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Lysosomes are highly dynamic degradation/recycling organelles that harbor sophisticated molecular sensors and signal transduction machinery through which they control cell adaptation to environmental cues and nutrients. The movements of these signaling hubs comprise persistent, directional runs-active, ATP-dependent transport along the microtubule tracks-interspersed by short, passive movements and pauses imposed by cytoplasmic constraints. The trajectories of individual lysosomes are usually obtained by time-lapse imaging of the acidic organelles labeled with LysoTracker dyes or fluorescently-tagged lysosomal-associated membrane proteins LAMP1 and LAMP2. Subsequent particle tracking generates large data sets comprising thousands of lysosome trajectories and hundreds of thousands of data points. Analyzing such data sets requires unbiased, automated methods to handle large data sets while capturing the temporal heterogeneity of lysosome trajectory data. This chapter describes integrated and largely automated workflow from live cell imaging to lysosome trajectories to computing the parameters of lysosome dynamics. We describe an open-source code for implementing the continuous wavelet transform (CWT) to distinguish trajectory segments corresponding to active transport (i.e., "runs" and "flights") versus passive lysosome movements. Complementary cumulative distribution functions (CDFs) of the "runs/flights" are generated, and Akaike weight comparisons with several competing models (lognormal, power law, truncated power law, stretched exponential, exponential) are performed automatically. Such high-throughput analyses yield useful aggregate/ensemble metrics for lysosome active transport.

Lysosomes—that is, acidic organelles known for degradation/recycling—move through the cytoplasm alternating between bursts of active transport and short, diffusive motions or even pauses. While their mobility is essential for lysosomes’ fusogenic and non-fusogenic interactions with target organelles, their movements have not been characterized in adequate detail. Here, large-scale statistical analysis of lysosomal movement trajectories reveals that lysosome trajectories in all examined cell types—both cancer and noncancerous ones—are superdiffusive and characterized by heavy-tailed distributions of run and flight lengths. Consideration of Akaike weights for various potential models (lognormal, power law, truncated power law, stretched exponential, and exponential) indicates that the experimental data are best described by the lognormal distribution, which, in turn, can be related to one of the space-search strategies particularly effective when “thorough” search needs to balance search for rare target(s) (organelles). In addition, automated, wavelet-based analysis allows for co-tracking the motions of lysosomes and the cargos they carry—particularly the nanoparticle aggregates known to cause selective lysosome disruption in cancerous cells. The methods we describe here could help study nanoparticle assemblies, viruses, and other objects transported inside various vesicle types, as well as coordinated movements of organelles/particles in the cytoplasm. Custom-written code that includes integrated workflow for our analyses is made available for academic use.