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Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells
Published on: February 18, 2014
Restructured endoplasmic reticulum generated by mutant amyotrophic lateral sclerosis-linked VAPB is cleared by the
Giulia Papiani1, Annamaria Ruggiano, Matteo Fossati
1Consiglio Nazionale delle Ricerche Institute of Neuroscience and Department of Pharmacology, University of Milan, Milano, Italy.
Abstract:
VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56S-VAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein.
Insights
Mutant VAPB protein forms unique cellular inclusions linked to ALS8. These inclusions are cleared by the proteasome, not autophagy, and their formation and clearance mechanisms differ from other neurodegenerative disease aggregates.
Area of Science:
- Cell Biology
- Neuroscience
- Protein Biochemistry
Background:
- Vesicle-associated membrane protein-associated protein B (VAPB) is an ER-resident protein crucial for lipid exchange.
- A mutant form, P56S-VAPB, is associated with amyotrophic lateral sclerosis type 8 (ALS8) and forms intracellular inclusions.
- The exact role and cellular handling of these P56S-VAPB inclusions in ALS pathogenesis remain unclear.
Purpose of the Study:
- To investigate the dynamic processes of P56S-VAPB inclusion formation and degradation.
- To elucidate the cellular mechanisms involved in the clearance of these unique protein aggregates.
- To compare the biogenesis and clearance of P56S-VAPB inclusions with those found in other neurodegenerative diseases.
Main Methods:
- Utilized stable HeLa-TetOff cell lines for inducible expression of wild-type and mutant VAPB.
- Employed microinjection techniques in non-transfected cells to study inclusion dynamics.
- Analyzed the role of the proteasome, macro-autophagy, and the AAA ATPase p97/VCP in inclusion clearance.
Main Results:
- P56S-VAPB rapidly forms small, polyubiquitinated clusters post-synthesis, which aggregate juxtanuclearly.
- Inclusion clearance occurs rapidly upon cessation of mutant protein synthesis, with degradation rates higher than wild-type VAPB.
- Inclusion clearance primarily involves the proteasome, with no significant role for macro-autophagy; p97/VCP appears crucial for aggregate extraction.
Conclusions:
- P56S-VAPB inclusions exhibit distinct formation and clearance mechanisms compared to other neurodegenerative disease aggregates.
- The proteasomal degradation pathway and p97/VCP activity are critical for resolving these inclusions.
- Understanding these unique cellular dynamics may offer new insights into the pathogenic mechanisms of ALS8.
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