Guy Tanentzapf

Gap junction channels are intercellular transmembrane proteins that play an essential role in the development of tissues. They are a complex of proteins that can form homomeric or heteromeric channels between coupled cells to allow the passage of ions and small molecules. In Drosophila melanogaster, gap junctions are composed of innexins (Inx), which are structural homologues of vertebrate connexins. In Drosophila testes, Inx proteins contribute to spermatogenesis via bi-directional soma-germline communication. Inx2 on the somatic cells, which support the developing germline, and Zpg (Inx4) on the germline cells form a coupled heterotypic transmembrane channel in the fly testis. While the significance of these proteins is established, little is known about the nature of this intercellular communication. Our lab has previously demonstrated that flies lacking innexin expression have rudimentary gonads and impaired germline and somatic cell differentiation. The recently determined 3D protein structure of an innexin protein predicts that the N-terminus may be involved in regulating channel permeability. We are undertaking a detailed structure function analysis of innexin function in the context of the Drosophila testes using site-directed mutagenesis to generate mutations within the N-terminal and C-terminal domains that are predicted to disrupt functionally important residues. The effects on spermatogenesis are analysed using immunostainings and various functional assays. A 4 amino-acid truncation of the N-terminal results in a loss-of-function phenotype, and a deletion of the C-terminal disrupts the subcellular localization of the Zpg protein. This provides a mechanistic insight into the function of structurally important domains in innexins and further our understanding of germline stem cell regulation and maintenance during spermatogenesis.

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Morphogenesis is the process by which cells rearrange to form complex three dimensional structures. Cell to extracellular matrix (ECM) adhesion, primarily mediated by Integrins, is essential for the formation and maintenance of tissue architecture. A critical way to regulate cell-ECM adhesion is by modulating the turnover of Integrins and their adhesion complex, and thereby modulating the stability of Integrin-based adhesions. We previously showed that mechanical force stabilizes Integrin-based adhesions during development by modulating Integrin turnover. Here, we extend our studies to understand how mechanical stress impacts the dynamics of the cytoplasmic adaptor protein Talin, a critical regulator of Integrin function. Using Fluorescence Recovery After Photobleaching (FRAP) analysis in combination with a newly developed mathematical model that encompasses the complexities of Talin turnover, we determined that mechanical force stabilizes cell-ECM adhesion by increasing the rate of assembly of Talin-mediated adhesion complexes. To dissect the mechanisms that regulate Talin turnover downstream of mechanical force, we used point mutations of Talin which abrogate specific functions of the Integrin adhesion complex and measured turnover kinetics. We found that the activation of Integrins, resulting in increased affinity for ECM ligands, is a crucial process to regulate adhesion complex turnover. To further investigate the role of Integrin activation in regulating adhesion stability, we introduced small molecules known to induce “outside-in activation” of Integrins in vitro into live, intact embryos. This approach revealed that outside-in activation stabilizes cell-ECM adhesion by decreasing Integrin endocytosis; similarly to what we have previously seen when mechanical force is increased. Based on this finding, we propose that mechanical force may induce changes in Integrin activation in order to stabilize cell-ECM adhesions. Overall, we show that Integrin activation is a key mechanism that regulates cell-ECM adhesion stabilization during embryogenesis.

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During the morphogenesis of multicellular organisms, cells undergo rearrangements and morphological changes to generate three dimensional structures and thus give rise to tissues and organs. Robust morphogenesis requires connections between neighbouring cells and to the extracellular environment in order to generate dynamic large-scale tissue rearrangements. Cellular adhesion to the extracellular matrix (ECM) is primarily mediated by the integrin family of adhesion receptors. Integrins are transmembrane heterodimeric receptors that bind ECM ligands extracellularly, and on their intracellular sides bind a diverse group of proteins known as the integrin adhesion complex (IAC). Integrins are involved in many fundamental biological processes and they contribute to animal development through two distinct mechanisms; first, via dynamic short-term adhesions such as those driving cell migrations and cellular rearrangements, and second via long-term stable adhesions such as those involved in tissue maintenance. Thus, the activity of integrins needs to be finely regulated during morphogenesis. In this thesis, I aim to investigate the regulation of integrins during Drosophila morphogenesis in the context of both stable long-term adhesions, and short-term dynamic adhesions. First, I investigate the role of talin, a large scaffolding protein which provides a direct link between integrins and the actin cytoskeleton, in the context of muscle development during fly embryogenesis. Importantly, talin also functions as an integrin regulator by regulating the affinity of integrin for its extracellular ligands, a process known as inside-out activation. I describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering in a mechanism distinct from its role in supporting inside-out activation. Secondly I investigate a process known as dorsal closure (DC), an integrin-dependent dynamic morphogenetic event during fly embryogenesis that is characterized by rapid tissue movements, and the generation of biophysical forces. I find that integrins play a key role in regulating cell behaviours critical for DC via two main mechanisms: through modulating the localization of cell-cell adhesion proteins, and by regulating the dynamics of force-generating actomyosin machinery. Overall, my work highlights the diverse functions of integrins during development, and necessity for fine-tuning of their regulation during morphogenesis.

