Jan Korb

Caveolae Are Involved in the Trafficking of Mouse Polyomavirus Virions and Artificial VP1 Pseudocapsids toward Cell Nuclei

ABSTRACT Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids (“empty” or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both “empty” vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

Engagement of phospholipid scramblase 1 in activated cells; Implication for phosphatidylserine externalization and exocytosis

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca2+-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcϵRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcϵRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.

Signaling assemblies formed in mast cells activated via Fcε receptor I dimers

Although aggregation of the Fcϵ receptor I (FcϵRI) is necessary for Ag‐mediated mast cell triggering, the relationship between the extent of the FcϵRI aggregation and subsequent biochemical and topographical events is incompletely understood. In this study, we analyzed the activation events induced by FcϵRI dimers, elicited by binding of anti‐FcϵRI mAb to rat basophilic leukemia cells. We found that, in contrast to extensively aggregated FcϵRI, receptor dimers (1) induced a less extensive association of FcϵRI with detergent‐resistant membranes, (2) delayed the tyrosine phosphorylation and membrane recruitment of several signaling molecules, (3) triggered a slower but more sustained increase in concentration of free cytoplasmic calcium, (4) induced degranulation which was not inhibited at higher concentrations of the cross‐linking mAb, and (5) failed to produce clusters of FcϵRI, Syk kinase and Grb2 adapter in osmiophilic membranes, as detected by immunogold electron microscopy on membrane sheets. Despite striking differences in the topography of FcϵRI dimers and multimers, biochemical differences were less pronounced. The combined data suggest that FcϵRI‐activated mast cells propagate signals from small signaling domains formed around dimerized/oligomerized FcϵRI; formation of large FcϵRI aggregates in osmiophilic membranes seems to promote both strong receptor triggering and rapid termination of the signaling responses.

Topography of plasma membrane microdomains and its consequences for mast cell signaling

Thy‐1 (CD90) is a glycoprotein bound to the plasma membrane by a GPI anchor. Aggregation of Thy‐1 in mast cells and basophils induces activation events independent of the expression of Fcϵ receptor I (FcϵRI). Although we and others have previously suggested that plasma membrane microdomains called lipid rafts are implicated in both Thy‐1 and FcϵRI signaling, properties of these microdomains are still poorly understood. In this study we used rat basophilic leukemia cells and their transfectants expressing both endogenous Thy‐1.1 and exogenous Thy‐1.2 genes and analyzed topography of the Thy‐1 isoforms and Thy‐1‐induced signaling events. Light microscopy showed that both Thy‐1 isoforms were in the plasma membrane distributed randomly and independently. Electron microscopy on isolated membrane sheets and fluorescence resonance energy transfer analysis indicated cross‐talk between Thy‐1 isoforms and between Thy‐1 and FcϵRI. This cross‐talk was dependent on actin filaments. Thy‐1 aggregates colocalized with two transmembrane adaptor proteins, non‐T cell activation linker (NTAL) and linker for activation of T cells (LAT), which had been shown to inhabit different membrane microdomains. Thy‐1 aggregation led to tyrosine phosphorylation of these two adaptors. The combined data indicate that aggregated GPI‐anchored proteins can attract different membrane proteins in different clusters and thus can trigger different signaling pathways.

Regulation of Ca2+ Signaling in Mast Cells by Tyrosine-Phosphorylated and Unphosphorylated Non-T Cell Activation Linker

Engagement of the FcεRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcεRI-induced tyrosine phosphorylation of the FcεRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcεRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcεRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.

Micromorphology of cytoplasmic nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA

Nucleoprotein (NP) complexes constituting the three basic components (A, B, C) of the postmicrosomal sediment (POMS) of chicken leukemic myeloblasts (CHLMs) which contain extrachromosomal DNA closely related to avian myeloblastosis virus DNA were analyzed electron microscopically. It was shown that these NP complexes resemble micromorphologically, depending on the origin of their POMS components, NP structures involved in three successive stages of early DNA synthesis. Nucleic acids harbored in these NP complexes exhibited micromorphological features typical for replicative structures. It was confirmed electron microscopically that the extrachromosomal DNA of CHLMs replicative in nature and of three length classes is organized into special NP complexes, each of which, as demonstrated, represents a unique reaction machinery of early DNA synthesis.

Ori-somes, nucleoprotein complexes descending from origin regions of animal chromosomal DNA replication. A micromorphological study

Abstract Micromorphology of nucleoprotein (NP) complexes designated according to their descent and shape as Ori-somes is presented. These NP complexes of three different types harbor molecules of cytoplasmic „small” polydisperse DNA, which descend from origin regions of chromosomal DNA replication and are equipped, as shown previously, with early DNA-synthesizing activities. By negative staining the Ori-somes are visualized as particles of irregular shape, sometimes of a subunit-like structure. Micromorphological differences in size and structural compactness noted among individual Ori-somes are dependent on their type similarly as earlier shown physico-chemically and biochemically. Such differences were also confirmed by two different spreading techniques. The most unravelled structures with electron diffuse centers belong to Ori-somes of component B associated with most active DNA synthesis. In contrast, the Ori-somes of components A and C, associated with pronounced RNA synthesis, revealed large electron-dense centers. The incidence of replicative structures present in Ori-somes corresponds with the level of their DNA- synthesizing activities.