January 29, 2014

The term “pathogen propulsion” sounds like an awesome technique for defeating the evil squid overlords. In fact, a lot of concepts involving propulsive actin comets sound awesomely science fictional, but thankfully they are not. Today’s image is from a paper describing how several viruses use actin comet tails to propel themselves to other cells.

Several pathogens such as baculovirus, Listeria, and Shigella hijack their host cell’s own actin cytoskeleton in order to propel themselves into other cells and spread infection. Behind each pathogen is a comet made of actin filaments and associated actin regulators, but the mechanism of propulsion and the structure of the actin comet have been debated. A recent paper in PLoS Biology by Mueller and colleagues describes the use of electron tomography to show a fishbone-like array of actin filaments behind baculovirus, the smallest pathogen known to use propulsive actin comets. These comets use an average of four actin filaments at any one time to propel the virus. Using these results, the researchers ran computer simulations that support a model of propulsion in which actin filaments are continuously tethered to the pathogen. The image above shows a negatively-stained actin comet tail behind a baculovirus particle (BV), and the 3D projection of the image shows branch points of the actin tail as red dots. Insets in top image show details of the branch points, and grey tube is a microtubule.

BONUS! Below is a movie of baculovirus propelling itself around a cell. Virus particles are red, and actin comet tails can be seen in green behind the particles.

Mueller J, Pfanzelter J, Winkler C, Narita A, Le Clainche C, et al. (2014) Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion. PLoS Biol 12(1): e1001765. doi:10.1371/journal.pbio.1001765.

May 6, 2013

Our bodies have multiple fronts for battling viruses, and it’s impressive that any of those suckers manage to invade our bodies at all.  When virus particles do make their way into a cell, it’s important for biologists to understand their pathway through a cell in order to create drug therapies and vaccines.  Today’s image is from a paper describing the use of high resolution imaging to understand this process.

The polarized cells that line our digestive and respiratory tracts form a tight barrier that protects our bodies from viruses.  Understanding how viruses are able to breach these polarized epithelial cells is important in guiding the development of therapeutics and vaccines, yet previous research has focused mainly on in vitro studies of virus entry into nonpolarized cells.  Microscopy advances have recently allowed the high-resolution imaging of virus entry in polarized epithelial cells.  Boulant and colleagues used live-cell spinning-disk confocal microscopy to follow the uptake of single mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs) in polarized Madin–Darby canine kidney cells.  Both virus particles were internalized by clathrin-mediated endocytosis at the apical surface.  MRV virions reached early and late endosomes, while ISVPs escaped the endocytic pathway prior to reaching early endosomes.  In the images above, the tight-junction protein ZO-1 (red, left) shows the typical belt pattern surrounding the polarized cells (side views are also shown in the top and side strips).  The MRV cell surface receptor JAM-A (red, right) is localized near tight junctions and on the apical side of the cells.

Boulant, S., Stanifer, M., Kural, C., Cureton, D., Massol, R., Nibert, M., & Kirchhausen, T. (2013). Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin-Darby canine kidney cells Molecular Biology of the Cell, 24 (8), 1196-1207 DOI: 10.1091/mbc.E12-12-0852

July 2, 2012

Microscopy can truly be a religious experience for some of us. We get to see the beauty of life unfold before our eyes, often in a dark room with the white-noise hum of equipment, all while being humbled by the mysteries in front of us. No matter your education, your amazing research pedigree, or the fancy-shmancy technology in front of you, you still don’t know how the heck it all happens…even in the tiniest of organisms. I’ll drink a bottle of immersion oil if that doesn’t bring your ass down a peg. Today’s image is from a paper describing the identification of a microtubule-like cytoskeleton in a bacteriophage.

Bacteriophages are viruses that infect bacteria. They are very common, found in dirt, sea water, and any place bacteria are found, and are very diverse. While bacteria are known to have cytoskeletal structures similar to our own cells, actin- or tubulin-like structures were not previously described in bacteriophages. A research group recently identified a tubulin-like protein called Phu-Z. Like the microtubules formed from tubulin in our own cells, Phu-Z assembles into filaments that surround the bacteriophage DNA and helps to position it within the infected bacterial cell. In the image above, Phu-Z is expressed in a bacterium and is able to assemble into filaments.

Kraemer JA, Erb ML, Waddling CA, Montabana EA, Zehr EA, Wang H, Nguyen K, Pham DS, Agard DA, & Pogliano J (2012). A Phage Tubulin Assembles Dynamic Filaments by an Atypical Mechanism to Center Viral DNA within the Host Cell. Cell, 149 (7), 1488-99 PMID: 22726436
Copyright ©2012 Elsevier Ltd. All rights reserved.

