Exploring the developmental functions of fat cadherins in Drosophila and mammals

The large cadherin Fat has important functions in morphogenetic processes during development of Drosophila and mammals. Yet, its molecular partners and signaling pathways are poorly understood. Here I studied a physical link between Drosophila Fat and the Hippo pathway regulator Expanded. I found that Expanded interacts with distinct domains of Fat through its FERM domain. This finding offers a possible explanation of how Fat regulates the apical localization of Expanded and its activity in the Hippo pathway. In order to better understand Expanded signaling, I further studied its phosphorylation and determined novel Expanded interactors, including the Yorkie co-factor Mask. The size and transmembrane nature of the mammalian Fat cadherin Fat4 had previously hindered the use of biochemical approaches to gain insight into its molecular functions. Here I developed several cell culture tools that allowed me to overcome some of these limitations and to study Fat4 localization and function. I performed proximity-dependent biotin identification (BioID) and identified an array of potential novel Fat4 interactors that will serve as a useful resource for future studies. Beside a variety of developmental defects, Fat4 mutant mice exhibit prenatal renal cysts with regions of abnormal primary cilia. Therefore, to understand if Fat4 regulates primary cilia, as has been suggested for several PCP proteins, I tested the effect of Fat4 depletion on cilia formation and maintenance in cell culture. Dramatic effects on cilia maintenance and centrosome positioning and coherence were found by knockdown with several independent siRNAs. However, CRISPR/Cas9-mediated Fat4 knockout did not confirm a requirement of Fat4 in these processes and indicated that cilia and centrosome defects were likely RNAi off-target effects. This highlights the potential pitfalls of RNAi and should be regarded as a cautionary tale

The DNA-dependent protease AtWSS1A suppresses persistent double strand break formation during replication

The protease WSS1A is an important factor in the repair of DNA-protein crosslinks in plants. Here we show that the loss of WSS1A leads to a reduction of 45S rDNA repeats and chromosomal fragmentation in Arabidopsis. Moreover, in the absence of any factor of the RTR (RECQ4A/TOP3α/RMI1/2) complex, which is involved in the dissolution of DNA replication intermediates, WSS1A becomes essential for viability. If WSS1A loss is combined with loss of the classical (c) or alternative (a) nonhomologous end joining (NHEJ) pathways of double-strand break (DSB) repair, the resulting mutants show proliferation defects and enhanced chromosome fragmentation, which is especially aggravated in the absence of aNHEJ. This indicates that WSS1A is involved either in the suppression of DSB formation or in DSB repair itself. To test the latter we induced DSB by CRISPR/Cas9 at different loci in wild-type and mutant cells and analyzed their repair by deep sequencing. However, no change in the quality of the repair events and only a slight increase in their quantity was found. Thus, by removing complex DNA-protein structures, WSS1A seems to be required for the repair of replication intermediates which would otherwise be resolved into persistent DSB leading to genome instability

Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway

The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth

Understanding consumers’ perception of the end-of-life of a garment : Applying the Theory of Planned Behavior to consumers’ disposal intention

