio, suggesting that upon transfer of the methyl group, the resulting S-adenosylhomocysteine is not accumulated in the Golgi. SAM incorporation was also found to be inhibited by S-adenosylhomocysteine, whereas UDP-GalA, UDP-GlcA, and acetyl-CoA had no effect. DIDS, a compound that inhibits nucleotide sugar transporters, also had little effect upon SAM incorporation. Interestingly, the combination of UDP-GalA + acetyl-CoA or UDP-GlcA + acetyl-CoA produced a slight increase in the uptake of SAM. These results support the idea that a SAM transporter is required for HGA biosynthesis.