skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Super‐resolution analyzing spatial organization of lysosomes with an organic fluorescent probe
Abstract Lysosomes are multifunctional organelles involved in macromolecule degradation, nutrient sensing, and autophagy. Live imaging has revealed lysosome subpopulations with dynamics and characteristic cellular localization. An as‐yet unanswered question is whether lysosomes are spatially organized to coordinate and integrate their functions. Combined with super‐resolution microscopy, we designed a small organic fluorescent probe—TPAE—that targeted lysosomes with a large Stokes shift. When we analyzed the spatial organization of lysosomes against mitochondria in different cell lines with this probe, we discovered different distance distribution patterns between lysosomes and mitochondria during increased autophagy flux. By usingSLC25A46mutation fibroblasts derived from patients containing highly fused mitochondria with low oxidative phosphorylation, we concluded that unhealthy mitochondria redistributed the subcellular localization of lysosomes, which implies a strong connection between mitochondria and lysosomes.  more » « less
Award ID(s):
1955358
PAR ID:
10445420
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Exploration
Volume:
2
Issue:
3
ISSN:
2766-2098
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Ginsberg, Stephen D. (Ed.)
    Lysosomes play important roles in catabolism, nutrient sensing, metabolic signaling, and homeostasis. NPC1 deficiency disrupts lysosomal function by inducing cholesterol accumulation that leads to early neurodegeneration in Niemann-Pick type C (NPC) disease. Mitochondria pathology and deficits in NPC1 deficient cells are associated with impaired lysosomal proteolysis and metabolic signaling. It is thought that activation of the transcription factor TFEB, an inducer of lysosome biogenesis, restores lysosomal-autophagy activity in lysosomal storage disorders. Here, we investigated the effect of trehalose, a TFEB activator, in the mitochondria pathology of NPC1 mutant fibroblastsin vitroand in mouse developmental Purkinje cellsex vivo. We found that in NPC1 mutant fibroblasts, serum starvation or/and trehalose treatment, both activators of TFEB, reversed mitochondria fragmentation to a more tubular mitochondrion. Trehalose treatment also decreased the accumulation of Filipin+cholesterol in NPC1 mutant fibroblasts. However, trehalose treatment in cerebellar organotypic slices (COSCs) from wild-type andNpc1nmf164mice caused mitochondria fragmentation and lack of dendritic growth and degeneration in developmental Purkinje cells. Our data suggest, that although trehalose successfully restores mitochondria length and decreases cholesterol accumulation in NPC1 mutant fibroblasts, in COSCs, Purkinje cells mitochondria and dendritic growth are negatively affected possibly through the overactivation of the TFEB-lysosomal-autophagy pathway. 
    more » « less
  2. GM1 gangliosidosis is a lysosomal storage disorder caused by deficiency of β-galactosidase (βgal) and subsequent accumulation of GM1 ganglioside in lysosomes. One of the pathological aspects of GM1 gangliosidosis, and other storage disorders, is impaired autophagy, i.e., a reduced fusion of autophagosomes and lysosomes to degrade cellular waste. Enzyme replacement therapy (ERT) can effectively treat systemic deficiency but is limited by immunogenicity and shortened half-life of intravenously administered enzyme. In this paper, we report a hyaluronic acid-b-polylactic acid (HA-PLA) polymersome delivery system that can achieve an enzyme-responsive and sustained delivery of βgal to promote the cell’s self-healing process of autophagy. HA-PLA polymersomes have an average diameter of 138.0 ± 17.6 nm and encapsulate βgal with an efficiency of 77.7 ± 3.4%. In the presence of model enzyme Hyaluronidase, HA-PLA polymersomes demonstrate a two-fold higher release of encapsulant than without enzyme. We also identified reduced autophagy in a cellular model of GM1 Gangliosidosis (GM1SV3) compared to healthy cells, illustrated using immunofluorescence. Enhanced autophagy was reported in GM1SV3 cells treated with βgal-loaded polymersomes. Most notably, the fusion of lysosomes and autophagosomes in GM1SV3 cells returned to normal levels of healthy cells after 24 h of polymersome treatment. The HA-PLA polymersomes described here can provide a promising delivery system to treat GM1 Gangliosidosis. 
    more » « less
  3. Abstract Eukaryotic cells have evolved membrane-bound organelles, including the endoplasmic reticulum (ER), Golgi, mitochondria, peroxisomes, chloroplasts (in plants and green algae) and lysosomes/vacuoles, for specialized functions. Organelle quality control and their proper interactions are crucial both for normal cell homeostasis and function and for environmental adaption. Dynamic turnover of organelles is tightly controlled, with autophagy playing an essential role. Autophagy is a programmed process for efficient clearing of unwanted or damaged macromolecules or organelles, transporting them to vacuoles for degradation and recycling and thereby enhancing plant environmental plasticity. The specific autophagic engulfment of organelles requires activation of a selective autophagy pathway, recognition of the organelle by a receptor, and selective incorporation of the organelle into autophagosomes. While some of the autophagy machinery and mechanisms for autophagic removal of organelles is conserved across eukaryotes, plants have also developed unique mechanisms and machinery for these pathways. In this review, we discuss recent progress in understanding autophagy regulation in plants, with a focus on autophagic degradation of membrane-bound organelles. We also raise some important outstanding questions to be addressed in the future. 
    more » « less
  4. Abstract Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1. 
    more » « less
  5. This study introduces a rapid, volumetric live-cell imaging technique for visualizing autofluorescent sub-cellular structures and their dynamics by employing high-resolution Fourier light-field microscopy. We demonstrated this method by capturing lysosomal autofluorescence in fibroblasts and HeLa cells. Additionally, we conducted multicolor imaging to simultaneously observe lysosomal autofluorescence and fluorescently-labeled organelles such as lysosomes and mitochondria. We further analyzed the data to quantify the interactions between lysosomes and mitochondria. This research lays the foundation for future exploration of native cellular states and functions in three-dimensional environments, effectively reducing photodamage and eliminating the necessity for exogenous labels. 
    more » « less