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Tissue morphogenesis requires force-generating mechanisms to drive the organization of cells into complex three-dimensional structures. Although such mechanisms have been characterized across the metazoan lineage, we know little about how force transmission across a tissue is regulated. Here, using Drosophila melanogaster as a model system, I provide evidence that integrin-mediated cell-ECM adhesion is required for the regulation and transmission of forces in tissues. Specifically I show that during Dorsal Closure (DC), an integrin-dependent morphogenetic process that occurs during Drosophila embryogenesis, failure to regulate the level of cell-ECM adhesion results in abnormal levels of tension in the amnioserosa (AS), an extra-embryonic epithelium that is essential for DC. Integrin-containing adhesive structures were identified on the basal surface of the AS that share many features with focal adhesions. Using mutations that either increase or decrease integrin-based Cell-ECM adhesion, I show that DC is defective in both cases, and that the level of adhesion is inversely correlated with the mobility of cells in the AS. Mathematical modeling, quantitative image analysis, and in vivo laser ablation experiments reveal a relationship between cell mobility and the magnitude, distribution and transmission of tension in the AS. Finally, I provide evidence that mechanical coupling exists between AS cells and their substrate, the underlying ECM and the yolk membrane. Overall, my data shows that integrins regulate the transmission of forces across the AS, and thereby control a critical component of DC. I propose that modulating Cell-ECM adhesion could provide control over force transmission within developing tissues to promote specific outcomes.

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Cells in multicellular organisms are arranged in complex three-dimensional patterns. To achieve such complexity, cells must form adhesive contacts with the extracellular matrix (ECM). The most common adhesion receptors that mediate cell-ECM adhesions are the integrins. A large cytoplasmic network of proteins, namely the integrin adhesion complex (IAC) is recruited to the site of adhesions. Regulated assembly and disassembly, or turnover, of the IAC is essential for dynamic cell movements and tissue maintenance. In this project, I sought to investigate the role of mechanical force on the turnover of talin, a core component of the IAC and an essential linker between integrins and the actin cytoskeleton.To investigate the turnover of talin in vivo, I performed fluorescence recovery after photobleaching (FRAP) on the myotendionous junctions (MTJs) in live Drosophila embryos and larvae. I used temperature sensitive mutants to alter the force acting on the MTJs. To better understand talin turnover, I collaborated with people from Dan Coombs’ lab (Department of Mathematics, UBC) to develop a mathematical model for the turnover of cytoplasmic adhesion proteins. This model is parametrized by four rate constants: talin binding on and off the adhesion complex at the plasma membrane, talin delivery to the plasma membrane due to the assembly of the IAC and talin removal from the plasma membrane due to the disassembly of the IAC.I hypothesized that changes in force would affect talin turnover and certain functional domains in talin would be required for mechanosening at the MTJs. I used targeted point mutations in the functional domains of talin to investigate their role in mechanosensing. Consistent with my hypothesis, I found out that disrupting functional domains in talin either abrogates or severely affect the ability of talin to respond to changes in force. First, these results provide direct evidence on how force is sensed at the adhesion complex. Secondly, this is the first in vivo study where four rate constants are used to characterize the turnover of a cytoplasmic protein.

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Animal gonads contain two types of cells, the soma and the germline. Interactionsbetween these two tissues regulate cell proliferation, differentiation, patterning, andhomeostasis in the gonad during development and throughout the life of the organism. Inparticular, interactions between the soma and germline in the gonads regulate thebehavior of germline stem cells (GSCs). Disruption of germline-soma interactions hassevere consequences for gametogenesis and especially on GSC function, resulting insterility, formation of somatic and germline tumors, and defective sexual determinationof the germline. The Drosophila melanogaster gonads provide a powerful, established,model system to study how soma-germline interactions regulate GSC function as well asspermatogenesis. Soma-germline interactions are initiated at the stem cell niche whereboth germline and somatic stem cells are housed. Upon exit from the stem cell niche, thesoma encapsulates the germline. The association and communication between the somaand germline ensures proper differentiation of the germline into mature sperm. Signalingevents between the germline and soma regulate germline stem cell self-renewal,displacement from the niche, and encystment of germ cells. The cell biologicalmechanisms that set up the milieu in which these signaling events take place are poorlyunderstood. In order to better understand the regulation of soma-germline interactions,we are performing a genetic screen using tissue-specific RNAi and fertility assays toidentify genes involved in this process. We have identified over 200 genes necessary inthe soma for spermatogenesis. Here I describe the characterization and classification ofgenes identified in the screen, as well as initial attempts to understand their role in the flytestis. The identification and characterization of the novel genes that mediate soma-germline interactions will provide crucial insight into the basic mechanisms that regulate spermatogenesis.

