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Listeria monocytogenes cell-to-cell spread in epithelia is heterogeneous and dominated by rare pioneer bacteria.
Listeria monocytogenes hijacks host actin to promote its intracellular motility and intercellular spread. While L. monocytogenes virulence hinges on cell-to-cell spread, little is known about the dynamics of bacterial spread in epithelia at a population level. Here, we use live microscopy and statistical modeling to demonstrate that L. monocytogenes cell-to-cell spread proceeds anisotropically in an epithelial monolayer in culture. We show that boundaries of infection foci are irregular and dominated by rare pioneer bacteria that spread farther than the rest. We extend our quantitative model for bacterial spread to show that heterogeneous spreading behavior can improve the chances of creating a persistent L. monocytogenes infection in an actively extruding epithelium. Thus, our results indicate that L. monocytogenes cell-to-cell spread is heterogeneous, and that rare pioneer bacteria determine the frontier of infection foci and may promote bacterial infection persistence in dynamic epithelia. Editorial note:This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)
Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry
The RNase E family is renowned for being central to
the processing and decay of all types of RNA in
many species of bacteria, as well as providing the
first examples of endonucleases that can recognize
50
-monophosphorylated ends thereby increasing
the efficiency of cleavage. However, there is
increasing evidence that some transcripts can be
cleaved efficiently by Escherichia coli RNase E via
direct entry, i.e. in the absence of the recognition of
a 50
-monophosphorylated end. Here, we provide
biochemical evidence that direct entry is central to
the processing of transfer RNA (tRNA) in E. coli, one
of the core functions of RNase E, and show that it is
mediated by specific unpaired regions that are
adjacent, but not contiguous to segments cleaved
by RNase E. In addition, we find that direct entry at a
site on the 50 side of a tRNA precursor triggers a
series of 50
-monophosphate-dependent cleavages.
Consistent with a major role for direct entry
in tRNA processing, we provide additional evidence
that a 50
-monophosphate is not required to
activate the catalysis step in cleavage. Other
examples of tRNA precursors processed via direct
entry are also provided. Thus, it appears increasingly
that direct entry by RNase E has a major role
in bacterial RNA metabolism
Spatiotemporal analysis of axonal autophagosome–lysosome dynamics reveals limited fusion events and slow maturation
Macroautophagy is a homeostatic process required to clear cellular waste. Neuronal autophagosomes form constitutively in the distal tip of the axon and are actively transported toward the soma, with cargo degradation initiated en route. Cargo turnover requires autophagosomes to fuse with lysosomes to acquire degradative enzymes; however, directly imaging these fusion events in the axon is impractical. Here we use a quantitative model, parameterized and validated using data from primary hippocampal neurons, to explore the autophagosome maturation process. We demonstrate that retrograde autophagosome motility is independent of fusion and that most autophagosomes fuse with only a few lysosomes during axonal transport. Our results indicate that breakdown of the inner autophagosomal membrane is much slower in neurons than in nonneuronal cell types, highlighting the importance of this late maturation step. Together, rigorous quantitative measurements and mathematical modeling elucidate the dynamics of autophagosome-lysosome interaction and autophagosomal maturation in the axon
Tension-dependent structural deformation alters single-molecule transition kinetics
We analyze the response of a single nucleosome to tension, which serves as a prototypical biophysical measurement where tension-dependent deformation alters transition kinetics. We develop a statistical-mechanics model of a nucleosome as a wormlike chain bound to a spool, incorporating fluctuations in the number of bases bound, the spool orientation, and the conformations of the unbound polymer segments. With the resulting free-energy surface, we perform dynamic simulations that permit a direct comparison with experiments. This simple approach demonstrates that the experimentally observed structural states at nonzero tension are a consequence of the tension and that these tension-induced states cease to exist at zero tension. The transitions between states exhibit substantial deformation of the unbound polymer segments. The associated deformation energy increases with tension; thus, the application of tension alters the kinetics due to tension-induced deformation of the transition states. This mechanism would arise in any system where the tether molecule is deformed in the transition state under the influence of tension
Effective Learning and Teaching in Medical, Dental and Veterinary Education
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/76336/1/AIAA-2004-3963-196.pd