 

June 25, 2012

Sometimes the scary side of life sobers you up and wipes that stupid grin off your face. I just read that as of 2008, an estimated 1,178,350 people over the age of 13 were living with HIV in the United States. And, get this…20% were undiagnosed. So, let me stand up and clap loudly and throw flowers around the necks of the scientists trying to uncover all of HIV’s nasty secrets. Today’s image is from a paper describing how one of our own proteins binds to an HIV receptor, and negatively regulates HIV infection.

The HIV virus attaches to the membrane of a T-cell with the help of the HIV receptor proteins CD4 and CXCR4, which induce actin-mediated rearrangements that enhance entry of the virus into the cell. A recent paper identified a role for a protein called syntenin-1 in HIV entry. The adaptor protein syntenin-1 is implicated in many processes that involve polarization of the actin cytoskeleton, such as cell migration. According to Gordón-Alonso and colleagues, syntenin-1 is recruited to CD4 at the site of virus attachment to the cell, and negatively regulates virus entry by regulating actin reorganization. Overexpression of syntenin-1 inhibits HIV cell fusion and production, while depletion of syntenin-1 increases HIV entry. In the images above, T-cells were incubated with (bottom row) or without (top) HIV virus. In the presence of HIV virus, syntenin-1 (green) colocalizes with the cap of CD4 (red), which forms as a result of clustering of the receptor and enhances virus entry.

Gordón-Alonso M, Rocha-Perugini V, Alvarez S, Moreno-Gonzalo O, Ursa A, López-Martín S, Izquierdo-Useros N, Martínez-Picado J, Muñoz-Fernández MA, Yáñez-Mó M, & Sánchez-Madrid F (2012). The PDZ-adaptor protein syntenin-1 regulates HIV-1 entry. Molecular biology of the cell, 23 (12), 2253-2263 PMID: 22535526

January 30, 2012

As the flu pins you to your bed this winter, take a feverish minute to thank the biologists who help us understand the virus that causes it, the influenza virus. And maybe make a promise to yourself that next year you’ll spend 10 minutes to get the vaccine.

Influenza is an RNA virus that causes fever, chills, pain, fatigue, and general misery. After the virus replicates inside a host cell, it assembles at the cell’s plasma membrane. Virus particles then bud from the cell’s plasma membrane, taking some of the membrane with it, and search for the next cell to invade. A recent paper describes the membrane composition of recently produced influenza virus particles, and suggests lipid raft involvement in influenza virus assembly. Lipid rafts are specialized membrane domains that float freely in the plasma membrane, and have a distinct composition of proteins and lipids (specifically sphingolipids and cholesterol) compared to the rest of the plasma membrane. In this paper, Gerl and colleagues quantified the lipid compositions for the host cell’s total membrane, the host cell’s apical membrane (where virus particles bud from), and influenza particles budded from these cells. The virus particles contained more sphingolipids and cholesterol than the host cell’s total or apical membrane, consistent with a model of virus budding from lipid rafts on the apical membrane. The electron micrographs above show purified spherical influenza virus particles recently budded from host cells.

Gerl, M., Sampaio, J., Urban, S., Kalvodova, L., Verbavatz, J., Binnington, B., Lindemann, D., Lingwood, C., Shevchenko, A., Schroeder, C., & Simons, K. (2012). Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane originally published in the Journal of Cell Biology, 196 (2), 213-221 DOI: 10.1083/jcb.201108175

December 1, 2011

There are a lot of times when I wish I was a fly on the wall during a totally awesome experiment I’ve read about. This is not one of them, but purely for my own safety. Today, I share with you a humbling and stunning image of the deadly Marburg virus, a virus so pathogenic it requires intense special handling and facilities.

The filovirus family is made of the Ebola and Marburg viruses, which cause deadly hemorrhagic fevers in humans and non-human primates. All filoviruses are single-stranded RNA viruses, yet their range of morphologies is an obstacle in understanding the structure and assembly of these viruses. In addition, the intense containment conditions (called biosafety level 4) required to work with these viruses make many techniques either difficult or impossible. A group of biologists recently jumped over all of these hurdles to image Marburg virus particles using a variety of electron microscopy techniques. Bharat and colleagues are the first to show a detailed 3-D structure of a biosaftely level 4 pathogen, both after release and during virus assembly within an infected cell. In addition, Bharat and colleagues determined the location of different viral proteins within the virus. Above is a cryo-electron microscopy image of Marburg virus particles. All three different morphologies—filamentous, 6-shaped, and round—have spine-like protrusions coming from the virus particle membrane (arrowheads). Inset image shows striations just under the membrane in a filamentous virus particle.

