Abstract
Subcellular protein delivery is especially important in signal transduction and cell behavior, and is typically achieved by localization signals within the protein. However, protein delivery can also rely on localization of mRNAs that are translated at target sites. Although once considered heretical, RNA localization has proven to be highly conserved in eukaryotes. RNA localization and localized translation are especially relevant in polarized cells like neurons where neurites extend dozens to hundreds of centimeters away from the soma. Local translation confers dendrites and axons the capacity to respond to their environment in an acute manner without fully relying on somatic signals. The relevance of local protein synthesis in neuron development, maintenance and disease has not been fully acknowledged until recent years, partly due to the limited amount of locally produced proteins. For instance, in hippocampal neurons levels of newly synthesized somatic proteins can be more than 20-30 times greater than translation levels of neuritic proteins. Thus local translation events can be easily overlooked under the microscope. Here we describe an object-based analysis used to visualize and quantify local RNA translation sites in neurites. Newly synthesized proteins are tagged with puromycin and endogenous RNAs labeled with SYTO. After imaging, signals corresponding to neuritic RNAs and proteins are filtered with a Laplacian operator to enhance the edges. Resulting pixels are converted into objects and selected by automatic masking followed by signal smoothing. Objects corresponding to RNA or protein and colocalized objects (RNA and protein) are quantified along individual neurites. Colocalization between RNA and protein in neurites correspond to newly synthesized proteins arising from localized RNAs and represent localized translation sites. To test the validity of our analyses we have compared control neurons to Aβ1 - 42-treated neurons. Aβ is involved in the pathology of Alzheimer's disease and was previously reported to induce local translation in axons and dendrites which in turn contributes to the disease. We have observed that Aβ increases the synthesis of neuritic proteins as well as the fraction of translating RNAs in distal sites of the neurite, suggesting an induction of local protein synthesis. Our results thus confirm previous reports and validate our quantification method.
Keywords:
FIJI/ImageJ analyses; RNA localization; colocalization analyses; fluorescence microscopy; local protein synthesis; neurites.