Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Abstract

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

Characterisation of Aspergillus fumigatus Endocytic Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative Confocal Microscopy

“…Lysosomes are cytoplasmic organelles which play an essential function in the recognition and killing of intracellular infective microorganisms [56]. Recently a significant number of live-cell imaging technologies have been developed to quantify lysosome moieties using fluorescence probes (for review [57]), but little is known about lysosome dynamics during active fungal infection. Using GFP-LAMP-1 epithelial cell transduction, we determined for the first time the rate of lysosome depletion upon A. fumigatus spore infection.…”

Early onset effects of single substrate accumulation recapitulate major features of LSD in patient-derived lysosomes

“…In order to assess whether such a secretion defect could be due to an altered lysosome motility, HeLa cells stably expressing the lysosomal marker Lamp1-GFP were treated with LEU-ME for 2 hr and the movement of the lysosomes was tracked by time-lapse microscopy ( Tsuruta et al, 2017 ). Consistent with a previously reported study aimed at analyzing the dynamic properties of late endosomes and lysosomes ( Falcon-Perez et al., 2005 ), the behavior of Lamp1-GFP positive structures appeared as a combination of short- and long-range movement events in control cells ( Figure 4 A), with an average speed-track ranging from 0.3 to 1.3 μm/s ( Figure 4 B).…”

“…The entrapment of the enlarged lysosomes will hinder them from reaching the plasma membrane and in this way contribute to the lysosomal secretion defect observed in several LSDs, as well as in other different hereditary pathologies (Brandt et al, 1975;Park et al, 2016;Samie and Xu, 2014). In addition, the endo-lysosomal compartments are able to modulate their activities as a function of their positioning in the cell (Cabukusta and Neefjes, 2018;Wandinger-Ness and Zerial, 2014).…”

A MATLAB-based program for three-dimensional quantitative analysis of micronuclei reveals that neuroinflammation induces micronuclei formation in the brain

“…DAPI was purchased from DOJINDO. Primary neurons were prepared from E13.5–14.5 Crl:CD1(ICR) mice as previously described 37 . Briefly, mouse cortical neurons were plated on coverslips coated with 0.01875% Poly (ethylenimine) solution (SIGMA) and cultured in Neurobasal medium (Thermo Fisher Scientific) containing B-27 supplement (Thermo Fisher Scientific) and 100 units penicillin/100 mg streptomycin (P/S) (Thermo Fisher Scientific).…”