Actin filaments and microtubules are involved in cell development and morphogenesis. Plant Class II formins regulate both cytoskeletal polymers. However their function has not yet been fully described. This study examines effects of LOF mutations in Arabidopsis thaliana FH13 (AT5G58160) and FH14 (AT1G31810) genes on early root system development using a pharmacological approach. Since measuring root length of numerous mutant lines in multiple conditions is laborious and time consuming, this thesis also involves optimization of this process with the aim to establish a reliable method of fast visualisation and measurement of Arabidopsis seedlings in a time series in the laboratory. Furthermore, statistical analysis for a large amount of data gathered in multiple conditions had to be optimized. While no significant phenotype in terms of root length was found in fh13, fh14 and double fh13 fh14 LOF mutants under standard conditions, treatment with cytoskeletal drugs revealed possible changes in lateral root branching in an fh14 mutant. Nevertheless, specific function of FH13 and FH14 remains a question.

Insulin stimulates glucose uptake in adipose and muscle cells via translocation of the intracellular vesicles containing GLUT4. It was largely unknown whether and/or how the signaling molecules such as PI 3-kinase and Akt regulate the mechanical movements of the GLUT4-containing vesicles. Hence, this study was performed to test the hypothesis that actin and microtubules function in translocating GLUT4 vesicles. Treatments of insulin as well as endothelin-1 (ET-1), an insulin-mimicking peptide which does not act through PI 3-kinase, induced polymerization of actin without affecting the microtubular network. By mass spectrometry, the tyrosine kinase PYK2 was identified to be tyrosine phosphorylated specifically by ET-1 but not by insulin. Expression of the carboxyl-terminal fragment (CRNK) PYK2, but not wild type nor kinase-deficient PYK2 mutants, inhibited ET-1-stimulated actin polymerization while expression of all three PYK2 constructs had no effect on insulin-stimulated actin polymerization. More importantly, expression of CRNK, but not wild type nor kinase-deficient PYK2 constructs, blocked ET-1- but not insulin-stimulated GLUT4 translocation to the plasma membrane. These suggest that ET-1 and insulin stimulate actin polymerization via distinct signaling pathways, and that the actin polymerization is required for GLUT4 vesicle translocation. In order to test the possible involvement of microtubule in GLUT4 vesicle translocation, time lapse imaging of 3T3-L1 adipocytes expressing GLUT4-YFP and tubulin-CFP was performed. GLUT4-YFP vesicles move long-range bi-directionally on microtubules, which suggests the presence of molecular motors on the vesicles. Moreover, insulin increased the number of vesicle movements on microtubules without changing the velocities. Interestingly, the stimulatory action of insulin appears to be independent of PI 3-kinase activation. Conventional kinesin was identified as a highly expressed kinesin isotype in adipocytes. Notably, expression of dominant negative mutants but not wild type kinesin inhibited insulin-stimulated long-range GLUT4 vesicle movements and GLUT4 translocation to the plasma membrane in live and fixed cells, respectively. These data indicate that insulin signaling induces the movement of GLUT4 vesicles on microtubule which is mediated by conventional kinesin. Overall, the data presented here provide evidence supporting the hypothesis that actin and microtubule cytoskeletons are required for insulin to mobilize GLUT4 vesicles in adipocytes.