Purpose The purpose of this master’s thesis is to investigate consumers’ perceptions of the end-of-life stage of a garment. Overall, the objective is to determine influencing factors that impact consumers’ disposal decisions to conceive a better understanding of post-purchase consumer behavior and formulate implications for both business and society. Design/Methodology/Approach The research of this study follows a deductive approach, whereby hypotheses are derived from existing literature and the Theory of Planned Behavior. A single quantitative data collection method is applied to collect primary data, namely, a cross-sectional self-administered online questionnaire. An effective sample size of 398 respondents is statistically analyzed using Structural Equation Modeling. The conducted descriptive research design investigates the causal relationships between the latent variables and the Behavioral Intention. Findings The empirical findings reveal that consumers’ Attitude positively and individuals’ Subjective Norm negatively influences the intention to dispose of garments. The Personal Value of consumers highly negatively impacts the Attitude and therefore indicates an indirect relationship to an individual’s behavioral intention. There is no significant correlation between consumers’ Endeavor to Change to their Attitude towards garment disposal and between Perceived Behavioral Control and an individual’s disposal intention. Implications Fashion enterprises are advised to act as educators to raise awareness of the adverse effects of frequent garment disposal and elucidate lifetime-extension measures, such as creating emotional attachment through customization or co-creation. Furthermore, emphasizing longevity during the production phase is crucial to hinder the influential factor of disposal due to damage. Therefore, policymakers’ importance is decisive in establishing industry-wide standards regarding garment production and lifetime-extension practices. Furthermore, societal education about garment disposal opportunities should be provided, for example, through implementation in the general curriculum of schools and governmental or nongovernmental organizations’ campaigns. Originality/Value By taking the general development toward a throwaway society into account, a connection to the textile industry is drawn in this thesis. When looking at the literature, it is visible that increasing attention is placed on the post-consumer phase. However, the technical constitution and the actual disposal approaches were mostly covered in this context. Therefore, the authors of this thesis examine the particular consumers’ determination of the end-of-life stage of garments to contribute to current circumstances and related literature

The repair of topoisomerase 2 cleavage complexes in Arabidopsis

DNA–protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2–DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that “clean” DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc

mTORC2 affects the maintenance of the muscle stem cell pool

Abstract Background The mammalian target of rapamycin complex 2 (mTORC2), containing the essential protein rictor, regulates cellular metabolism and cytoskeletal organization by phosphorylating protein kinases, such as PKB/Akt, PKC, and SGK. Inactivation of mTORC2 signaling in adult skeletal muscle affects its metabolism, but not muscle morphology and function. However, the role of mTORC2 in adult muscle stem cells (MuSCs) has not been investigated. Method Using histological, biochemical, and molecular biological methods, we characterized the muscle phenotype of mice depleted for rictor in the Myf5-lineage (RImyfKO) and of mice depleted for rictor in skeletal muscle fibers (RImKO). The proliferative and myogenic potential of MuSCs was analyzed upon cardiotoxin-induced injury in vivo and in isolated myofibers in vitro. Results Skeletal muscle of young and 14-month-old RImyfKO mice appeared normal in composition and function. MuSCs from young RImyfKO mice exhibited a similar capacity to proliferate, differentiate, and fuse as controls. In contrast, the number of MuSCs was lower in young RImyfKO mice than in controls after two consecutive rounds of cardiotoxin-induced muscle regeneration. Similarly, the number of MuSCs in RImyfKO mice decreased with age, which correlated with a decline in the regenerative capacity of mutant muscle. Interestingly, reduction in the number of MuSCs was also observed in 14-month-old RImKO muscle. Conclusions Our study shows that mTORC2 signaling is dispensable for myofiber formation, but contributes to the homeostasis of MuSCs. Loss of mTORC2 does not affect their myogenic function, but impairs the replenishment of MuSCs after repeated injuries and their maintenance during aging. These results point to an important role of mTORC2 signaling in MuSC for muscle homeostasis

mTOR controls embryonic and adult myogenesis via mTORC1

The formation of multi-nucleated muscle fibers from progenitors requires the fine-tuned and coordinated regulation of proliferation, differentiation and fusion, both during development and after injury in the adult. Although some of the key factors that are involved in the different steps are well known, how intracellular signals are coordinated and integrated is largely unknown. Here, we investigated the role of the cell-growth regulator mTOR by eliminating essential components of the mTOR complexes 1 (mTORC1) and 2 (mTORC2) in mouse muscle progenitors. We show that inactivation of mTORC1, but not mTORC2, in developing muscle causes perinatal death. In the adult, mTORC1 deficiency in muscle stem cells greatly impinges on injury-induced muscle regeneration. These phenotypes are because of defects in the proliferation and fusion capacity of the targeted muscle progenitors. However, mTORC1-deficient muscle progenitors partially retain their myogenic function. Hence, our results show that mTORC1 and not mTORC2 is an important regulator of embryonic and adult myogenesis, and they point to alternative pathways that partially compensate for the loss of mTORC1