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Published on: November 3, 2015
Localization of smoothelin in avian smooth muscle and identification of a vascular-specific isoform
X H Wehrens1, B Mies, M Gimona
1Department of Molecular Cell Biology and Genetics, University Maastricht, The Netherlands.
Abstract:
Smoothelin is a smooth muscle-specific protein of minor abundance first identified via a monoclonal antibody obtained using an avian gizzard extract as antigen. Dual labelling of ultrathin sections with antibodies to smoothelin together with antibodies to other smooth muscle proteins showed that smoothelin was co-distributed with filamin and desmin in the cytoskeleton domain of the smooth muscle cell. From the finding that smoothelin, unlike desmin, was readily extracted by Triton X-100 as well as under conditions that solubilized myosin, beta-actin and filamin, we conclude that smoothelin is most likely associated with the actin cytoskeleton. Western blot analysis of gizzard smooth muscle tissue revealed an immunoreactive protein band with an apparent molecular weight of 59 kDa that separated into 3-4 isolated variants, while avian vascular muscle showed a polypeptide band of 95 kDa. These results point to the presence of specific isoforms in visceral and vascular smooth muscles. The 59 kDa isoform was shown to be distinct from the 60 kDa filamin-binding protein, described by Maekawa and Sakai (FEBS Lett. 221, 68-72, 1987). As compared to other smooth muscle markers, such as calponin and SM22, smoothelin appeared very late during differentiation in the chick gizzard, on about the 18th embryonic day.
Insights
Smoothelin is a smooth muscle protein identified in avian gizzard. This protein is associated with the actin cytoskeleton and shows distinct isoforms in visceral and vascular smooth muscles.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Smoothelin is a smooth muscle-specific protein.
- It was initially identified using a monoclonal antibody against avian gizzard extract.
- Its precise function and localization within smooth muscle cells were not fully understood.
Purpose of the Study:
- To investigate the cellular localization and biochemical properties of smoothelin.
- To identify different isoforms of smoothelin in avian smooth muscle.
- To determine the developmental expression pattern of smoothelin.
Main Methods:
- Immunofluorescence microscopy using dual labeling with antibodies against smoothelin and other smooth muscle proteins (filamin, desmin).
- Biochemical extraction using Triton X-100 and other solubilizing agents.
- Western blot analysis of avian gizzard and vascular smooth muscle tissue.
- Comparison with known smooth muscle markers like calponin and SM22.
Main Results:
- Smoothelin co-distributes with filamin and desmin in the cytoskeleton of smooth muscle cells.
- Smoothelin is associated with the actin cytoskeleton, being extractable with Triton X-100.
- Western blots revealed a 59 kDa smoothelin isoform in gizzard smooth muscle and a 95 kDa isoform in vascular smooth muscle, indicating tissue-specific isoforms.
- Smoothelin expression appears late during embryonic development (around day 18 in chick gizzard).
Conclusions:
- Smoothelin is an actin cytoskeleton-associated protein in avian smooth muscle.
- Distinct isoforms of smoothelin are present in visceral and vascular smooth muscle.
- Smoothelin is a late-emerging marker of smooth muscle differentiation.
