Characterization of somatic nuclear actin and its role in the DNA damage response

• Main Author: Belin, Brittany J
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2014
• Subjects: Biology; Biophysics; Cellular biology
• ISBN: 1303849011; 9781303849015

Actin is an abundant eukaryotic protein with diverse and well-characterized cytoplasmic functions. Actin is also present in the nucleus, but its roles in that compartment have long been a subject of dispute. The primary reason for this debate is that common tools for labeling actin filaments in cells have yielded little information about the distribution, dynamics and functional form (e.g. filamentous or monomeric) of nuclear actin. To provide the first visualization of actin structures in the somatic nucleus, we generated and validated nuclear-localized fluorescent actin reporters. Monomeric actin probes concentrate at nuclear speckles, where they may participate in mRNA processing. Filamentous actin probes recognize discrete, punctate filaments with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: (1) diffusive, with an average diffusion coefficients of 0.06-0.08 μm 2/s or (2) subdiffusive, with a mobility coefficient of 0.015 μm 2/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh, suggesting that actin filaments form part of a large, viscoelastic structure in the nucleoplasm, and may act as scaffolds that help organize nuclear contents. Following induction of DNA damage, elongated actin filaments and actin filament aggregates form throughout the nucleoplasm and at peri- and intranucleolar regions. DNA damage-induced filaments are regulated by the poorly characterized formin-family nucleator, formin-2, and are required for efficient double-strand break signaling and nuclear oxidation during acute genotoxic stress. In addition to developing novel nuclear actin reporter constructs, we performed a systematic comparison of common actin reporters used to detect cytoplasmic filaments in vivo. Our analysis indicates that reporter binding preferences are governed largely by filament regulatory mechanisms rather than actin dynamics. We have also uncovered the previously undetected, yet often suggested, Golgi-associated actin filaments in Drosophila S2 cells.