The role of septins during vaccinia virus spread

Septins are highly conserved components of the cytoskeleton found in animals and fungi. They play a variety of roles in key cellular processes including cell division, cell migration and membrane trafficking. During host-pathogen interactions, septins inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella. In addition, two recent genome-wide RNAi screens demonstrated that septins play an undefined role during vaccinia virus replication. Utilizing cell-based assays and microscopy I set out to determine the role of septins in vaccinia infected cells. I found that septins are recruited to vaccinia virus immediately following its fusion with the plasma membrane during viral egress. Live cell imaging reveals that septins are lost from beneath the virus once the virus stimulates Arp2/3 complex-dependent actin polymerization to enhance its cell-to-cell spread. Virus-induced actin polymerization involves the phosphorylation of the viral protein A36, leading to the recruitment of Cdc42, Nck, Grb2, WIP and N-WASP, which activate the Arp2/3 complex. Chemical or genetic inhibition of A36 phosphorylation dramatically increases the number of virus particles co-localizing with septins. Further experiments demonstrate that the recruitment of Nck and subsequently dynamin, but not Grb2, WIP:N-WASP or the Arp2/3-complex, promote the loss of septins from virions. RNAi-mediated depletion of septins increases virus release, accelerates cell-to-cell spread, and induces more robust actin tails. Collectively, my results demonstrate that septins limit the spread of vaccinia infection in cell monolayers and the recruitment of dynamin downstream of Nck enables the virus to overcome septin-mediated restriction. This is the first example of septins having an anti-viral effect and my work identifies a new role for septins in host defence

Fairness and transparency throughout a digital humanities workflow: Challenges and recommendations

How can we achieve sufficient levels of transparency and fairness for (humanities) research based on historical newspapers? Which concrete measures should be taken by data providers such as libraries, research projects and individual researchers? We approach these questions from the vantage point that digitised newspapers are complex sources with a high degree of heterogeneity caused by a long chain of processing steps, ranging, e.g., from digitisation policies, copyright restrictions to the evolving performance of tools for their enrichment such as OCR or article segmentation. Overall, we emphasise the need for careful documentation of data processing, research practices and the acknowledgement of support from institutions and collaborators

Mitochondria mediate septin cage assembly to promote autophagy of Shigella.

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria

Septins suppress the release of vaccinia virus from infected cells.

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization

Historyblogosphere. Bloggen in den Geschichtswissenschaften

For the first time in the German language historical disciplines, a book has been subject to open peer review as it was developed. All contributions were discussed online with comments. The ‘history blogosphere’ has thereby engaged in understanding an especially timely topic– namely, the medium of the blog itself

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

<div><p>Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells <i>in vivo</i>. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since <i>Listeria monocytogenes</i>, <i>Shigella flexneri</i>, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.</p></div

Cryo-electron tomography of a baculovirus actin comet tail <i>in vivo</i>.

<p>(A) Cryo-electron tomogram of a baculovirus comet tail in a B16 melanoma cell. Image shows 19 nm sections of the tomogram. Since the virus tail was not in one plane in the ice layer, the tomogram is shown in three images, separated by white lines, taken at different z-levels. Insets show details of branch junctions from the squares in the overview image. (B) Projection of 3D model derived from the tomogram in (A) showing the branch points as red dots and actin filaments as translucent lines. Yellow region indicates the core of the tail previously traversed by the cross-section of the virus, used for deriving (C). (C) Plot of the number of filaments transecting the core region in (B), taken as the number involved in pushing. (D) Projection from the rear of the complete comet tail model with actin filaments of the host cytoskeleton (translucent) as well as one microtubule (grey tube) superimposed. See also Movie S3. Bars (A, B), 100 nm; inset, 10 nm.</p

Mathematical simulation of comet tail architecture.

<p>(A–C) Schematic representation of the three different models considered. (A). Tethered model. (B) Tethered during branching model. (C) Untethered model. Actin filaments are in green, branchpoints in red, and the virus is depicted in grey. Polymerizing filaments can push (1). The virus surface is continuously tethered to the barbed ends (A, 2) or to the Arp2/3 complex during branching (B, 2) or not at all (C). Tethering is modeled by a spring connection. Branches (3) are initiated by Arp2/3 complex (red) recruited to the plus ends of actin filaments at the virus surface. Elongation of filaments that can no longer push (4) continues until they are capped (5). Filaments at the rear of the tail become de-branched and depolymerize from their minus ends (6). In (B) and (C) filaments lagging behind are not tethered (7). (D) Baculovirus actin comet tail in a B16 melanoma cell observed in vitreous ice, shown in two z-sextons, 18 nm thick. (E) Model derived from tomogram in (D) showing branch points in red and actin filaments in green. The angles of filaments to the core axis are shown for three examples ξ₁–ξ₃. (F–I) Simulated comet tails for the different model scenarios: (F) tethered actin filaments; (G) filaments tethered during branching; (H) untethered filaments; (I) tethered filaments but with branching towards and away from the virus surface. Filaments in different colors belong to different subsets. (J) Histograms of angles of filaments to the core trajectory from three cryo-tomograms (<i>n</i> = 485) compared to the model simulations with tethered filaments (<i>n</i> = 49,321), tethered filaments only during branching (<i>n</i> = 58,895), untethered filaments (<i>n</i> = 51,168), and filaments with random branching (<i>n</i> = 155,556). Measured versus tethered n.s. (<i>p</i> = 0.8270); measured versus tethered during branching **** (<i>p</i><0.0001); measured versus untethered **** (<i>p</i><0.0001); Measured versus tethered random branching **** (<i>p</i><0.0001); tethered versus tethered during branching **** (<i>p</i><0.0001); tethered versus untethered **** (<i>p</i><0.0001); tethered versus tethered random branching **** (<i>p</i><0.0001). Data nonparametric, by Kruskal-Wallis test. (K) Histogram of filament lengths for the experimental data (measured) and the “tethered” simulation compared. Bars (A–C), 50 nm, (D, E) 100 nm, (F–I) 300 nm.</p