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Evidence for fusion between multilamellar endosomes and autophagosomes in HeLa cells
Abstract:
The organelles of the endocytic and autophagic pathways were studied in HeLa cells using immunoelectron microscopy and stereological techniques. In the absence of autophagic stimulation, characteristic particulate structures containing closely packed layers of membrane received the fluid-phase marker horseradish peroxidase after 25 min uptake. These multilamellar endosomes contained the majority of the cellular immunogold labeling obtained by using a monoclonal antibody (1B5) directed against a lysosomal glycoprotein. Lysosomes with homogeneous dense content were only poorly labeled. After stimulation of autophagy, two classes of autophagosome profile appeared. One had a double limiting membrane and content that resembled the surrounding cytoplasm. The other most abundant type possessed a single limiting membrane and contained multilamellar structures which were strikingly similar to multilamellar endosomes, not only in form, but also in volume and membrane packing density. Immunogold labeling showed that now the majority of 1B5 labeling was located in the class of autophagosomes which contained multilamellar structures. Stereological methods showed that, after autophagic stimulation, multilamellar endosomes had become depleted, while multilamellar structures had appeared within the autophagosomes. Taken together, these data provide evidence that autophagosomes of HeLa cells fuse with preexisting multilamellar endosomes.
Insights
Autophagosomes in HeLa cells fuse with existing multilamellar endosomes, particularly after autophagy stimulation. This process involves the transfer of cellular components and is crucial for cellular recycling.
Area of Science:
- Cell Biology
- Molecular Biology
- Endosomal-Autophagic Pathways
Background:
- The endocytic and autophagic pathways are critical for cellular homeostasis and material degradation.
- Understanding the interplay between these pathways is essential for comprehending cellular function and disease.
Purpose of the Study:
- To investigate the relationship and potential fusion events between endosomes and autophagosomes in HeLa cells.
- To characterize the morphology and content of these organelles under basal and stimulated autophagic conditions.
Main Methods:
- Immunoelectron microscopy was employed to visualize cellular structures.
- Stereological techniques were used for quantitative analysis of organelle morphology and distribution.
- Horseradish peroxidase served as a fluid-phase marker to trace endocytic uptake.
- Immunogold labeling with a monoclonal antibody (1B5) against a lysosomal glycoprotein identified specific cellular components.
Main Results:
- Multilamellar endosomes were identified as major recipients of fluid-phase markers and contained significant lysosomal glycoprotein labeling under basal conditions.
- Upon stimulation of autophagy, a distinct class of autophagosomes emerged, characterized by a single membrane and containing multilamellar structures resembling endosomes.
- These autophagosomes with multilamellar structures showed increased lysosomal glycoprotein labeling, while preexisting multilamellar endosomes became depleted.
Conclusions:
- Autophagosomes in HeLa cells demonstrably fuse with preexisting multilamellar endosomes.
- This fusion event is a significant mechanism for the transfer of endosomal content into the autophagic pathway for degradation.
- The findings elucidate a key interaction between the endocytic and autophagic machinery in cellular material processing.