Measuring Intracellular Vesicle Density and Dispersion Using Fluorescence Microscopy and ImageJ/FIJI

Background

Intracellular vesicles are important components of the endocytic and secretory pathways, which are responsible for maintaining several critical cellular functions. For this reason, many studies have focused on the pathways that regulate the distribution of vesicles in both physiological and pathological conditions, while others have examined the consequences of aberrant vesicle distribution in many important cellular processes.

In 1988, Aunis and Bader showed that two pools of secretory vesicles exist in secretory cells (Aunis and Bader, 1988). The first pool was located just below the plasma membrane and the secretion of this pool was not regulated by the cytoskeleton, but attached to the plasma membrane as a result of being bound to elements of the cytoskeleton; the second pool was attached to actin filaments, slightly away from the membrane, which could be mobilized to the inner leaflet of the plasma membrane after the depletion of the first pool. This was achieved by depolarization of the membrane, rearrangement of the actin filaments and dissolution of the cytoskeleton barrier, leading to its detachment from the cytoskeleton to reach the exocytic sites.

Koseoglu et al. (2011) also provided evidence for the existence of different pools of secretory vesicles and that membrane cholesterol content is important for exocytosis of these pools. The authors observed that membrane cholesterol sequestration from chromaffin cells of the adrenal medulla did not alter either the kinetics or the amount of release of the first pool of vesicles, which was already pre-anchored to the plasma membrane. However, the subsequent release of the remaining vesicles, belonging to the second slow-releasing pool, also called the reserve pool, was compromised by changes in membrane cholesterol levels. The authors further suggested that the change in the polymerization of actin filaments induced by cholesterol depletion could be compromising the mobilization of the slow releasing or reserve vesicles.

Mobilization of lysosome pools is described in several situations (Brito et al., 2019; Lawrence and Zoncu, 2019). One example is plasma membrane repair (PMR), a mechanism widely used by nucleated cells to maintain plasma membrane integrity. Briefly, when a cell suffers a micro-injury, its membrane must be sealed to avoid depolarization, cytoplasm leakage and cell death. Upon injury, extracellular Ca²⁺ flows through the lesions into the cytoplasm, increasing cytoplasmic Ca²⁺ concentration, which in turn induces lysosomal exocytosis. Lysosomal exocytosis is then followed by a massive endocytosis, which reseals the plasma membrane by carrying wounded portions in endosomes into the cell. The whole process involves the participation of lysosomal hydrolases, which act on the extracellular leaflet of the plasma membrane and facilitates the endocytic process by inducing plasma membrane invagination and its inward budding (Idone et al., 2008; Tam et al., 2010; Castro-Gomes et al., 2016).

Some parasites, such as Trypanosoma cruzi and Leishmania amazonensis, can subvert PMR to invade different host cells (Horta et al., 2020). Tardieux et al. (1992) verified that the mobilization of lysosomes is a key event for effective cell infection by T. cruzi, which subverts PMR mechanism to invade cells. Hissa et al. (2012), studying how the protozoan parasite T. cruzi enters cardiomyocytes, showed that depletion of membrane cholesterol led to a decrease in host cell invasion by interfering with lysosome recruitment and fusion during parasite-host cell interaction. More recently, Cavalcante-Costa et al. (2019) showed that lysosomal positioning is also crucial for L. amazonensis invasion to non-phagocytic cells, using basically the same mechanism previously described for T. cruzi.

Therefore, lysosomal positioning is crucial for PMR and cell invasion by some pathogens, as well as many other cellular mechanisms. These vesicles can be found around cell nuclei or spread throughout the cell, moving fast along cell microtubules towards the cell periphery in a stop-and-go manner (Cabukusta and Neefjes, 2018), depending on cell type or special conditions, such as the presence of certain drugs or plasma membrane injury by cytotoxins. Since lysosomes function as Ca²⁺ sensitive vesicles (Rodriguez et al., 1997), their positioning and exocytosis are finely regulated by the presence of this ion in the cytosol.

The examples above show that the distribution of intracellular vesicles can be disturbed by several conditions and that their correct positioning is pivotal for physiological cellular functions. Thus, it is critical to have a reliable method to measure the distribution and movement of intracellular vesicles. Here we describe a method to quantify lysosomes in cells that takes advantage of immunofluorescence, a commonly-used technique, and open source software ImageJ/FIJI (Schneider et al., 2012; Schindelin et al., 2012). This protocol provides a way for researchers to compare changes in the distribution of lysosomes in diverse processes and it can be adapted to studies involving different types of intracellular vesicles such as endosomes, Golgi vesicles or secretory granules.