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Animal muscles must maintain their function while bearing substantial mechanical loads and undergoing numerous contraction/extension cycles. How muscles withstand persistent mechanical strain is presently not well understood. The basic unit of muscle is the sarcomere, which is primarily composed of cytoskeletal proteins. I hypothesized that cytoskeletal proteins undergo renewal via protein turnover and that this is required to maintain muscle function. Using the adult flight muscles of the fruit fly, Drosophila melanogaster, I confirmed that the sarcomeric cytoskeleton undergoes turnover throughout the life of the organism. To uncover which cytoskeletal components are specifically required to maintain adult muscle function I performed an RNAi-meditated knockdown screen in adult D. melanogaster targeting the entire fly “cytoskeletome”, the set of known cytoskeletal and cytoskeletal-associated proteins. Systematic gene knockdown was restricted to adult flies and muscle function was analyzed with behavioural assays. This approach identified 47 genes required for maintaining muscle function, 40 of which had no previously known role in this process. Detailed analysis of the role of candidate genes in adult muscles using confocal and electron microscopy showed that while muscle architecture was largely maintained after gene knockdown, maintenance of sarcomere length was disrupted. Specifically, I found that the ongoing synthesis and turnover of the key structural sarcomere component Projectin (bent) was required to maintain M-line integrity. Together, these results provide direct in vivo evidence of muscle protein turnover and identify possible roles for this process by uncovering specific functional defects associated with reduced expression of a subset of cytoskeletal proteins in the adult animal.

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Cell-extra cellular matrix (ECM) adhesion through the integrin family of receptors is required for metazoan development, and throughout adult life. Elucidating the mechanisms that regulate this adhesion is fundamental to understanding how animals create and maintain tissue architecture. Modulating adhesion assembly and disassembly is one of the key ways in which adhesion strength and integrity is regulated. We concentrate on analyzing the dynamics of three important components of the integrin adhesion complex (IAC), talin, tensin, and ILK, to determine how they function as mechano-sensory components of cell-ECM adhesions in the context of a living, multicellular organism, Drosophila melanogaster. We utilize fluorescently-tagged proteins under conditions of altered mechanical force, combined with a specialized fluorescence recovery after photobleaching (FRAP) protocol, to examine the dynamics of talin, tensin, and ILK. We subsequently use advanced mathematical modeling to gain mechanistic insight into how protein turnover is modified by tensile force. Furthermore, we attempt to clarify the role of key talin domains in mechanosensation, using FRAP and Drosophila homologs of previously characterized talin mutations, under conditions of altered force. The results outlined in this work show that talin mobility is directly regulated by force in an intact, complex organism at sites of stable adhesion between integrins and the ECM. Moreover, the results indicate that the mobility change due to increased force is a robust process, and not easily disrupted by mutating talin domains. Changes in talin dynamics when force is reduced is an active process, and is dependent on both the physical linkage of talin to integrin, and the ability of talin to auto-inhibit. Furthermore, studies of talin, tensin, and ILK turnover with high-temporal resolution uncover the intricacies of adhesion regulation in response to changing environmental conditions, with talin primarily regulated on the level of recycling, tensin regulated by a mix of both recycling and binding, and ILK regulated through control of binding.

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Integrins are essential mediators of cell-ECM adhesion and they are, therefore, important to animal viability. Integrin-mediated transient (short-term) cell adhesion underlies dynamic processes such as cell migration while integrin-mediated stable (long-term) cell adhesion is essential for maintaining tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is unknown whether turnover is also required for maintenance of long-term adhesion. Fluorescence Recovery After Photobleaching (FRAP) was used to analyze the dynamics of the Integrin Adhesion Complex (IAC) in a model for long-term cell-ECM adhesion, Myotendinous Junctions (MTJs), in fly embryos and larvae. It was found that the IAC undergoes turnover in the MTJs and that this process is mediated by clathrin-dependent endocytosis but not lateral diffusion. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover and altering Rab5 activity weakened MTJs such that it leads to muscle attachment defects. In addition, growth of the MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover and it is possible that this growth-dependent decrease is regulated by the mechanical tension exerted on MTJs by muscle contraction. Experiments using mutations that result in increased mechanical tension exhibited lower IAC turnover. In contrast, mutations that lower mechanical tension exhibited higher IAC turnover with the exception of integrins. Therefore, we propose that IAC turnover is regulated during development by mechanical tension in long-term cell-ECM adhesions to allow normal tissue growth and maintenance.

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