463 research outputs found

    A ced-3 caspase – ect-2 RhoGEF axis coordinates functional interactions between the apoptotic pathway and cell size in Caenorhabditis elegans

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    Programmed cell death via apoptosis is a common cell fate during animal development and its mis-regulation can have serious implications in diseases and disorders. Therefore, it is of high importance to study to apoptosis. The highly conserved central apoptotic pathway was initially discovered in C. elegans and consists of four genes acting in a sequence – egl-1 BH3-only, ced-9 Bcl-2, ced-4 Apaf-1 and ced-3 caspase. The most downstream gene in the pathway, ced-3, encodes for a cysteine protease called caspase, and is essential in the execution of apoptosis. A previous study in our lab had uncovered a novel non-apoptotic role of ced-3 caspase in promoting asymmetric division of C. elegans neuroblasts (Mishra et al., 2018). However, the mechanism by which ced-3 caspase promotes asymmetric division still remained to be elucidated. Thus, in my study, I aimed to decipher the mechanism(s) by which the apoptotic gene, ced-3 caspase, promotes asymmetric cell division. To that end, I first demonstrated that CED-3 caspase protein physically and directly interacts with a regulator of actomyosin contractility, called ECT-2 RhoGEF (Rho guanine-nucleotide exchange factor). Furthermore, using the NSM (neurosecretory motor neuron) lineage in C. elegans, I found that ECT-2 RhoGEF is asymmetrically enriched in the NSM neuroblast, which is the mother of the apoptotic cell. I also found that the asymmetric enrichment of ECT-2 RhoGEF depends on ced-3 caspase activity. Next, by analysing the cell size ratios of the daughters of the NSM neuroblast, my colleagues and I found that genetically, ced-3 caspase acts upstream of ect-2 RhoGEF to promote the asymmetric division by size of the NSM neuroblast. We refer to this as the ced-3-ect-2 axis. Based on these findings, we propose that the ced-3-ect-2 axis promotes polar actomyosin contractility in the NSM neuroblast, which results in its asymmetric division by size and thereby the formation of its smaller apoptotic daughter cell called the NSMsc (NSM sister cell). Molecularly, we propose that CED-3 de-recruits ECT-2 from the dorsal cortex of the NSM neuroblast before metaphase, and that this de-recruitment of ECT-2 is important for the NSM neuroblast to divide asymmetrically. 6 Next, my colleagues and I investigated the effect of the size of the smaller daughter cell, the NSMsc, on its apoptotic fate. We found that increasing the size of the NSMsc by reducing ect-2 activity decreases its probability to undergo apoptosis. Conversely, for the first time, we showed that decreasing the size of the NSMsc by hyperactivation of ect-2 can increase its probability to undergo apoptosis. Thus, we propose that cell size and apoptosis are inversely corelated – larger cells are more prone to survive and smaller cells are more prone to die. Taken together, the findings from this study have found reciprocal interactions between the apoptotic pathway and cell size. In the NSM neuroblast, the apoptotic pathway acts upstream of cell size i.e. ced-3 promotes asymmetric division of the NSM neuroblast and the formation of a smaller NSMsc. Conversely, in the NSMsc, cell size acts upstream of the apoptotic pathway i.e. the small size of the NSMsc promotes the activation/activity of CED-3 and thereby its apoptosis

    Opportunities and challenges for deep learning in cell dynamics research

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    With the growth of artificial intelligence (AI), there has been an increase in the adoption of computer vision and deep learning (DL) techniques for the evaluation of microscopy images and movies. This adoption has not only addressed hurdles in quantitative analysis of dynamic cell biological processes, but it has also started supporting advances in drug development, precision medicine and genome-phenome mapping. Here we survey existing AI-based techniques and tools, and open-source datasets, with a specific focus on the computational tasks of segmentation, classification, and tracking of cellular and subcellular structures and dynamics. We summarise long-standing challenges in microscopy video analysis from the computational perspective and review emerging research frontiers and innovative applications for deep learning-guided automation for cell dynamics research