Bharat, T., Riches, J., Kolesnikova, L., Welsch, S., Krähling, V., Davey, N., Parsy, M., Becker, S., & Briggs, J. (2011). Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells PLoS Biology, 9 (11) DOI: 10.1371/journal.pbio.1001196

November 17, 2011

A virus is both crafty and determined…pretty good for a non-living thing, right? Today’s image is from a paper discussing transport of the simian virus 40, which has some clever tricks up its little capsid sleeve to make the host cell help out with the viral infection.

Simian virus 40 (SV40) is a DNA virus that does not have its own membrane envelope, making transport into and around a cell a non-trivial task. SV40 first binds to a cell’s membrane, then uses endocytosis to enter the cell and travel to the endoplasmic reticulum (ER). From the ER, the virus penetrates the membrane to have access to the cytosol of the cell, where it can then reach the nucleus and replicate its genome. A recent paper describes this ER membrane penetration step, and reports that SV40 particles go through a major structural change within the lumen of the ER. According to Geiger and colleagues, this conformational change exposes a part of the minor viral protein VP2, which then embeds the virus into the ER membrane. This change attracts the cell’s own ERAD machinery, normally used to find and degrade misfolded proteins in the ER, which then allows the escape of the virus particles into the cytosol. Images above are electron micrographs of cells infected with SV40 for 2 (left), 6 (right), and 19 (bottom) hours. Early in the infection, virus particles are bound to endosomal or vesicle membranes (left, arrows). In the ER, the virus appears more compact (right, red arrows), indicating the structural change. Later, the ER membrane appears flattened around the virus particles (bottom, arrows).

Geiger, R., Andritschke, D., Friebe, S., Herzog, F., Luisoni, S., Heger, T., & Helenius, A. (2011). BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol Nature Cell Biology, 13 (11), 1305-1314 DOI: 10.1038/ncb2339
Adapted by permission from Macmillan Publishers Ltd, copyright ©2011

October 4, 2010

Today’s image is a first for HighMag…I’m showing a second image from the paper that provided Thursday’s image. This is a fantastic paper, and has great images using different techniques to show the authors’ take-home message.

M2 is a protein from the influenza virus that is able to shape and release a newly-replicated budded virus particle out of an infected cell, allowing the infection of more cells. Thursday’s image showed the ability of a domain of the M2 protein to induce budding on a synthesized vesicle. Today’s image shows the localization of M2 at the neck of budding virus particles (red arrows). The authors determined the localization of M2 using electron microscopy and immuno-gold labeling, which means that tiny gold beads are attached to antibodies able to find M2. The little beads represent M2 localization in the image above, with bottom images showing a higher magnification.

Reference: Jeremy S. Rossman, Xianghong Jing, George P. Leser and Robert A. Lamb. Cell 142 (17), 902-913. ©2010 Elsevier Ltd. All rights reserved. Paper can be found here.

September 30, 2010

I love proteins that bend membranes. Bending a membrane is an amazing feat…if we could relate the work of one of these proteins to a large scale human example, I’m sure we’d all be impressed. Today’s post is about one of these proteins, and it comes from a virus.

A recent paper looks at the mechanism that influenza virus uses when a newly replicated virus particle buds out of the cell’s plasma membrane, in order to infect other cells. While many viruses use proteins hijacked from the infected cell for this budding process, influenza virus uses its own protein called M2. M2 alters the curvature of the plasma membrane in order to shape the bud, and mediates membrane scission which is required for release of the virus particle. Images are of synthesized vesicles without (left) or with (right) the domain of M2 thought to bend membrane, and shows that this domain alone is able to induce budding.

Bonus!! Video of entire M2 protein inducing budding in a synthesized vesicle can be found here.

Reference: Jeremy S. Rossman, Xianghong Jing, George P. Leser and Robert A. Lamb. Cell 142 (17), 902-913. ©2010 Elsevier Ltd. All rights reserved. Paper can be found here.

August 26, 2010

Pathogens such as viruses and bacteria must move within infected cells in order to replicate and spread to other cells. Viruses typically hijack the cell’s microtubule cytoskeleton for motility within the cell, while bacteria typically use actin-based mechanisms. Authors of a recent paper have demonstrated how a baculovirus type species uses actin polymerization to first move to the cell’s nucleus, then later move to the cell surface in order to quickly spread to nearby cells. Image above shows an infected cell with the virus (red) translocated to spikes at the cell’s surface. Actin is in green.

Reference: Taro Ohkawa, Loy E. Volkman, and Matthew D. Welch, 2010. Originally published in Journal of Cell Bioloy. doi: 10.1083/jcb.201001162. Paper can be found here.