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Updated: Mar 30, 2026
Cell Membrane Repair Assay Using a Two-photon Laser Microscope
Published on: January 2, 2018
Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair
Chie Matsuda1, Kazuyuki Kiyosue2, Ichizo Nishino3
1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan; Department of Neurophysiology, Tokyo Medical University, Shinjuku, Tokyo 160-8402, Japan; Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan.
Background:
Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy.
Objectives:
The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy.
Methods:
Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts.
Results:
Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation.
Conclusion:
Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy.
Insights
Dermal fibroblasts from dysferlinopathy patients and SJL mice exhibit impaired membrane repair, offering a viable model for studying dysferlinopathies. This research highlights fibroblasts as a valuable tool for understanding these muscular dystrophies.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Dysferlin, a sarcolemmal protein, is crucial for membrane repair and its defect causes Miyoshi myopathy and limb-girdle muscular dystrophy type 2B.
- Myoblasts and myotubes from patients are commonly used to study dysferlinopathy mechanisms.
Purpose of the Study:
- To investigate the potential of using dermal fibroblasts from dysferlin-deficient patients and SJL mice as a model system for dysferlinopathy research.
Main Methods:
- Dysferlin protein expression was assessed in patient and mouse fibroblasts via immunoblotting and immunocytochemistry.
- Membrane repair capacity was evaluated using a membrane wound-healing assay with confocal microscopy.
- Membrane blebbing response to hypotonic shock was analyzed in human and mouse fibroblasts.
Main Results:
- Fibroblasts from dysferlinopathy patients and SJL mice showed significantly reduced membrane repair capabilities and failed to exhibit membrane blebbing under hypotonic stress.
- Proteasomal inhibition partially restored membrane blebbing in fibroblasts with missense or truncated dysferlin mutations but not with nonsense or frameshift mutations.
Conclusions:
- Dermal fibroblasts from dysferlinopathy patients and SJL mice demonstrate impaired plasma membrane repair.
- These fibroblasts represent a promising and accessible cellular model for investigating dysferlinopathy.
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