Motor properties of Myosin 5c are modulated by tropomyosin isoforms and inhibited by pentabromopseudilin.

Myosin 5c (Myo5c) is a motor protein that is produced in epithelial and glandular tissues, where it plays an important role in secretory processes. Myo5c is composed of two heavy chains, each containing a generic motor domain, an elongated neck domain consisting of a single α-helix with six IQ motifs, each of which binds to a calmodulin (CaM) or a myosin light chain from the EF-hand protein family, a coiled-coil dimer-forming region and a carboxyl-terminal globular tail domain. Although Myo5c is a low duty cycle motor, when two or more Myo5c-heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments. We describe the purification and functional characterization of human Myo5c-HMM co-produced either with CaM alone or with CaM and the essential and regulatory light chains Myl6 and Myl12b. We describe the extent to which cofilaments of actin and Tpm1.6, Tpm1.8 or Tpm3.1 alter the maximum actin-activated ATPase and motile activity of the recombinant Myo5c constructs. The small allosteric effector pentabromopseudilin (PBP), which is predicted to bind in a groove close to the actin and nucleotide binding site with a calculated ΔG of -18.44 kcal/mol, inhibits the motor function of Myo5c with a half-maximal concentration of 280 nM. Using immunohistochemical staining, we determined the distribution and exact localization of Myo5c in endothelial and endocrine cells from rat and human tissue. Particular high levels of Myo5c were observed in insulin-producing ß-cells located within the pancreatic islets of Langerhans.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins.

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin ß4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity.

According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2-16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8(+) T cell immunity.

Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8(+) T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming "nodular inflammatory foci" (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

Single cell detection of latent cytomegalovirus reactivation in host tissue.

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

The mouse cytomegalovirus immediate-early 1 gene is not required for establishment of latency or for reactivation in the lungs.

The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV Deltaie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the Deltaie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Deltaie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Deltaie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV.

Anti-HLA antibodies and pulmonary valve allograft function after the Ross procedure.

BACKGROUND AND AIM OF THE STUDY: Rejection is a plausible cause of failure of allograft valves, but has not been demonstrated unequivocally in humans. A cross-sectional study has been conducted on the frequency of anti-human leukocyte antigen (HLA) antibodies in order to identify any correlation with allograft function in adult patients, following the Ross procedure. METHODS: Anti-HLA antibodies were determined during regular follow up (median 1.1 years postoperatively) in a random sample of 197 patients (151 males, 46 females; mean age 46 +/- 13 years). Panel-reactive antibodies (PRA) were determined by cytotoxicity testing; anti-HLA class 2 antibodies (HLA2AB) were determined by ELISA in a subgroup of 94 patients. Echocardiographic examinations were performed during the first visit and at a median of 6.8 years postoperatively. RESULTS: The prevalence of positive antibody tests was 47% for PRA and 52% for HLA2AB. A slight deterioration of allograft valve function occurred between the two examinations (median maximal pressure gradient increased from 9 mmHg to 13 mmHg, p < 0.001; median degree of regurgitation increased from zero to trivial, p = 0.020). The degree of regurgitation was slightly, but significantly, higher in PRA-positive patients at both examinations (p = 0.008 and p = 0.038). No relationship was observed between PRA status and pressure gradients, nor between HLA2AB status and allograft valve function. Neither were any other risk factors for allograft valve deterioration identified. CONCLUSION: Subtle, clinically irrelevant and temporally stable differences with regard to regurgitation across the allograft were observed between PRA-positive and -negative patients. These findings neither proved nor disproved rejection, but rather suggested that a slow deterioration of allograft valve function was complex, and most likely multifactorial.