Within 2-6 hours after infection by vesicular stomatitis virus (VSV), newly assembled VSV particles are released from the surface of infected cells. In that time, viral ribonucleoprotein (RNP) particles (nucleocapsids) travel from their initial sites of synthesis near the nucleus to the edge of the cell, a distance of 5-10 μm. The hydrodynamic radius of RNP particles (86 nm) precludes simple diffusion through the mesh of cytoskeletal fibers. To reveal the relative importance of different transport mechanisms, movement of GFP-labeled RNP particles in live A549 cells was recorded within 3 to 4 h postinfection at 100 frames/s by fluorescence video microscopy. Analysis of more than 200 RNP particle tracks by Bayesian pattern recognition software found that 3% of particles showed rapid, directional motion at about 1 μm/s, as previously reported. 97% of the RNP particles jiggled within a small, approximately circular area with Gaussian width σ = 0.06 μm. Motion within such "traps" was not directional. Particles stayed in traps for approximately 1 s, then hopped to adjacent traps whose centers were displaced by approximately 0.17 μm. Because hopping occurred much more frequently than directional motion, overall transport of RNP particles was dominated by hopping over the time interval of these experiments. Movement of cellular components is critical for their functions. However, this movement faces a number of barriers, including the dense network of the cytoskeleton 1. For particles too large to diffuse in the cytoplasm (hydrodynamic radius> approximately 50 nm) 2 , movements consist of a mixture of seemingly random motions and directional motion driven by molecular motors on microtubules and actin filaments 3. Viruses face the same hurdles during their intracellular replication cycles and use the same cellular mechanisms to distribute viral components throughout the cell. The purpose of the experiments presented here was to determine the transport modes of the ribonucleoprotein (RNP) core (nucleocapsid) of vesicular stomatitis virus (VSV) across the cytoplasm using a variational Bayesian approach to analyze single particle tracks in living cells 4. Vesiculovirus Indiana, commonly referred to as VSV (Indiana serotype), is one of the prototypes for the large group of viruses with nonsegmented negative strand RNA genomes (order Mononegavirales). These viruses include many human pathogens, such as measles, Ebola, and rabies viruses, as well as many other viruses that infect both animals and plants. These viruses encode an RNA-dependent RNA polymerase that is responsible for transcription of viral mRNAs and replication of genome RNA, and for most of these viruses, the replication cycle occurs entirely in the cytoplasm 5. The VSV RNP core consists of the 11 kb RNA genome encapsidated by approximately 1200 copies of the viral N protein. The RNP also contains approximately 400 copies of the viral P protein and 50 copies of the L protein, which together are responsible for the RNA polymerase activity 6. The resultant mass of t...
Diffusion of macromolecules and higher-order structures in the crowded interior of cells frequently shows an anomalous behavior with the mean-square displacement (MSD) increasing nonlinearly in time, MSDft a . Here we have probed to which extent also larger organelle structures show such an anomalous diffusion and how non-equilibrium contributions affect their diffusional motion [Phys. Rev. E 98, 012406 (2018), in press: Biophys. J. 115 ( 2018)]. In particular, we have employed single-particle tracking to monitor the motion of tubular junctions in the endoplasmic reticulum (ER) network and of long-lived membrane domains on the ER, called ER exit sites (ERES). Our results show that both, ER junctions and ERES show a distinct anomalous diffusion with a significant anti-correlation of successive step increments that is reminiscent of fractional Brownian motion. Disrupting the microtubule cytoskeleton significantly altered the subdiffusive characteristics of both entities, highlighting that even anomalous diffusion is an actively driven process in living cells. While the diffusion behavior of ER junctions was seen to be directly dependent on the presence and activity of microtubules, ERES were only indirectly affected. ERES therefore can be seen as mobile membrane domains that perform a quasi-one-dimensional random walk on the shivering ER backbone.
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