Probing the role of nonmuscle tropomyosin isoforms in intracellular granule movement by microinjection of monoclonal antibodies.

Abstract

Abstract. Chicken embryo fibroblast (CEF) cells were microinjected with several different monoclonal antibodies that recognize certain nonmuscle isoforms of tropomyosin. Immediately after injection, cells were recorded with a time-lapse video imaging system; later analysis of the tapes revealed that particles in cells injected with one of these antibodies (CG1, specific for CEF tropomyosin isoforms 1 and 3) showed a dramatic decrease in instantaneous speed while moving, distance moved per saltation, and proportion of time spent in motion. Injection of Fab fragments of CG1 resulted in similar changes in the pattern of granule movement. This inhibition of granule movement by CG1 antibody was reversible; at 2.5 h after injection, granules in injected cells had already reached threefourths of normal speed. The speed of granule movement in cells injected either with antibody specific for tropomyosin isoforms not present in CEF cells, or with CG1 antibody preabsorbed with tropomyosin, was not significantly different from the speed of granules in uninjected cells. When cells were injected with CG1 or Fab fragments of CG1, fixed, and counter-stained with rabbit antibodies to reveal the microtubule, microfilament, and intermediate filament systems, no obvious differences from the patterns normally seen in uninjected cells were observed. Examination of the ultrastrncture of injected cells by EM confirmed the presence of apparently intact and normal microtubule, actin, and intermediate filament networks.These experiments suggest that tropomyosin may play an important role in the movement of vesicles and organelles in the cell cytoplasm. Also, we have shown previously that the CG1 determinant can undergo a motility-dependent change in reactivity, that may be important for the regulatory function of nonmuscle tropomyosin (Hegmann, T. E., J. L.-C. Lin, and J. J.-C. Lin. 1988. J. Cell Biol. 106:385-393). Therefore, in addition to postulated microtubule-based motors, microfilaments may play a critical role in regulating granule movement in nonmuscle cells.

Caldesmon and the Regulation of Cytoskeletal Functions

“…It was reported 197 that a monoclonal antibody against the C-terminal region of CaD inhibited granular movement in fibroblast cells, similar to the effect of anti-Tm antibodies, 198 once again indicating that both CaD and Tm are involved in the organization of the cytoskeleton and trafficking. Furthermore, CaD is associated with Grb2, Shc and Sos; in this complex CaD was found to be tyrosine-phosphorylated.…”

Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells

“…Monoclonal antibodies, CG1 and CG-␤6, were generated against chicken gizzard Tms and characterized previously (15,16,28,30). In addition to chicken Tms, the CG1 specifically recognizes human Tm isoform, hTm1, whereas the CG-␤6 recognizes the epitope in the COOH-terminal region of hTM2 and hTM3 (7,29,30).…”

“…For permeabilizing the cells we used 0.1% Triton X-100 in HBSS for 10 min. After blocking with 10% goat serum (for Tm1, ␣-actin), respectively, donkey serum (for Tm6), 0.05% Triton X-100, and 1% BSA for 30 min, cells were incubated, depending on the experiment, with the sheep polyclonal anti-Tm6 antibody (Sh X Tropomyosin, Smooth Muscle; Millipore) at 1:200 dilution, the mouse monoclonal anti-Tm1 antibody [CG1; (7,15,16,28)] at 1:500 dilution, the mouse monoclonal anti-␣-actin antibody (Sigma) at 1:10,000 dilution, or the mouse monoclonal anti-␤-actin antibody (Sigma) at 1:400 dilution overnight. For immunofluorescence staining of ␤-actin (single staining) and cytoplasmic ␥-actin, differentiated vascular smooth muscle cells were fixed with 1% paraformaldehyde for 20 min followed by ice-cold methanol for 3 min.…”

“…Residual protein-binding sites on the membrane were blocked with blocking buffer (Odyssey, LI-COR Biosciences, Lincoln, NB). The membranes were incubated either with the sheep polyclonal anti-Tm6 antibody (Sh X Tropomyosin) at 1:300 dilution, the mouse monoclonal anti-Tm1 antibody [CG1; (7,15,16,28)] at 1:2,000 dilution, the mouse monoclonal anti-Tm2 and Tm3 antibody [CG-␤6; (28,30)] at 1:500 dilution, the mouse monoclonal anti-Tm4 antibody [LC24; (57)] at 1:500 dilution, the mouse monoclonal anti-Tm5NM1 antibody [LC1; (53,57)] at 1:500 dilution, the sheep polyclonal ␣9d used at 1:400, the sheep polyclonal ␣2a used at 1:100, the sheep polyclonal ␥9d used at 1:100, the rabbit polyclonal ␦9d used at 1:250 , the rabbit monoclonal anti-␣-actin antibody (Epitomics) at 1:3,000 dilution, the mouse monoclonal anti-␤-actin antibody (Sigma) at 1:100 dilution or the mouse monoclonal anti-␥-actin antibody (Santa Cruz) at 1:500 dilution. As secondary antibodies, IRDye680-conjugated donkey anti-sheep IgG (Odyssey, LI-COR Biosciences) or IRDye800CM-conjugated goat anti mouse IgG (Odyssey, LI-COR Biosciences) were used at 1:1,000 dilution.…”

“…To reduce background, the antibodies used in this assay were cross-linked to protein G-coupled dynabeads (Invitrogen) using the cross-linking reagent Bis(sulfosuccinimidyl)suberate (BS 3) following the manufacturer's instructions. The precleared cell lysates were incubated for 16 h at room temperature with either the sheep polyclonal anti-Tm6 antibody (Sh X Tropomyosin) or the mouse monoclonal anti-Tm1 antibody [CG1; (7,15,16,28)], cross-linked to protein G-coupled dynabeads. As negative control, the mouse monoclonal anti-green fluorescent protein antibody (Clontech) cross-linked to protein G-coupled dynabeads was used.…”