    The roles of Wnt signaling during mitosis

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    Wnt signaling is crucial for embryonic patterning, the formation of tissues during development, and tissue maintenance in the adult organism. The canonical Wnt pathway induces the transcription of β-catenin target genes, thereby controlling cell cycle progression during G1/S phase, stem cell self-renewal, and stem cell differentiation. However, besides its transcriptional roles, it emerged that Wnt signaling also takes post-translational functions during mitosis. Accordingly, a misregulation of the pathway causes severe mitotic defects, such as chromosome misalignments and chromosome segregation errors, which can ultimately lead to aneuploidy. In this work, I aimed to identify targets of Wnt signaling, which safeguard the correct progression through mitosis, and characterize the underlying molecular mechanisms to explain the emergence of mitotic defects upon Wnt disturbance. First, I revealed that the mitotic kinesin KIF2A is recruited by the Wnt component DVL. DVL localizes KIF2A to the mitotic spindle poles, where it regulates microtubule minus-end dynamics to ensure chromosome alignment before anaphase, both in somatic cells and pluripotent stem cells. This process is supported by the phosphorylation of KIF2A at serine 100 and the interaction with PLK1, which is positively regulated by active Wnt signaling and LRP6 signalosome formation. Second, I verified an S phase-dependent mechanism of Wnt signaling, ensuring the equal segregation of chromosomes in pluripotent stem cells during anaphase. At this, I hypothesize that Wnt signaling contributes to the error-free replication of DNA in S phase, which mediates microtubule plus-end assembly in mitosis, and thereby facilitates faithful chromosome segregation. The validation of both mechanisms in pluripotent stem cells emphasizes their relevance for the understanding of developmental defects, tissue degeneration, and cancer progression, which is often characterized by chromosomal instability. Besides, KIF2A was recruited by DVL also in interphase, indicating that the Wnt-mediated regulation of KIF2A may contribute to processes beyond mitosis, namely ciliogenesis and neurogenesis. Taken together, in my work, I revealed two novel Wnt-dependent mechanisms, which function directly in mitosis or through S phase to control microtubule minus- or plus-end dynamics respectively, ensuring the faithful progression through mitosis, preservation of euploidy, and possibly further post-mitotic processes

    The role of WNT/STOP signaling during neurogenesis and ciliogenesis

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    WNT signaling is an evolutionarily conserved pathway that plays essential roles in development and disease. A recently described branch of WNT signaling, known as WNT/STOP (stabilization of proteins), promotes cell growth and division by stabilizing target proteins from glycogen synthase 3 (GSK3)-mediated degradation. Key regulators of WNT/STOP are Cyclin Y (CCNY) and Cyclin Y like 1 (CCNYL1), conserved cyclins that, together with their associated cyclin dependent kinase 14 (CDK14) and 16 (CDK16), phosphorylate and prime the WNT co-receptor LRP6, thereby inhibiting GSK3. Most of the biological functions of WNT/STOP signaling have been described in vitro, or in post-transcriptional germ cells. However, whether or not WNT/STOP signaling plays essential roles in dividing somatic cells is incompletely understood. In order to study the in vivo functions of WNT/STOP signaling in somatic cells, Ccny/Ccnyl1 double knockout (DKO) mice were generated and analyzed. Strikingly, mutant embryos displayed severe defects in the neocortex characterized by a thinner lateral cortex and reduced basal progenitors and post mitotic neurons. Mechanistically, WNT/STOP is shown to promote asymmetric cell division by regulating the levels of apical-basal astral microtubules, and the differentiation of neural progenitors by stabilizing Sox4 and Sox11, two neurogenic transcription factors that are characterized as direct GSK3 substrates in this thesis. Apart from the neurogenesis defects, Ccny/l1 deficiency also led to defects in primary cilia formation in apical progenitors. Primary cilia are microtubule-based organelles involved in transducing cell signaling pathways and defects in cilia formation are associated with human disease. A detailed phenotypic analysis of DKO embryos and cell lines deficient or mutant for Ccny/l1 revealed that the regulation of ciliogenesis by WNT/STOP signaling was also extended to developing kidneys, 293T cells and adult mouse preadipocytes. Mechanistically, CCNY and LRP6 concentrate in primary cilia and LRP6 becomes phosphorylated following WNT stimulation, suggesting that cilia act as WNT-responsive organelles. WNT/STOP signaling activates the Protein phosphatase 1 regulatory subunit PPP1R2 in the cilia, which then inhibits the negative ciliary regulator protein phosphatase 1 (PP1). In summary, the findings in this thesis unveil crucial in vivo roles for WNT/STOP signaling in neocortex development and primary cilia formation, with important implications for embryonic development and disease

