Structure, Dynamics, and Regulation of Collective Cell Migration

Collective migration is the process by which cells organize individual motions to productively migrate as a group and plays a fundamental role in organism development, tissue regeneration, and cancer invasion. In development, coordinated migration facilitates the formation of complex organ structures and is required for proper dissemination of neural crest cells throughout an organism. After injury, this process allows breaches in epithelial layers to be repaired while maintaining tissue integrity, and in cancer, collective behavior enhances invasion of tumor cells into the surrounding tissue. Chapter 1 provides an introduction for the role of collective migration across an organism’s lifespan, the mechanisms used by cells to generate motile force, and the emergence of collective behavior. Chapter 2 dissects the intertwined roles of three fundamental parameters often altered in collective migration processes: cell density, cell adhesion, and cell-cell contractility through the Rho-ROCK-Myosin II signaling axis. Through quantitative analysis of large-scale time-lapse imaging and mathematical modeling, I identify force-sensitive contractility and cell packing as mediators of two distinct classes of collective migration. From these results, I formulate a phase-diagram of collective cell migration and test predictions in an in-vivo epithelium using genetic manipulations to drive collective motion between predicted migratory phases. In Chapter 3, the effect of phenotypic heterogeneity on the organization of cells is examined, providing insight into the effects of early cancer progression on epithelial dynamics. I find that mutant cells within an otherwise wild-type tissue impact organization through local and field-effects, disrupting normal dynamics and leading to cell-type segregation. Chapter 4 provides a theoretical framework for quantitatively understanding and predicting the dynamics of protein interactions underlying biological processes including collective migration. Traditional chemical kinetics approaches break down in situations where components are slow diffusing or in countable numbers, requiring the formulation of new models that take into account this level of complexity. Here I develop an event-driven algorithm that bridges well-mixed and unmixed systems and use it to predict the effect of apparent changes in enzymatic efficiency due to alterations in mobility that may be caused by protein complex formation. Overall the work in this dissertation advances our understanding of the structure and dynamics of collective migration and the parameters governing this process by combining quantitative statistical analysis, mathematical modeling, and in-vivo live imaging

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin–containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin–containing vesicular trafficking during AJ remodeling in live epithelia

Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl polarity complex promotes epithelial proliferation via overlapping mechanisms

The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin

Cell density and actomyosin contractility control the organization of migrating collectives within an epithelium

The mechanisms underlying collective migration are important for understanding development, wound healing, and tumor invasion. Here we focus on cell density to determine its role in collective migration. Our findings show that increasing cell density, as might be seen in cancer, transforms groups from broad collectives to small, narrow streams. Conversely, diminishing cell density, as might occur at a wound front, leads to large, broad collectives with a distinct leader–follower structure. Simulations identify force-sensitive contractility as a mediator of how density affects collectives, and guided by this prediction, we find that the baseline state of contractility can enhance or reduce organization. Finally, we test predictions from these data in an in vivo epithelium by using genetic manipulations to drive collective motion between predicted migratory phases. This work demonstrates how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease

Assessing discards in an illegal small-scale fishery using fisher-led reporting

Funding: Newton Fund (IL 2018-Grant Agreement 414695818 James PER), Fondo Nacional de Desarrollo Científico, Tecnológico y de Innovación Tecnológica (PE) (FONDECYT 2018-222).About a third of all marine fish in the world are caught in Small-Scale Fisheries (SSF). SSF are increasingly recognised as essential for food security and livelihoods for vulnerable and economically fragile communities globally. Although individual SSF vessels are usually perceived as having little impact on the ecosystem, the cumulative impact of gear type and number of vessels may be substantial. Bottom trawling is a common fishing method that can greatly influence the marine ecosystem by damaging the seafloor and generating high levels of discards. However, appropriate sampling coverage using on-board observer programmes to collect these data from SSF are rare, as they are expensive and pose logistical constraints. A mobile App was used to assess whether self-reporting by fishers could provide reliable fine-scale information on fishing effort and discards over time in an illegal shrimp trawling fishery in northern Peru. Maps depicting the spatial distribution of trawling effort and the proportion of discards from observers and fishers were compared using the Similarity in Means (SIM) Index, which ranges from 0 when spatial patterns differ completely to 1 when spatial patterns are very similar. High levels of agreement between spatio-temporal patterns of effort (SIM Index = 0.81) and discards (0.96) were found between fisher and observer maps. Moreover, far greater spatial coverage was accomplished by fishers, suggesting that self-reporting via an App represents a useful approach to collect reliable fisheries data as an initial step for effective monitoring and management of these fisheries.Publisher PDFPeer reviewe

Mutations in <i>N</i>-acetylglucosamine (<i>O</i>-GlcNAc) transferase in patients with X-linked intellectual disability

Contains fulltext : 177227.pdf (publisher's version ) (Open Access)N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system

The immune gene repertoire encoded in the purple sea urchin genome

Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology

RA-MAP, molecular immunological landscapes in early rheumatoid arthritis and healthy vaccine recipients

Rheumatoid arthritis (RA) is a chronic inflammatory disorder with poorly defined aetiology characterised by synovial inflammation with variable disease severity and drug responsiveness. To investigate the peripheral blood immune cell landscape of early, drug naive RA, we performed comprehensive clinical and molecular profiling of 267 RA patients and 52 healthy vaccine recipients for up to 18 months to establish a high quality sample biobank including plasma, serum, peripheral blood cells, urine, genomic DNA, RNA from whole blood, lymphocyte and monocyte subsets. We have performed extensive multi-omic immune phenotyping, including genomic, metabolomic, proteomic, transcriptomic and autoantibody profiling. We anticipate that these detailed clinical and molecular data will serve as a fundamental resource offering insights into immune-mediated disease pathogenesis, progression and therapeutic response, ultimately contributing to the development and application of targeted therapies for RA.</p