Fluorescent staining of the actin cytoskeleton in human lymphocytes, monocytes and polymorphonuclear cells using a DNAse 1/anti-DNAse 1 immunoglobulin fluorescein conjugated system.

The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric "G") remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.

Cell surface energy and membrane associated actin in lymphocytes.

We have shown previously that membrane associated actin correlates with the migratory abilities of lymphocytes during recirculation, and that cell surface energy correlates with the adhesiveness of lymphocytes to other cells. In this study, measurements of actin content and cell surface energy have been made for various lymphocyte subpopulations to examine the possibility that recirculation ability may be related to nonspecific adhesiveness. We have found that: both cell surface energy and actin content combine to provide a consistent explanation for the relative rates of recirculation of various lymphocyte subpopulations, and cell surface energies and actin contents vary independently in these lymphocyte subpopulations. Comparison of the actin contents and cell surface energies of metastatic and nonmetastatic lymphoma cell lines indicated that the differences in metastatic potential were more likely attributable to specific receptor-ligand interactions than to nonspecific adhesiveness. Cell surface energy and actin content are consistent with the greater adhesiveness of B cells than T cells to nylon wool, providing a physical basis for this common cell separation technique.

Polymerized actin in lymphoid cells: functional interpretation.

Nitrobenzoxadiazol (NBD) phallacidin, an active fluorescent derivative of the actin-binding mushroom toxin phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeleton structures. Topographical fluorescent microscopy images of lymphoid cells obtained with NBD-phallacidin staining reveal that the major feature of the cellular cytoskeleton characterized by actin are mainly associated with cell membrane, a pattern that correlates strikingly with their DNAse 1 inhibition. Such actin pools may thus be involved in a membrane-associated protein network controlling membrane viscoplastic deformation and cell motility.

Actin activity is high in the immunocompetent fraction of thymocytes.

We have shown previously that actin activity of lymphocytes correlates with their migratory abilities. To determine whether any subpopulation of thymocytes would possess the potential for peripheral migration, sheep thymocytes were separated into four fractions by Percoll discontinuous gradient centrifugation and actin activity of each fraction was determined. The fraction enriched for immunocompetent medullary-type cells showed the highest actin activity among thymocytes, but the activity was significantly lower than that of peripheral lymphocytes. The implications of the observation in relation to thymocyte migration is discussed.

Distribution of membrane-associated actin in sheep lymphocytes. Functional implications.

Using the DNAase I inhibition assay, we titrated two forms of actin, monomeric (G) and total (G + F) actin of various populations of lymphocytes in adult and fetal sheep. Lymphocytes leaving a lymph node always showed higher actin activities than lymphocytes resident in the lymph node at the time of examination. T cells had significantly higher actin activities than B cells. A subpopulation of T cells which is present in the blood but not in the lymph showed much lower activities than total T cell populations in the blood or efferent lymph. Thymocytes showed the lowest actin activities among the lymphoid cell populations examined. A proposal is made that DNAase I inhibitions per cell can be considered as an intrinsic parameter of lymphocyte recirculating abilities.

DNase I inhibitions in tumors of different metastasizing capacities: a possible index of invasiveness.

Tumor pairs, selected on the basis of their different capacities to metastasize in vivo (SP73/AS and ASML from the rat, Eb/ESb from the mouse), have been assayed for their membrane associated actin through the DNase inhibition assay. It is found that, provided inhibitions per cell are corrected for the influence of gross heterogeneities in size distributions, the more metastatic tumor cells have significantly higher DNase I inhibitions than their less invasive counterparts. This observation, which extends our previous study of normal recirculating lymphocytes, is rationalized by postulating a participation of these actin pools to a property critical for both normal recirculation and metastatic spreading, arguments are presented which favor cell surface deformability as a possible candidate.

Distribution of actin content in human B and T lymphocytes by DNAse 1 inhibition test.

The DNAse inhibition assay, which allows for the titration of two forms of soluble actin in cell extracts, has been applied to normal human lymphocytes from peripheral blood. Actin-like activities associated with the membrane-rich fraction of extracts from thymus dependent (T) lymphocytes are significantly higher than those from bone marrow derived (B) lymphocytes, on a per cell basis, provided care is paid to the elimination of monocytic and polymorphonuclear cells which produce significantly higher DNAse 1 inhibition. The apparent discrepancy with a recent report that normal T and B lymphocytes do not differ in their total actin content is discussed with respect to the functional uniqueness of membrane-associated DNAse 1 inhibitions and the importance of using purified lymphocyte populations.

Actin content in lymphocytes: a proposed correlation with their recirculating properties.

The DNAase inhibition assay, which allows for the titration of monomeric and total actin content per cell, has been applied to various subpopulations of rat lymphocytes. We found that characteristic values are associated with functionally distinct subpopulations. Thus, thymus-dependent (T) lymphocytes have significantly higher monomeric and total actin per cell than bone marrow-derived (B) lymphocytes, irrespective of the separation procedure used (in vitro or in vivo). Moreover, these values appear to increase with the differentiation state of the cells within the T compartment (i.e., from thymus to lymph node to thoracic duct lymphocytes). The fact that the observed trends overlap the known in vivo recirculating abilities of the populations tested prompted us to discuss the possible involvement of intracellular actin in the control of the cell-surface deformability.

Lipid fluidity and membrane protein monitoring using 1,6-diphenyl-1,3,5-hexatriene.

Experiments have been designed to challenge the use of stedy-state fluorescence polarizaiton with 1,6-diphenyl-1,3,5-hexatriene as an evaluator of the fluidity of cell plasma membranes. We used paraffinic systems, of defined structure and composition (liquid paraffin, soap bilayers and phospholipid liposomes--with and without incorporated proteins), to demonstrate that corresponding polarizaiton values cannot be interpreted in terms of the overall fluidity of the labeled medium. In homogeneous structured paraffinic media (lipid bilayers), knowledge of the location of the probe is essential for a consistent interpretation of the observed fluorescence polarization. Due to the highly polarizable electronic structure of the diphenylhexatriene molecule, the presence of heterogeneities with potential sites for interaction (e.g., C18-coated Si particles, albumin molecules, etc.) can lead to relatively high polarization values, even in isotropic media. In cellular systems, translocation experiments from labeled cells to added proteins show a rather localized peripheral distribution of the probe as well as its high affinity for hydrophobic sites of proteins. This and other arguments presented here suggest that although cellular polarization values represent an intricate average over all labeled hydrophobic regions of the cell (phospholipid bilayers, membrane proteins, etc.), these values might reflect, to a large extent, interactions of the probe with proteins from the inner periphery of the cell.

Membrane associated proteins and malignancy: an experimental hypothesis.

Normal human lymphocytes and human tumor cell lines were labeled with an apolar fluorescent probe (1, 6-diphenyl-1, 3, 5-hexatriene), and fluoresence polarization (P) values were determined. The arguments presented in this study suggest that although P values represent an intricate average of all labeled hydrophobic regions of the cell (phospholipid bilayers, membrane proteins and enzymes, etc.), to a large extent, interactions of the probe with membrane proteins are of primary importance. The lower P values obtained with the tumor cell lines, compared with the normal lymphocytes, were interpreted as indicating alterations in either the structure or concentration of a membrane associated protein(s).

Study of platelet membrane proteins through fluorescence polarization of diphenyl hexatriene.

Fluorescence polarization (FP) of diphenylhexatriene (DPH) has been currently interpreted as cell membrane fluidity. We studied here FP of DPH labelled platelets under activation of membrane enzymes and concluded that FP of DPH could rather reveal interactions with hydrophobic pockets of membrane proteins.