    Mesostoma ehrenbergii spermatocytes provide an unusual and exciting model to investigate chromosome movement, cleavage furrows and tethers during cell division

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    For my PhD thesis I studied and tracked chromosome movement in primary spermatocytes from a species of aquatic flatworm, the Mesostoma ehrenbergii, with the hope to better understand what determines the force behind these intricate and highly co-ordinated movements during meiosis. Canonical theories of chromosome movement state that microtubules are the main producers of force for chromosome movement in the meiotic spindle, however my research has shown chromosomes can not only move but can move even faster in the absence of microtubules. I also tracked the cleavage furrow and showed it was able to simultaneously move and ingress in the absence of microtubules. I worked on determining how the force is produced by using a laser microbeam to sever various components in the cell and by adding various myosin and actin inhibiting and enhancing drugs to see how chromosome movement may be altered. In addition, I discovered elastic tethers are present in Mesostoma during metaphase and anaphase and my research on functionally disabling tethers suggests they play a role in coordinating or maintaining non-random chromosome movement in these cells. In sum, my PhD research has added evidence to the non-microtubule model of chromosome movement by showing actin and myosin is involved in movement and that tethers may play a role in coordinating or maintaining non-random chromosome movement in these cells

    A New Dimension of Cancer Treatment: Analysing and Targeting High-Grade Glioma Invasion Using Three-Dimensional In Vitro Models

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    High-Grade Gliomas (HGG) are an extremely aggressive form of brain cancer with limited targeted treatments and dire prognosis. Despite global improvements in overall cancer survival, HGG survival rates have stagnated over the last three decades. This can be partly attributed to HGG’s characteristically invasive progression into the brain, where far-invaded cells are often missed by first-line therapies leading to tumour recurrence. The use of anti-invasive agents may limit extensive cell spread and improve efficacy of adjuvant anti-cancer therapies, thus, this study has investigated the novel anti-invasive PTU (p-tolyl-ureidopalmitic acid), an omega-3-derivative with the ability to cross the blood brain barrier. PTU was tested in primary patient-derived HGG cells cultured in 3D models designed to replicate discrete aspects of the 3D in vivo brain microenvironment. Across models, PTU treatment decreased the number of invading HGG cells and mitigated both single and collective invasion phenotypes. Importantly, this data demonstrates that 3D in vitro culture can reliably represent a whole-picture of HGG invasion. Additionally, this study involved the development of a dynamic, temporal reporter of transcriptional co-activators Yes-associated Protein and Transcriptional co-Activator with PDZ-binding motif (YAP/YAZ), which are mediators of a number of malignant processes and regulated by bio-mechanical signalling. This was achieved with an Adeno-associated-virus (AAV)-mediated gene delivery method, which also revealed a number of AAV capsid variants with tropism to primary patient-derived HGG cells. This reporter has potential future uses in revealing YAP/TAZ activity within the 3D biologically-relevant in vitro models. Together, these findings reveal potential successful therapeutic strategies for HGG and provide multiple insights into future research possibilities in the hopes of mitigating the extent of this truly debilitating and devastating disease
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