The organization of molecules within a cell is central to cellular processes ranging from metabolism and damage repair to migration and replication. Uncovering the emergent properties of this biomolecular organization can improve our understanding of how organisms function and reveal ways to repurpose their components outside of the cell. This dissertation focuses on the role of organization in two widely studied systems: enzyme cascades and active cytoskeletal filaments.Part I of this dissertation studies the emergent properties of the spatial organization of enzyme cascades. Enzyme cascades consist of a series of enzymes that catalyze sequential reactions: the product of one enzyme is the substrate of a subsequent enzyme. Enzyme cascades are a fundamental component of cellular reaction pathways, and spatial organization of the cascading enzymes is often essential to their function. For example, cascading enzymes assembled into multi-enzyme complexes can protect unstable cascade intermediates from the environment by forming tunnels between active sites. We use mathematical modeling to investigate the role of spatial organization in three specific systems. First, we examine enzyme cascade reactions occurring in multi-enzyme complexes where active sites are connected by tunnels. Using stochastic simulations and theoretical results from queueing theory, we demonstrate that the fluctuations arising from the small number of molecules involved can cause non-negligible disruptions to cascade throughput. Second, we develop a set of design principles for a compartmentalized cascade reaction with an unstable intermediate and show that there exists a critical kinetics-dependent threshold at which compartments become useful. Third, we investigate enzyme cascades immobilized on a synthetic DNA origami scaffold and show that the scaffold can create a favorable microenvironment for catalysis. Part II of this dissertation focuses on the organization of active cytoskeletal filaments. Many mechanical processes of a cell, such as cell division, cell migration, and intracellular transport, are driven by the ATP-fueled motion of motor proteins (kinesin, dynein, or myosin) along cytoskeletal filaments (microtubules or actin filaments). Over the past two decades, researchers have been repurposing motor protein-driven propulsion outside of the cell to create systems where cytoskeletal filaments glide on surfaces coated with motor proteins. The study of these systems not only elucidates the mechanisms of force production within the cell, but also opens new avenues for applications ranging from molecular detection to computation. We examine how microtubules gliding on surfaces coated with kinesin motor proteins can generate collective behavior in response to mutualistic interactions between the filaments and motors, thereby maximizing the utilization of system components and production. To this end, we used a microtubule-kinesin system where motors reversibly bind to the surface. In experiments, microtubules gliding on these reversibly bound motors were unable to cross each other and at high enough densities began to align and form long, dense bundles. The kinesin motors accumulated in trails surrounding the microtubule bundles and participated in microtubule transport. In conclusion, our study of the emergent properties of the spatial organization of enzyme cascades and the mutualistic interactions within active systems of motor proteins and cytoskeletal filaments provides insight into both how these systems function within cells and how they can be repurposed outside of them.

Cytoskeletal tension plays an important role in numerous biological functions of adherent cells, including mechanosensing of the cell’s microenvironment, mechanotransduction, cell spreading and migration, cell shape stability, and in stem cell lineage. It is believed that for normal biological functions the cell must maintain its cytoskeletal tension stable, at a preferred set-point level, under external perturbations. This is known as tensional homeostasis. Any breakdown of tensional homeostasis is closely associated with disease progression, including cancer, atherosclerosis, and thrombosis. The exact mechanism and the relevant environmental conditions for the maintenance of tensional homeostasis are not yet fully understood. This thesis investigates the impacts of substrate stiffness, availability of functional cadherin junctions and steady shear stress on tensional homeostasis of cells and cell clusters. We define tensional homeostasis as the ability of cells to maintain a consistent level of tension with low temporal traction field fluctuations. Traction forces of isolated cells, multicellular clusters, and monolayer are measured using micropattern traction microscopy. Temporal fluctuations of the traction field are calculated from time-lapsed traction measurements. Results demonstrated that substrate stiffness, cadherin cell-cell junctions and shear stress all impact tensional homeostasis. In particular, we found that stiffer substrates promoted tensional homeostasis in endothelial cells, but were detrimental to tensional homeostasis in vascular smooth muscle cells. We also found that E-cadherins were essential for tensional homeostasis of gastric cancer cells and that extracellular and intracellular mutations of E-cadherin had domain-specific effects on tensional homeostasis. Finally, laminar flow-induced shear stress led to increased traction field fluctuations in endothelial cell monolayers, contrary to reports of physiological shear promoting vascular homeostasis. A possible reason for this discrepancy might be the limitation of our approach which could not account for mechanical balance of traction forces in the monolayers. Through the exploration of these environmental factors, we also found that tensional homeostasis was a length scale-dependent and cell type-dependent phenomenon. These insights suggest that future studies need to take a more comprehensive approach and aim to make observations of different cell types on multiple length scales, in order decipher the mechanism of tensional homeostasis and its role in (patho)physiology. / 2021-08-30T00:00:00Z

