In particular, microtubule (MT) cytoskeleton and its associated proteins play crucial roles during this process 6,7. Neuronal morphogenesis, like other cellular events in which dynamic cellular asymmetries must be established and maintained, depends on the organization of multiple cytoskeleton systems 1–5. A functional nervous system depends on the intricate connections between neurites originated from different neurons. These polarized neurons contain long cellular protrusions called neurites that will later develop into axons or dendrites. During neuronal morphogenesis, post-mitotic neurons transform from their symmetrical shapes into highly polarized ones. Together, these data suggest a model in which Ran-dependent TPX2 activation promotes acentrosomal MT nucleation in neurons. Finally, applying a Ran-importin signaling interfering compound phenocopies the effect of TPX2 depletion on MT dynamics. Furthermore, overexpressing the downstream effector of Ran, importin, compromises MT formation and neuronal morphogenesis. Depleting TPX2 decreases MT formation frequency specifically at the tip and the base of the neurite, and these correlate precisely with the regions where active GTP-bound Ran proteins are enriched. In addition, TPX2 localizes to the centrosome and along the neurite shaft bound to MTs. First, neurite elongation is compromised in TPX2-depleted neurons. Here we provide evidence showing that TPX2 is an important component of this acentrosomal MT organizing center. The existence of the acentrosomal MT organizing center in neurons has been proposed but its identity remained elusive. Use cellular imaging analysis software to run quantitative analysis of the neuronal cell images to characterize several parameters including number of processes per cell, length of neurite outgrowth, branching, and number of cells.The microtubule (MT) cytoskeleton is essential for the formation of morphologically appropriate neurons. Analyze neuronal network – High content analysis provides a quantitative method to determine effects of positive and negative factors on neurite outgrowth.Acquire images with large field-of-view optics so more cells can be sampled with fewer sites per well, leading to dramatically faster plate acquisition times. Acquire neuronal images – High-content imaging of neurons allows scientists to both characterize and measure changes in neuronal networks such as neurite number, length, and branching, as well as to determine gross or specific toxicity reactions.Immunostaining protocols with fluorescently-conjugated antibodies can also be performed post-cell fixation. Stain for markers – After compound treatment is complete, live cell stains can be added directly to the media.Treat with compounds – the cells were then exposed to toxic compounds for 48 hours.Culture neuronal cells – cells were grown and allowed to form neurite networks in 96- or 384-well microplates.Inhibition or stimulation of neurite outgrowth is implicated in a broad range of CNS disorders or injuries including stroke, Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries. Understanding the signaling mechanisms driving neurite outgrowth provides valuable insight for interpreting neurotoxic responses and compound screening data and for interpreting factors influencing neural development and regeneration. Importantly, the development of neurons can be affected by neurotoxic chemicals. The growth of neurites can be stimulated or inhibited by neurotrophic factors. Development of neurites requires a complex interplay of both extracellular and intracellular signals. Neurite outgrowth is a commonly used assay to study neuronal development and neuronal degeneration in vitro. This biological phenomenon is referred to as neurite outgrowth and is regulated by complex intracellular signaling events. Neurons create connections via extensions of their cellular body called axons and dendrites, which are commonly referred to as “neurites” or “processes”. HIGH-THROUGHPUT, HIGH CONTENT SCREENING.PROTEIN DETECTION, QUANTITATION, ANALYSIS.NUCLEIC ACID (DNA/RNA) DETECTION & ANALYSIS.
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