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Fluorescent Protein Imaging Approaches Uncover Dynamic Subcellular Distribution of Core Clock Proteins in Neurospora crassa
No full textCircadian clocks are endogenous self-sustained timekeeping systems that allow organisms to anticipate and adapt to daily environmental cycles. Found in diverse forms of life, these molecular clocks regulate the expression of ~ 40% of the genome in animals, fungi, and plants. In animals and fungi, circadian oscillators are built on transcription-translation feedback loops (TTFLs), in which positive regulators drive the expression of negative regulators which, once synthesized and imported into the nucleus, inhibit their own activators. This precisely regulated negative feedback incorporates multiple steps of delays that are essential for generating ~24-hour rhythms. Through these mechanisms, circadian clocks temporally coordinate biological processes such as metabolism, growth, and behavior with predictable environmental cycles and thus provide evolutionary benefits.
Despite this conservation, how circadian clocks function in syncytial systems, where nuclei share a common cytoplasm, remains poorly understood. In filamentous fungi such as Neurospora crassa, the clock must generate coherent rhythms across a multinucleated network while maintaining the autonomy of individual nuclei. This presents unique challenges not encountered in single-cell systems and calls for new approaches to investigate the spatiotemporal organization of clock proteins. Therefore, the central question of this thesis is: how does a circadian clock operate within the syncytial environment of the Neurospora crassa hyphae and mycelium?
To address this, the research first developed technical platforms that enable visualization of core clock proteins at endogenous levels to allow long-term live-cell imaging over multiple circadian cycles. Codon-optimized fluorescent proteins were introduced to overcome the intrinsic challenges of low protein abundance and fungal-specific expression constraints. We provide a toolkit for multi-color and photoconvertible imaging of circadian regulators, establishing a foundation for quantitative single-cell circadian biology in filamentous fungi. Building on these tools, optimized culture conditions and microfluidic systems were established to support continuous imaging across circadian timescales. These advances extended the observation window from hours to days and allowed tracking of proteins within individual nuclei as the syncytial network developed.
The resulting platform revealed how nuclei within a common cytoplasm produce coherent circadian outputs despite variability in transcriptional activity. Direct visualization demonstrated that nuclear localization of FRQ, the central negative regulator, cycles robustly in across all nuclei. Local sharing of multiple clock proteins among nuclei was documented, and this facilitates coordinated timing within the complex syncytial environment. Within nuclei, circadian proteins form small, dynamic nuclear bodies that occasionally co-localize, suggesting a role for subnuclear organization in coordinating rhythmic regulation.
Finally, the mechanisms of nucleocytoplasmic transport were investigated as a conserved step important for circadian rhythms. FRQ nuclear import was found to be strongly regulated across the circadian cycle, peaking in the early subjective day. The relatively slow import rate parallels that of mammalian PER proteins, suggesting shared delay strategies for generating ~24-hour periodicity. FRQ import depends on rhythmic binding with Importin α but not through the previously predicted nuclear localization sequence, and the nuclear entry of WC-1 also requires Importin α. Distinct Importin βs contributes uniquely to clock function, including a potential connection between Importin β3 and a phosphatase, highlighting selective transport as a key regulatory step in circadian clocks.
Together, this work establishes one of the first systematic cell biological investigations of a syncytial circadian clock. By building the imaging platforms necessary to monitor clock proteins in living fungal networks and applying these platforms to mechanistic questions of nuclear coordination and transport, the thesis provides both technical resources and conceptual insights. Research advances in Neurospora crassa deepen our understanding of fungal biology, reveal general principles of coherent temporal regulation in multinucleated systems, and continue to contribute to chronobiology research
Regulation and Disruption of Microtubule Cytoskeleton Dynamics in Peripheral Sensory Neuropathy
No full textPeripheral neuropathy is a wide-spread, growing public health issue, with poor clinical outcomes and a lack of available regenerative treatments. A major limitation to the development of novel therapies is poor understanding of the molecular mechanisms driving regeneration in peripheral neurons. One factor of interest in peripheral neuropathy is the tumor-suppressor phosphatase Pten. Pten knockout has been previously shown to promote axon regeneration in a variety of neuronal subtypes, and to promote functional recovery in animal models of peripheral nerve injury. One of Pten\u27s downstream targets is the microtubule cytoskeleton, and past work from our lab has found Pten-KO accelerates microtubule polymerization rates in the axonal growthcone. However, Pten has an extremely broad range of downstream effectors, and it remains unclear whether Pten-KO drives growth via the microtubule cytoskeleton or through an independent mechanism. Here, we isolate the cytoskeletal effects of Pten through a series of genetic manipulations, and find that Pten-KO appears to drive distal axonal elongation in peripheral sensory neurons via the microtubule cytoskeleton. We also show evidence for differential genetic regulation of microtubule dynamics across subaxonal compartments. Next, we extend these findings to a mouse model of diabetic neuropathy, the leading cause of peripheral neuropathy in the U.S. We build on previous studies showing that Pten is upregulated in diabetic neurons, and find that microtubule polymerization is suppressed in the growthcones of diabetic neurons. Interestingly, we find that Pten knockout has no effect on the microtubule cytoskeleton in these neurons, even though it still increases cellular-level growth. Our results suggest that diabetes impacts the microtubule cytoskeleton, which could contribute to the inhibited regeneration seen in diabetic neuropathy, via a Pten-independent mechanism. We also show that Pten-KO drives neuronal growth in diabetic neurons via a MT-independent mechanism. Overall, our results further elucidate the effects of Pten on the MT cytoskeleton in peripheral neurons, and highlight the MT cytoskeleton as a relevant, promising clinical target for the development of regenerative therapies in diabetic neuropathy
