Relative Importance of Beta and Gamma Cytoplasmic Actins to Cellular and Organismal Viability

University of Minnesota Ph.D. dissertation. May 2018. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: James Ervasti. 1 computer file (PDF); viii, 171 pages.The highly homologous Actb and Actg1 are ubiquitously expressed and are hypothesized to carry out both redundant and unique functions, but studies using genetic knockout and transcript knockdown have yielded conflicting data. To elucidate the cause of this discrepancy, I characterized actin transcript and protein levels, and cellular phenotypes in both gene- and transcript-targeted primary MEFs. Gene targeting of Actb, but not Actg1, led to decreased cell proliferation, decreased cellular ATP levels, and increased serum response factor signaling in primary MEFs. However, SV40 largeT antigen transformed MEFs supported proliferation in the absence of Actb. Consistent with in vivo mouse studies, both gene and transcript targeting approaches demonstrated the loss of Actb is more disruptive to primary MEF function than is the loss of Actg1. Previous mouse models showed that Actb KOs are embryonically lethal while Actg1 KOs are viable. To determine whether the four amino acid differences between the cytoplasmic actins are essential for life, we generated a mouse model where the Actb gene is edited to encode γ-actin protein instead, an allele referred as Actbc-g. In contrast to the lethal phenotype of Actb KOs, homozygous Actbc-g mice were born at Mendelian ratios, do not exhibit early lethality, and Actbc-g MEFs displayed proliferation and random migration rates similar to WT. Nonetheless, Actbc-g mice showed progressive high frequency hearing loss and stereocilia degeneration as previously reported in the hair-cell specific Actb knockout mice. Thus β-actin protein is not universally required for normal cellular function, but is necessary for maintenance of auditory stereocilia

Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts

The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin

Essential nucleotide- and protein-dependent functions of Actb/β-actin

The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions

Engineering a hyperactive TcBuster transposase for efficient gene delivery for cell therapy applications

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The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel–Gruber syndrome, Joubert syndrome, Senor–Loken syndrome and Bardet–Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290

A Haptic-Assisted Guidance System For Working Machines Based on Virtual Force Fields

The goal of this work is proposing a methodology that demonstrates the ability to use perceptual stimuli to optimize or make it easy and intuitive manipulation. In particular we are addressing the problem of driving a vehicle that carries on board a manipulator arm, we are faced with two fundamental problems: the problem of navigation and the problem of the manipulation that between them are not uncorrelated. In fact, depending on the task that the manipulator must make, the position of the base has a central role to improve performance, reduce work times and the cognitive load of the operator. The user defines the working points after which the system helps him to better positioning of the base, there is no degree of autonomy, but simply have the stimuli that provide the operator with greater awareness. We study how these stimuli with respect to the user is able to position itself well. In particular, we tackled the problems of: • navigation and safety, that is limits within which to navigate • comprehension the workspace • optimal manipulation • nonholonomic The innovative aspect is to find the right combination of haptic signals such as force fields, barriers, signals and vibrations that man is better able to understand during the various phases in which the vehicle is to be found, thus ensuring better performance for complete the assigned task

Modellazione termo-fluidodinamica di un sistema di accumulo di energia termica (TESS - Thermal Energy Storage System)

Sviluppo di un modello dedicato alla previsione delle prestazioni di un mezzo di accumulo solido, il quale scambia calore con un fluido termovettore che fluisce all'interno di tubi ricavati nel mezzo stesso. Si è eseguita una modellazione termo-fluidodinamica, tramite l'utilizzo del software MATLAB, che ha permesso di studiare il comportamento termodinamico di uno storage che subisce nel tempo vari cicli di carica, stand-by e scarica fino al raggiungimento di una condizione stazionaria nel tempo (stato di stazionario ciclico)

A Haptic-Assisted Guidance System For Working Machines Based on Virtual Force Fields

This paper presents a new approach to improve the remote navigation of a teleoperated demolition machine. To improve navigation usability of the teleoperated system the following topics will be covered: obstacle avoidance, wall alignment, and machine optimal positioning for demolition operations. In order to enhance the ability of the operator to navigate in a cluttered environment, a Haptic-Assisted Guidance System(HAGS) will be introduced. HAGS is a guidance system based on haptic simulation which improves the awareness of the operator on the surrounding environment. The solution proposed has been tested using a virtual environment scenario and a commercial Haptic interface, the results have been analyzed and discussed