Browsing by Subject "actin"
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Item Microtubule-based control of glioma cell migration mechanics(2018-08) Prahl, LouisCell migration underlies the extensive tissue invasion that drives brain tumor (glioma) progression. Glioma cell migration involves the coordinated mechanical functions of the actin cytoskeleton, myosin motors, and substrate adhesions through a biophysical motor-clutch model. Although computational forms of the motor-clutch model predict glioma cell migration behaviors as a function of tissue stiffness, less is known about how other cellular structures such as microtubules influence migration. Presently, a number of microtubule-targeting agents (MTAs) are used to treat various cancers (including gliomas) so understanding their mechanism of action is necessary in order to develop better therapies. In this dissertation, I show that two commonly used MTAs (paclitaxel and vinblastine) each have distinct and nearly opposite effects on traction forces that motor-clutch simulations predict, and which correlate with changes to microtubule organization and dynamics. Effects of MTAs are consistent with influencing F-actin assembly and nucleation rates of protrusions, which impairs the ability of glioma cells to spontaneously polarize and migrate. Microtubule-dependent signaling networks that are perturbed in MTA-treated cells support novel roles for receptor tyrosine kinase (RTK) signaling pathways in mediating these effects. In the final study, we use microfabricated channels that replicate geometric and mechanical features of brain tissue alongside simulation-based methods to study confined glioma cell migration. Simulations recapitulate the dynamics of glioma cell migration in microchannels, as well as accurate predictions of the effects of MTAs and other pharmacological inhibitors of motor-clutch system components. This provides novel evidence for motor-clutch-based cell migration in confinement. In summary, this dissertation identifies specific mechanisms by which microtubules regulate motor-clutch based migration of glioma cells, and outlines a systems-level physics-based approach for understanding anti-motility therapy.Item MyTH4-FERM myosin based filopodia initiation(2020-07) Arthur, AshleyFilopodia are thin actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba and Myo10 in animals, are essential for initiation. DdMyo7 is localized to dynamic regions of the ac-tin-rich cortex. Analysis of VASP with altered activity reveals that localized actin polymerization is required for myosin recruitment and activation in Dic-tyostelium. Targeting of DdMyo7 to the cortex is not sufficient for filopodia ini-tiation; VASP activity is required as well. The actin regulator locally produces new actin filaments which activates a MF myosin. Myosin then shapes or crosslinks the actin network so parallel bundles of actin can extend during filo-podia formation. This work reveals cooperativity of an actin binding protein and the actin cytoskeleton on mediating myosin activity during filopodia initia-tion.Item Nucleotide- and protein-dependent functions of the mammalian cytoplasmic actins(2022-12) Sundby, LaurenThe mammalian cytoplasmic β- and γ-actin proteins are 99% identical but carry out unique biological functions. Essential functions of β-actin have been linked to protein-independent functions of the Actb nucleotide sequence rather than the protein. I generated a novel mouse line to determine if the γ-actin gene, Actg1, also supports protein-independent functions. Mice with a knocked out (KO) Actg1 gene expressing γ-actin from Actb were viable but had increased mortality despite expressing γ-actin protein at levels no different than control cells with normal survival. A survival defect in Actg1 KO mice expressing a constant level of γ-actin protein revealed an important role for the Actg1 nucleotide sequence. Cells isolated from these mice had normal proliferation rates while Actg1 KO cells devoid of γ-actin have impaired proliferation, suggesting a role for the γ-actin protein in cell growth. Together, these data identified unique roles for the Actg1 nucleotide sequence and γ-actin protein.Actb KO mice are embryonic lethal and Actb KO cells fail to proliferate. In contrast, mice and cells specifically lacking β-actin protein are viable with normal survival and normal proliferation, respectively. To determine why the Actb nucleotide sequence is essential for organism development and cell function I used RNA-sequencing and mass spectrometry to analyze the transcriptome, proteome, and phosphoproteome of Actb KO cells. I observed large scale changes in transcription, translation, and protein- phosphorylation in Actb KO cells, suggesting an important role for Actb in gene expression and protein modification. Analysis of differentially expressed transcripts and proteins in Actb KO cells identified the cell cycle as a significantly dysregulated pathway, implicating Actb as an important regulator of the cell cycle.Item The Role of Cellular Architecture in Vascular