Cell-substrate interactions are important to study for development of accurate in vitro research platforms.  Recently it has been demonstrated that physical microenvironment of cells directly affects cellular motility and cytoskeletal arrangement.  Specifically, previous studies have explored the role of material stiffness (Young's modulus: N/m2) on cell behavior including attachment, spreading, migration, cytoskeleton arrangement (stress fiber and focal adhesion distribution) and differentiation. In this study using our recently described non-electrospinning fiber manufacturing platform, customized scaffolds of suspended nanofibers are developed to study single cell behavior in a tunable structural stiffness (N/m) environment.  Suspended fibers of three different diameters (400, 700 and 1200 nm) are deposited in aligned configurations in two lengths of 1 and 2 mm using the previously described STEP (Spinneret based Tunable Engineered Parameters) platform.  These fibers present a gradient of structural stiffness to the cells at constant material stiffness.   Single cells attached to fibers are constrained to move along the fiber axis and with increase in structural stiffness are observed to spread to longer lengths, put out longer focal adhesions, have elongated nucleus with decreased migration rates. Furthermore, more than 60% of cell population is observed to migrate from areas of low to high structural stiffness. Additionally dividing cells are observed to round up and daughter cells are observed to migrate away from each other after division. Interestingly, dividing rounded cells are found to be anchored to the fibers through thin protrusions emanating from the focal adhesion sites. These results indicate a substrate stiffness sensing mechanism that goes beyond the traditionally accepted modulus sensing that cells have been shown to respond to previously.  From this work, the importance of structural stiffness in cellular mechanosensing at the single cell-nanofiber scaled warrants consideration of the above factors in accurate design of scaffolds in future. / Master of Science

Student Number : 9502128Y - MSc dissertation - School of Anatomical Sciences - Faculty of Science / Retinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10-5M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.

Obesity, a disease characterized by excess adipose tissue (AT), is a growing worldwide epidemic. The Centers for Disease Control and Prevention (CDC), in 2017-2018, reported the prevalence of obesity in adults in the United States was 42.4% . Obesity increases the risk for many other serious health conditions such as type 2 diabetes, cardiovascular diseases, stroke, and some cancers. In individuals with obesity, the hypertrophic expansion of adipocytes, the main cell type within AT, is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Adipocytes are mechanoresponsive and affected by different microenvironmental cues, including hypoxia and mechanical (un)loading. Yet, no study has focused on the role of the aforementioned factors on the adipocyte mechanical response, including actin cytoskeletal remodeling. This dissertation aims to develop an in vitro model of healthy/diseased AT to explore the effect of microenvironmental cues on adipocyte function and actin cytoskeletal remodeling. The first aim is to study (1) the crosstalk between fibroblasts and adipocytes in a co-culture model and (2) the effect of hypoxia on the ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) mechanical pathway and actin cytoskeletal remodeling in adipocytes. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. The second aim explores the effects of mechanical unloading (simulated microgravity) on key adipocyte functions and actin cytoskeletal remodeling. This study demonstrated that mechanical unloading enhances adipocyte maturation via increased lipogenesis and lipolysis and cortical actin remodeling, which together further enhanced glucose uptake. However, disrupting cortical actin remodeling by using inhibitors or exposure to a high concentration of free fatty acids (FFAs) diminished enhanced adipocyte functions observed in simulated microgravity. Overall, the results of these studies support the importance of microenvironmental cues on adipocyte actin cytoskeletal remodeling. Therefore, targeting mechanical pathways that regulate actin cytoskeletal remodeling can be used to improve adipocyte function and AT metabolism and possibly treat related diseases such as type 2 diabetes and obesity. / Bioengineering

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