Jody Rosenblatt

Detection of hepatitis C viral sequences in non-A, non-B hepatitis

The role of hepatitis C virus (HCV) in post-transfusion non-A, non-B hepatitis (NANBH) was investigated by analysing clinical samples for both HCV RNA by cDNA/polymerase chain reaction and antibodies against C100-3 by radioimmunoassay. Of fifteen chronic NANBH patients and one patient with chronic cryptogenic liver disease, ten were positive for anti-C100-3 and seven of the ten had viral sequences in their livers. However, two patients negative for anti-C100-3 also had substantial levels of HCV RNA in their livers. In acute post-transfusion NANBH (one surgical patient and two experimentally infected chimpanzees), HCV RNA was detected in the absence of anti-C100-3. In addition, infectious plasma from a seronegative patient with acute post-transfusion NANBH and a seronegative pool of plasma from a chimpanzee with chronic post-transfusion NANBH had high levels of HCV. These findings show that anti-C100-3-positive patients with chronic post-transfusion NANBH are likely to be viraemic; confirm that antibodies to C100-3 are a marker for infectivity; and suggest that the prevalence of HCV infections may be underestimated from the frequency of antibodies to C100-3 alone.

Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins

Based on the flavi- and pestivirus model of genome organization for the hepatitis C virus (HCV) (1-5), the nucleotide and deduced amino acid sequences of the putative envelope (E1) and the junction between the E1 and NS1/envelope 2 (E2) region from six different human isolates of HCV were compared with the nucleotide and predicted amino acid sequences of the prototype hepatitis C virus (HCV-1) (5). The overall percentage of nucleotide and amino acid changes among all six isolates, including HCV-1, from nucleotide 713 to 1630 (amino acid 129 to 437) was between 3 and 7%, which is comparable to that seen in some flaviviruses (6-8). An analysis of the number of nucleotide and deduced amino acid sequence changes among all six isolates and HCV-1 revealed a moderately variable domain of approximately 40 amino acids in the E1 region and a hypervariable domain (Region V) of approximately 28 amino acids, which is directly downstream from a putative signal peptide sequence, in the junction between E1 and NS1/E2. A similar hypervariable domain is not found in the C-terminus of the envelope polypeptide or in the N-terminus of the NS1 polypeptide domain of the flaviviruses. These findings suggest that the mature NS1/E2 polypeptide starts about amino acid 380 and that the NS1/E2 domain may correspond to a second envelope glycoprotein as in the case of the pestivirus. The observed heterogeneity in the putative structural proteins of HCV may have important ramifications for future vaccine development.

Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15.

We have examined the role of phosphorylation in the regulation of human cyclin‐dependent kinase‐2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site‐directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.

An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism

BACKGROUND Simple epithelia encase developing embryos and organs. Although these epithelia consist of only one or two layers of cells, they must provide tight barriers for the tissues that they envelop. Apoptosis occurring within these simple epithelia could compromise this barrier. How, then, does an epithelium remove apoptotic cells without disrupting its function as a barrier? RESULTS We show that apoptotic cells are extruded from a simple epithelium by the concerted contraction of their neighbors. A ring of actin and myosin forms both within the apoptotic cell and in the cells surrounding it, and contraction of the ring formed in the live neighbors is required for apoptotic cell extrusion, as injection of a Rho GTPase inhibitor into these cells completely blocks extrusion. Addition of apoptotic MDCK cells to an intact monolayer induces the formation of actin cables in the cells contacted, suggesting that the signal to form the cable comes from the dying cell. The signal is produced very early in the apoptotic process, before procaspase activation, cell shrinkage, or phosphatidylserine exposure. Remarkably, electrical resistance studies show that epithelial barrier function is maintained, even when large numbers of dying cells are being extruded. CONCLUSIONS We propose that apoptotic cell extrusion is important for the preservation of epithelial barrier function during cell death. Our results suggest that an early signal from the dying cell activates Rho in live neighbors to extrude the apoptotic cell out of the epithelium.

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts

Within hours of Listeria monocytogenes infection, host cell actin filaments form a dense cloud around the intracytoplasmic bacteria and then rearrange to form a polarized comet tail that is associated with moving bacteria. We have devised a cell-free extract system capable of faithfully reconstituting L. monocytogenes motility, and we have used this system to demonstrate that profilin, a host actin monomer-binding protein, is necessary for bacterial actin-based motility. We find that extracts from which profilin has been depleted do not support comet tail formation or bacterial motility. In extracts and host cells, profilin is localized to the back half of the surface of motile L. monocytogenes, the site of actin filament assembly in the tail. This association is not observed with L. monocytogenes mutants that do not express the ActA protein, a bacterial gene product necessary for motility and virulence. Profilin also fails to bind L. monocytogenes grown outside of host cytoplasm, suggesting that at least one other host cell factor is required for this association.

Actin dynamics in vivo

Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.

Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. We present surprising new evidence that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. We propose that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex.

P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium.

Communication between microtubules and actin/myosin networks determine epithelial cell extrusion polarity.

Mechanical stretch triggers rapid epithelial cell division through Piezo1

Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.