Smooth Muscle Function and Mechanics(2017-08) Win, ZawRecently, there has been a push towards clinical translation of biomechanical models of tissues by developing patient-specific models to predict disease outcomes. To accomplish this, it is necessary to understand the functional and mechanical properties of all the tissue components, including individual cells. In vasculature, tissues and cells have different structures based on their functional role. The principle goal of this work is to determine how cellular architecture influences function and mechanical properties. To test our hypotheses, we have developed in vitro models to study the relationship between structure and function at the tissue and cellular scale. We have developed microfluidic capture array device (MCAD) technology to study cell structure and function in 2D engineered vascular smooth muscle tissue and have developed cellular micro-biaxial stretching (CμBS) microscopy to determine single cell mechanical properties. First, using MCAD technology we were able to vary initial cell-cell contact during seeding to bias the cellular architecture in confluent vascular smooth muscle tissues. We found that tissues seeded using initially higher cell–cell contact conditions yielded tissues with more elongated cellular architecture which lead to greater contractile function in engineered tissues. We then used CμBS microscopy to determine the elastic anisotropic mechanical properties of individual cells, given by the strain energy density (SED) function. We found that smooth muscle cells (VSMCs) with native-like architectures are highly anisotropic and can be described by a SED based on the actin cytoskeletal organization. Then, we utilized CμBS microscopy to characterize loading and unloading mechanics of VSMCs. We found that VSMCs exhibit architecture-dependent anisotropic hysteresis where highly structured VSMCs exhibit typical hysteresis associated with viscous loss when stretched in the direction of actin fiber alignment but exhibit reverse hysteresis when stretched in the direction orthogonal to actin fiber alignment. We then modeled the observed hysteresis using two models: a quasi-linear (QLV) model and a Hill-type active fiber model and found that the QLV model was insufficient to characterize the anisotropic hysteresis but the Hill-type active fiber model was able to predict the anisotropic hysteresis in highly-organized VSMCs.Item Structure-Function Analysis of Motor Proteins: Insights from Conventional and Unconventional Myosins(2016-12) Petersen, KarlMyosin motor proteins play fundamental roles in a multitude of cellular processes. Myosin generates force on cytoskeletal actin filaments to control cell shape, most dramatically during cytokinesis, and has a conserved role in defining cell polarity. Myosin contracts the actin cytoskeleton, ensuring prompt turnover of cellular adhesion sites, retracting the cell body during migration and development, and contracting muscle among diverse other functions. How myosins work, and why force generation is essential for their function, is in many cases an open question. Chapter 2 presents a structure-function analysis of the amoebozoan myosin 7 (DdMyo7) in live Dictyostelium discoideum cells. DdMyo7 bears structural resemblance to human Myosin 7 (a protein involved in maintenance of the retina, stereocilia of the ear, and gut microvilli) but has functional similarity to human Myosin 10, a regulator of cell adhesion that is also essential in formation of actin-based structures called filopodia. Phylogenetic analysis of these related proteins shows that DdMyo7 is not directly related to any human myosin but rather represents a molecular ancestor of several vertebrate myosins (Myo7, Myo10 and Myo15). Functional analysis focused on rescue of myo7– cells. The two MyTH4-FERM domains were fully redundant in rescuing formation of filopodia. A conserved Myo7 regulatory motif in the C-terminal FERM domain was found to stimulate filopodia formation when mutated, establishing DdMyo7 as a filopodial motor with features of Myo7 and Myo10. A molecular chimera of DdMyo7 motor/lever arm region fused to the MF domain of human Myo10 partially rescued filopodia formation, suggesting the MF domain plays a similar role in filopodia in divergent organisms. Structural information must be combined with physiological data to understand the mechanism of myosin motor function. Structural studies have long focused on conventional myosin 2 as a model due to ease of protein expression and purification. This approach has yielded considerable data regarding the static structures and in vitro kinetics of the myosin mechanochemical cycle; however, high-resolution methods to observe the dynamics of myosin activation in cells have been lacking. Chapter 4 introduces methods and instrumentation for rapid, precise measurement of fluorescence lifetime. This is a necessary step toward Myo2-based live cell FRET sensors described in Chapter 5. Implications of this work for future studies of myosin physiological function are discussed in Chapter 6.