In addition to filamentous actin (F-Actin) and intermediate filaments, microtubules (MTs) are core components of the cytoskeleton in all eukaryotic cell types, providing structure and shape to the cytoplasm (Pollard & Goldman, 2018). They are highly dynamic elements that mediate a variety of cellular functions, including cell division, cell migration, and transport (Fletcher & Mullins, 2010). MTs form the spindle apparatus during mitosis, which is essential to segregate the chromosomes (Petry, 2016). They constitute the internal structure of cilia and flagella (Loreng & Smith, 2017), underlying cell mobility
Microtubules are dynamic polymers of α/β-tubulin. They regulate cell structure, cell division, cell migration, and intracellular transport. However, functional contributions of individual tubulin isotypes are incompletely understood. The neuron-specific β-tubulin Tubb3 displays highest expression around early postnatal periods characterized by exuberant synaptogenesis. Although Tubb3 mutations are associated with neuronal disease, including abnormal inhibitory transmission and seizure activity in patients, molecular consequences of altered Tubb3 levels are largely unknown. Likewise, it is unclear whether neuronal activity triggers Tubb3 expression changes in neurons. In this study, we initially asked whether chemical protocols to induce long-term potentiation (cLTP) affect microtubule growth and the expression of individual tubulin isotypes. We found that growing microtubules and Tubb3 expression are sensitive to changes in neuronal activity and asked for consequences of Tubb3 downregulation in neurons. Our data revealed that reduced Tubb3 levels accelerated microtubule growth in axons and dendrites. Remarkably, Tubb3 knockdown induced a specific upregulation of Tubb4 gene expression, without changing other tubulin isotypes. We further found that Tubb3 downregulation reduces tubulin polyglutamylation, increases KIF5C motility and boosts the transport of its synaptic cargo N-Cadherin, which is known to regulate synaptogenesis and long-term potentiation. Due to the large number of tubulin isotypes, we developed and applied a computational model based on a Monte Carlo simulation to understand consequences of tubulin expression changes in silico. Together, our data suggest a feedback mechanism with neuronal activity regulating tubulin expression and consequently microtubule dynamics underlying the delivery of synaptic cargoes.
Cytoskeletal pattern formation and structural dynamics are key to a variety of biological functions and a detailed and quantitative analysis yields insight into finely tuned and well-balanced homeostasis and potential pathological alterations. High content life cell imaging of fluorescently labeled cytoskeletal elements under physiological conditions is nowadays state-of-the-art and can record time lapse data for detailed experimental studies. However, systematic quantification of structures and in particular the dynamics (i.e. frame-to-frame tracking) are essential. Here, an unbiased, quantitative, and robust analysis workflow that can be highly automatized is needed. For this purpose we upgraded and expanded our fiber detection algorithm FilamentSensor (FS) to the FilamentSensor 2.0 (FS2.0) toolbox, allowing for automatic detection and segmentation of fibrous structures and the extraction of relevant data (center of mass, length, width, orientation, curvature) in real-time as well as tracking of these objects over time and cell event monitoring.
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