Visualization of lysosomal membrane proteins by cryo electron tomography

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• Lysosomes are vital organelles implicated in cellular homeostasis and signaling.
• Lysosomal dysfunction is linked to neurological disorders, lysosomal storage diseases, and cancer.
• Structural characterization of lysosomal membrane proteins in native environments is challenging.

Purpose of the Study:

• To develop a workflow for visualizing lysosomal membrane proteins in native lysosomal membranes using cryo-electron tomography.
• To structurally characterize key proteins within the lysosomal membrane.
• To establish a platform for studying lysosomal membrane protein function in health and disease.

Main Methods:

• Developed a cryo-electron tomography (cryo-ET) workflow.
• Isolated endolysosomes by targeting specific membrane proteins (TRPML1 and TMEM192).
• Utilized sub-tomogram averaging for structural refinement of membrane proteins.

Main Results:

• Successfully visualized lysosomal membrane proteins within intact, native lysosomal membranes.
• Identified and structurally refined key proteins like V-ATPase, Flotillin, and Clathrin.
• Revealed heterogeneous distribution of these proteins across endolysosomal organelles.

Conclusions:

• The integrated proteomics and structural biology workflow provides a powerful platform for studying lysosomal membrane proteins.
• This approach advances understanding of lysosomal function in health and disease.
• Paves the way for future discoveries in membrane-associated lysosomal mechanisms.