Labeling Lysosomes in Live Cells with LysoTracker

Chazotte, Brad

doi:10.1101/pdb.prot5571

Cited by 105 publications

(94 citation statements)

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“…1D). Although we did not study in depth the intracellular localization of these exosomes in the HMECs, preliminary data using LysoTracker to stain lysosomes [53] indicated some colocalization, suggesting that some of the exosomes were in lysosomal compartments of HMECs after their uptake (data not shown).…”

Section: Resultsmentioning

confidence: 90%

Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

et al. 2014

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Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.

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Section: Resultsmentioning

confidence: 90%

Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

et al. 2014

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“…To do so, we incubated hippocampal slice cultures with LysoTracker red (50 nM, 40–60 min), a fluorescent dye that preferentially labels lysosomal compartments (Chazotte, 2011). We found LysoTracker-labeled puncta throughout the dendritic arbor of CA3 and CA1 neurons and in some dendritic spines (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

Activity-Dependent Exocytosis of Lysosomes Regulates the Structural Plasticity of Dendritic Spines

Padamsey

McGuinness

Bardo

et al. 2017

Neuron

151

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SummaryLysosomes have traditionally been viewed as degradative organelles, although a growing body of evidence suggests that they can function as Ca2+ stores. Here we examined the function of these stores in hippocampal pyramidal neurons. We found that back-propagating action potentials (bpAPs) could elicit Ca2+ release from lysosomes in the dendrites. This Ca2+ release triggered the fusion of lysosomes with the plasma membrane, resulting in the release of Cathepsin B. Cathepsin B increased the activity of matrix metalloproteinase 9 (MMP-9), an enzyme involved in extracellular matrix (ECM) remodelling and synaptic plasticity. Inhibition of either lysosomal Ca2+ signaling or Cathepsin B release prevented the maintenance of dendritic spine growth induced by Hebbian activity. This impairment could be rescued by exogenous application of active MMP-9. Our findings suggest that activity-dependent exocytosis of Cathepsin B from lysosomes regulates the long-term structural plasticity of dendritic spines by triggering MMP-9 activation and ECM remodelling.

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“…Aggregates of AO fluoresce bright red in the acidic environment of autophagic vacuoles, but fluoresce bright green in the cytoplasm and nucleus [Stankiewicz et al, 1996]. Lysotracker red labels acidic organelles (mainly lysosomes) [Chazotte, 2011]. Briefly, cells were incubated with 50 µmol/L MDC, 5 µg/ml AO, or 50 nmol/L lysotracker red for 15 min at 37°C, and then washed with PBS three times.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Luo

Wang

et al. 2012

J of Cellular Biochemistry

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Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert-butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12-Atg5 conjugate, Atg7 contents decreased but LC3-II accumulated in tBHP-treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ-1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions.

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Labeling Lysosomes in Live Cells with LysoTracker

Cited by 105 publications

References 0 publications

Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

Activity-Dependent Exocytosis of Lysosomes Regulates the Structural Plasticity of Dendritic Spines

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

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