Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground‐breaking discoveries on an organelle that continues to surprise and excite cell biologists.

We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS.We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS.AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS.We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

The skin acts as a barrier to environmental insults and provides many vital functions. One of these is to shield DNA from harmful ultraviolet radiation, which is achieved by skin pigmentation arising as melanin is produced and dispersed within the epidermal layer. This is a crucial defence against DNA damage, photo‐ageing and skin cancer. The mechanisms and regulation of melanogenesis and melanin transfer involve extensive crosstalk between melanocytes and keratinocytes in the epidermis, as well as fibroblasts in the dermal layer. Although the predominant mechanism of melanin transfer continues to be debated and several plausible models have been proposed, we and others previously provided evidence for a coupled exo/phagocytosis model. Herein, we performed histology and immunohistochemistry analyses and demonstrated that a newly developed full‐thickness three‐dimensional reconstructed human pigmented skin model and an epidermis‐only model exhibit dispersed pigment throughout keratinocytes in the epidermis. Transmission electron microscopy revealed melanocores between melanocytes and keratinocytes, suggesting that melanin is transferred through coupled exocytosis/phagocytosis of the melanosome core, or melanocore, similar to our previous observations in human skin biopsies. We, therefore, present evidence that our in vitro models of pigmented human skin show epidermal pigmentation comparable to human skin. These findings have a high value for studies of skin pigmentation mechanisms and pigmentary disorders, whilst reducing the reliance on animal models and human skin biopsies.

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred-either in a membrane-bound melanosome or as a melanosome core, that is, melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1, CtBP1/ BARS, Cdc42 or RhoA, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct routes of melanin internalization. Furthermore, we show that melanocore uptake induces protease-activated receptor-2 (PAR-2) internalization by keratinocytes to a higher extent than melanosomes. Because skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore-based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.

Background and Purpose: Although endovascular treatment (EVT) for acute ischemic stroke is classified as I evidence, outcomes after EVT in real-world practice appear to be less superior than those in randomized clinical trials (RCT). Additionally, the effect of EVT is unclear compared with medical treatment (MT) for patients with mild symptoms defined by National Institutes of Health Stroke Scale score <6 or with severe symptoms defined by Alberta Stroke Program Early CT Score <6. Methods: Literatures were searched in big databases and major meetings from December 6, 2009, to December 6, 2019, including RCTs and observational studies comparing EVT against MT for patients with acute ischemic stroke. Observational studies were precategorized into 3 groups based on imaging data on admission: mild stroke group with National Institutes of Health Stroke Scale score <6, severe stroke group with Alberta Stroke Program Early CT Score <6 or ischemic core ≥50 mL, and normal stroke group for all others. Outcome was measured as modified Rankin Scale score of 0 to 2, mortality at 90 days, and symptomatic intracranial hemorrhage (sICH) at 24 hours. Results: Fifteen RCTs (n=3694) and 37 observational studies (n=9090) were included. EVT was associated with higher modified Rankin Scale 0 to 2 rate and lower mortality in RCTs and normal stroke group, whereas EVT was associated with higher sICH rate in normal stroke group, and no difference of sICH rate appeared between EVT and MT in RCTs. In severe stroke group, EVT was associated with higher modified Rankin Scale 0 to 2 rate and lower mortality, whereas no difference of sICH rate was found. In mild stroke group, there was no difference in modified Rankin Scale 0 to 2 rate between EVT and MT, whereas EVT was associated with higher mortality and sICH rate. Conclusions: Evidence from RCTs and observational studies supports the use of EVT as the first-line choice for eligible patients corresponding to the latest guideline. For patients with Alberta Stroke Program Early CT Score <6, EVT showed superiority over MT, also in line with the guidelines. On the contrary to the guideline, our data do not support EVT for patients with National Institutes of Health Stroke Scale score <6.