The role of differentiated vascular myocytes in neointimal formation in canine carotid artery was investigated. Using antibodies and cDNA probes, cells were characterized in situ and after isolation. In situ characterization indicated the majority of medial cells expressed both smooth muscle myosin and alpha actin but many cells were negative to these markers. All adventitial cells were negative for these proteins. The muscle protein-positive cells were designated differentiated, vascular myocytes (VSMC). The others were designated type 2 cells. Sequential enzyme digestion from the lumenal surface yielded VSMC ( Ͼ 90%) while digestions from the adventitial surface yielded type 2 cells ( Ͼ 90%). VSMC were viable in culture but did not spread, proliferate, or alter expression of muscle proteins. Type 2 cells proliferated and increased their expression of muscle actin but did not express muscle myosin. Characterization of neointimal cells from injured carotid arteries indicated they were morphologically and immunologically identical to cultured type 2 cells. We concluded that: ( a ) canine carotid artery media consists of a heterogeneous cell population; ( b ) serum does not stimulate isolated VSMC to undergo phenotypic modulation or proliferate; and ( c ) type 2 cells may be responsible for neointimal formation because they proliferate and acquire a phenotype identical to in situ neointimal cells. ( J. Clin. Invest. 1996. 97:814-825.)
Recent studies on the mobility of membrane markers on crawling cells indicate that there is no long-range centripetal flow of membrane proteins or lipids during cell locomotion. In this article we reflect on the history of ideas about membrane flow in cells, and we discuss how these new findings will shift the focus of research in cell locomotion away from the cell surface to the molecular interactions and dynamics of the actin cytoskeleton.
The distribution in living cells of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been studied by use of monoclonal antibodies. The presence of the molecule throughout the plasma membrane and on the substrate attached surface of the cell was demonstrated by immunofluorescence. Cell growth kinetics were not altered and the cells remained motile in the presence of the antibody. The uniform distribution of the direct immunofluorescence stain persisted for long periods (>100 h), which indicates that the fluorescent monoclonal antibodies may be used to trace antigen surface distribution during cell functions. In motile cells, but not Go or confluent cells, the degree of fluorescent staining decreased toward the leading edge; this gradient increased markedly during the time that the antibody was bound to the cells. However, the gradation was not seen with the lipid probe, dihexadecylindocarbocyanine. The antigen was "patched" only by the application of a second antibody directed to the rat monoclonal antibody and the relationships of these patches to the underlying cytoskeleton were characterized.
Abstract.A recently introduced extension of videoenhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-#m colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK: cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 ttm2/s. A diffusion coefficient in the 0.1 #mVs range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2/zm in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lameUoplasm and perinuclear cytoplasm at a velocity of 0.5-1 #m/min by a directed ATPdependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.
Abstract. A characteristic feature of fibroblast locomotory activity is the rearward transport across the leading lamella of various materials used to mark the cell surface. The two processes most frequently inyoked as explanations for this transport phenomenon, called capping, are (a) retrograde membrane flow arising from directed membrane insertion and (b) rearward cortical cytoskeletal flow arising from cytoskeletal assembly and contraction. The retrograde lipid flow hypothesis, the most current form of the membrane flow scheme, makes explicit predictions about the movement of membrane proteins subjected to the postulated rearward lipid flow. Several of these predictions were tested by comparing the behavior of four membrane proteins, Pgp-1, Thy-1, H-2, and influenza HA0, identified by fluorescent antibodies. With the exception of Pgp-1, these proteins were uniformly distributed under nonaggregated conditions but were capped when aggregated into patches. In contrast, Pgp-1 was capped in similar time frames in both nonaggregated and aggregated states where the lateral diffusion coefficients were very different. Furthermore, the capping behavior of two tagged membrane proteins was markedly different yet both had similar diffusion coefficients. The results from these tests disprove the bulk membrane flow hypothesis and are at odds with explicit predictions of the retrograde lipid flow hypothesis for the mechanism of capping. This work, therefore, supports the alternative cytoskeletal-based mechanism for driving capping. Requirements for coupling cytoskeletal movement to membrane components are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.