ABSTRACT
The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.
Subject(s)
Mechanotransduction, Cellular , Nuclear Envelope/physiology , Actomyosin/metabolism , Animals , Cell Movement , Embryonic Development , HeLa Cells , Humans , Mice , Myosin Heavy Chains/metabolism , Neoplasm Invasiveness , Neoplasms/pathologyABSTRACT
In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.
Subject(s)
Colonic Neoplasms/therapy , Oncolytic Virotherapy , Oncolytic Viruses/genetics , Semliki forest virus/genetics , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bystander Effect , Cell Line, Tumor , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Genetic Vectors , Green Fluorescent Proteins/biosynthesis , Interferon Type I/pharmacology , Interferon Type I/therapeutic use , Interferon-beta/metabolism , Mice, Inbred BALB C , Necrosis , Neoplasm Transplantation , STAT1 Transcription Factor/metabolism , Transfection , Treatment OutcomeABSTRACT
Endogenous activities of phospholipases A and C in Ureaplasma urealyticum were assayed in cellular fractions of exponential-phase cells. Enzymatic studies indicated that ATPase activity was localized in the plasma membrane fraction and NADH and NADPH dehydrogenase activities were localized in the cytosol fraction. Studies with purified ureaplasma membranes demonstrated that, of three serovars tested, endogenous phospholipase A1, A2, and C activities were localized in the plasma membrane. Very low levels of activity were observed in the cytosol fractions. Phospholipase A2 activity in the plasma membrane was 3- to 5-fold higher than the activity in the lysates and 60- to 300-fold higher than the activity of phospholipase A1. Phospholipase C was localized mainly in the plasma membrane, with 20% found in the cytosol fraction. The levels of activity were comparable among the three serovars. There was a significantly lower level of activity in cells from the stationary growth phase than in the exponential phase. Significant differences were observed in the phospholipase A activities among the U. urealyticum serovars 3, 4, and 8. Phospholipase A2 activity was twofold higher in serovar 8 membranes, and phospholipase A1 activity was twofold higher in serovar 3 membranes. These results demonstrate that endogenous activities of phospholipase A and C are localized primarily in the plasma membrane fraction of U. urealyticum. The specific activities in the membranes of the phospholipases varied among the three serovars. Phospholipase enzymes may function as virulence factors in U. urealyticum and may vary among the serovars.
Subject(s)
Phospholipases A/metabolism , Type C Phospholipases/metabolism , Ureaplasma/enzymology , Cell Membrane/enzymology , Cytosol/enzymology , Histocytochemistry , Humans , Phospholipases A1 , Phospholipases A2 , Ureaplasma/pathogenicity , Ureaplasma/ultrastructure , Virulence/physiologyABSTRACT
A screening assay for phospholipase C using a chromogenic substrate incorporated into agar medium is described. The assay directly visualizes phospholipase C activity of mycoplasma lysates and membranes on agar plates, or the activity may be measured by spectrophotometry. The results from the assay confirm the presence in Ureaplasma urealyticum of phospholipase C, which is predominantly localized in the membrane fraction. The procedure has the potential to screen phospholipase C activity in other mycoplasmas and microorganisms in general.
Subject(s)
Type C Phospholipases/metabolism , Ureaplasma/enzymology , Culture Media , Substrate SpecificityABSTRACT
The results of recent studies support the concept that Ureaplasma urealyticum may be a major cause of perinatal infection in both term and preterm infants. It has been postulated that phospholipase degradation of placental phospholipids by microorganisms triggers the onset of premature labor. Since the presence of ureaplasmas in placentas is associated with pregnancy loss, prematurity, and neonatal morbidity, we assayed U. urealyticum for the presence of phospholipase A1, A2, and C activities. Phospholipase A1 activity was low in lysates of exponential-phase cells of U. urealyticum. Phospholipase A2 activity was present and was 100-fold higher than the activity of phospholipase A1 in serotypes 3,4, and 8. The total activity and specific activity of phospholipase A2 in serotype 8 were nearly threefold higher than the activities in serotypes 3 and 4. Cell lysates of all three serotypes showed the presence of phospholipase C activity during the exponential phase of growth, and no significant difference in activity was observed among the three serotypes. In stationary-phase cells the phospholipase C activity was 10-fold lower than the activity in exponential-phase cells. Our results demonstrate that phospholipase activities are present in U. urealyticum cells and that the specific activities of phospholipase A2 differed among the three serotypes tested, while the activities of phospholipases A1 and C were similar.
Subject(s)
Phospholipases A/metabolism , Phospholipases/metabolism , Type C Phospholipases/metabolism , Ureaplasma/enzymology , Humans , Phospholipases A1 , Phospholipases A2 , Phospholipids/metabolism , Substrate Specificity , Ureaplasma/growth & developmentABSTRACT
We have previously reported that synthesis of phospholipids increases 4-fold at the onset of chemotactic migration during development of Dictyostelium discoideum and that the newly synthesized phospholipids are preferentially incorporated into the plasma membrane (De Silva, N. S. and Siu, C-H. (1980) J. Biol. Chem. 255, 8489-8496). To test the hypothesis that the rapid transfer of phospholipids to the plasma membrane is mediated by vesicles, we isolated phospholipid-rich vesicles from cells at 6 h of development. These vesicles had an average size of 0.35 micrometer in diameter. They banded at a density of 1.097 g/cm3 and they had a phospholipid: protein (w/w) ratio of 2.25. The predominant classes of phospholipids in these vesicles were phosphatidylethanolamine and phosphatidylcholine. In pulse-labeling studies using [3H]glycerol, these low density vesicles had the highest phospholipid-specific activity, which was about 3 times higher than that of 6-h plasma membranes. Almost 80% of the incorporated radioactivity was found to be associated with phosphatidylethanolamine and phosphatidylcholine. When cells were chased with cold precursor after pulse labeling, the specific activity of these vesicles dropped by almost 20-fold in 90 min, while plasma membranes showed a 2.5-fold increase in 60 min. Addition of colchicine to 7-h cells inhibited the translocation of newly synthesized phospholipids to the plasma membrane. The low density vesicles were found in much reduced amounts in preaggregation stage cells or the aggregateless mutant WL3. These results indicate that transfer of newly synthesized phospholipids from their site of synthesis to the plasma membrane probably occurs through a special class of phospholipid-rich vesicles.
Subject(s)
Dictyostelium/metabolism , Membrane Lipids/metabolism , Phospholipids/metabolism , Cell Fractionation , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Dictyostelium/growth & development , Microscopy, ElectronABSTRACT
Synaptic membranes were isolated from the forebrains of rats of increasing postnatal ages. Developmentally related changes in the structure and concnetration of synaptic membrane glycoproteins were indicated by: (1) a 2--3 fold increase in glycoprotein sialic acid between 5 and 60 days; (2) a similar increase in the number of membrane receptors for the lectins concanavalin A and wheat germ agglutinin; (3) transient increases between 10 and 17 days in the receptors for lentil and castor bean lectins and (4) an age dependent stimulation of castor bean lectin binding by neuraminidase. Labelling of SDS polysacrylamide gels of synaptic membranes with [125I]concanavalin A or wheat germ agglutinin revealed specific, age dependent changes in the lectin binding properties of individual molecular weight classes of glycoproteins. Differences in the glycoproteins composition of synaptic junctional complexes isolated from 10 and 28-day-old brains were also revealed by lectin binding studies. The results indicate that the number and structure of oligosaccharides associated with synaptic membrane glycoproteins are under developmental regulation.