Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells.
This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca(2+) depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.
Eddinger
Thomas J
TJ
Biological Sciences, Marquette Univ, Milwaukee, WI 53233, USA. thomas.eddinger@marquette.edu
Schiebout
Jessen D
JD
Swartz
Darl R
DR
D022005:Adherens Junctions / Q000737:chemistry* / Q000502:physiology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods; D048430:Cell Shape / Q000187:drug effects / Q000502:physiology; D003106:Colon / Q000737:chemistry / Q000166:cytology; D003598:Cytoskeletal Proteins / Q000032:analysis*; D004285:Dogs; D005353:Fibronectins / Q000032:analysis; D007082:Ileum / Q000737:chemistry / Q000166:cytology; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000032:analysis*; D009130:Muscle, Smooth / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D051571:Protein Kinase C-alpha / Q000032:analysis; D011817:Rabbits; D013270:Stomach / Q000737:chemistry / Q000166:cytology; D016608:Talin / Q000032:analysis; D014132:Trachea / Q000737:chemistry / Q000166:cytology; D016596:Vinculin / Q000032:analysis
D003598:Cytoskeletal Proteins; D005353:Fibronectins; D008562:Membrane Glycoproteins; D016608:Talin; D016596:Vinculin; D015240:Phorbol 12,13-Dibutyrate; D002217:Carbachol; D051571:Protein Kinase C-alpha