Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Most of the mitochondrial proteins are imported from the cytoplasm, with only a small fraction (about 1%) encoded in the mitochondrial genome. Import is mediated by two complexes: TOM (transporter outer membrane) and TIM (transporter inner membrane). We have a pretty good idea about the players involved in mitochondrial protein import, but we have very little idea about the dynamics of TOM/TIM movement in the mitochondrial membrane.

We tried addressing this question using single molecule fluorescent microscopy in isolated yeast mitochondria. What we see is that Tom40, the central component of TOM complex, is highly confined (i.e. restricted in terms of aerea it can sample) but within its confinement it moves pretty rapidly.

References:

Kuzmenko et al., Scientific Reports (2011) 1:95

long memories of RelA

Enzymes have their cycles, catalytic ones: bind a substrate, catalyse some sort of reaction, release the product... then do it again. These cycles have memory effects: long turnover is likely to be followed by another long one, and short one is likely to be followed by another short one. This makes total sense: efficient act of catalysis is possible only when appropriate conformation is achieved and all the residues are aligned as they should be... and that is a recipe for one more efficient round!

Now let us look at RelA. Based on in vivo single molecule tracking investigations we recently proposed a model of RelA catalytic cycle: it sits on the ribosome, gets activated by arrival of deacylated tRNA to the A site, falls off and performs multiple acts of ppGpp synthesis from ATP and GDP. Importantly, RelA must go through the ribosome-bound stage in order to get activated. This seems to be an extreme cause of memory effects - while active off the ribosome, RelA remembers the activation event that happened on the ribosome!

It is quite a scary thought... what else does it remember? Does it remember the moment I started working on it? Well, surely not, even I don't remember that moment any more. Or may be I am just blocking out that memory.

References:

H. Peter Lu, Phys. Chem. Chem. Phys (2011), 13 pp. 6734-6749, PIMD 21409227

Brian P. English et al., PNAS (2011), 108(13) pp. E365-373, PIMD 21730169

Active role of the stringent response in antibiotic tolerance

From time to time we try killing bacteria with antibiotics. Most of the bugs die, but not all. These survivors fall into two categories: resistant bugs and tolerant bugs. Resistant bugs have specific mechanisms counteracting the drug: mutations in the target site, enzymes destroying the antibiotic, etc. Tolerant bugs are not getting killed using some more general approach, such as forming a biofilm efficiently shielding them from contact with the drug or shutting down its biosynthetic activity and waiting for the better days to come.

The stringent response is a mechanism rewiring the bacterial physiology under stress. It changes many things simultaneously, and, not surprisingly, functionality of the stringent response is linked to antibiotic tolerance. However, the big question here is the nature of this link: do bugs need functional stringent response in order to tolerate the drug just because relaxed bugs do not shut down their growth when needed and die, or does the stringent response induce production of certain specific enzymes protecting from the drug?

Recent report by Nguyen and colleagues seems to settle this question. Using series of in vivo experiments with E. coli knock-out strains deficient either in stringent response per se (knock-outs of RelA and SpoT) or in down-stream stringent response-regulated targets they show that the main source of antibiotic tolerance is not a general biosynthetic shut-down. Specifically, they identify two genes induced during the stringent response - superoxide dismutase (SOD) and catalase - to be crucial for bacterial survival in the presence of antibacterials.  What these do, they protect the bug from the hydroxyl radical. And build-up the latter was recently identified as a common mechanism causing the cell death during treatment by different unrelated antibacterials. 

References:

Nguyen at al., Science (2011) 334, pp. 982-986 PIMD 22096200

Kohanski et al. Cell (2007) 130, pp. 797-810 PIMD 17803904