Quantitative exploration of specific transcription factor binding in the presence of different levels of non specific binding.

<p>Quantitative exploration of specific transcription factor binding in the presence of different levels of non specific binding. Consider a TF ([TF] = 1 nM) that specifically recognizes 10 binding sites/nucleus. Specific recognition takes place with Ks' ranging between 10⁸ to 10¹⁴) while non-specific recognition takes place with much lower affinity. Intranuclear concentration of specific target sites is about 3.10⁻¹¹M (assuming a nuclear volume of 5.10⁻¹³L). The initial concentration of irrelevant DNA binding sites is assumed to be 7 orders of magnitude higher than sDNA. The color scale represents the ratio of the concentration of TF bound to specific site on DNA in the case WGD+ (leaving TF and its targets duplicated) over the concentration of TF bound to specific sites before WGD. For low non-specific binding the concentration of specifically bound TF targets in WGD+ is twice as much as in the case without WGD (blue zone). In presence of significant non-specific binding, the concentration of specifically bound sites can be as much as 4× higher than without duplication as the synthesis of TFs is doubled whereas non-specific binding sites available for sequestration are in identical concentration. The example of TF1 and TF2 (in balance) is displayed. TF1 and TF2 have the same global concentration but TF1 binds only specifically and TF2 has substantial non-specific binding. Under the scenario WGD+, TF2 might form as much as two times more complexes than TF1, which obviously would perturb their balance.</p

Kinetics effects of WGD and deletions.

<p>A) Outline of a minimal mitotic cell cycle model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008201#pone.0008201-Goldbeter1" target="_blank">[29]</a>, based on a cascade of post-translational modifications that modulates in the end a protease degrading a cyclin. Such a negative feedback loop generates oscillations. B) The blue curve is the periodic variation of cyclin with the set of parameters of Goldbeter (1991). The green curve (with faster cycling) corresponds to parameters for doubled enzyme concentrations resulting from a WGD followed by extensive DNA deletions.</p

The triple cost of polyploidy.

<p>A) Original diploid cell. Chromosomes are represented as blue lines. B) Cell after whole genome duplication (WGD). Notice that the cellular volume has doubled. C) After WGD superfluous gene copies can become junk DNA or be replaced by selfish DNA. This avoids paying the cost of transcription and translation of vast genomic regions and contributes to the rediploidization process.</p

Dynamics of the formation of the dimer MM (in balance with monomer N) and genome duplication.

<p>Formation of MM depends on the synthesis rate S, the degradation of the coding mRNA and monomers (D) and the interaction of the monomers. Blue curve: dimer formation after WGD (parameters S = D = 1) and red curve dimerization after WGD+deletions (leaving only M, N and the protease-encoding genes as duplicates, S = D = 2). Notice that the steady state is reached more rapidly in the latter system (red curve) than in the orginal tetraploid or diploid (blue curve). Such a kinetic difference can be crucial, especially especially if time delays (as in the mitotic clock of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008201#pone-0008201-g003" target="_blank">figure 3</a>) are important. If MM is in balance with monomers N, there might be a problem before reaching the steady state.</p

Non-specific protein-DNA interactions and WGD.

<p>A) Original diploid cell. Blue lines: chromosomes, green segments on the chromosomes: TF-encoding gene, yellow chromosomal segments: specific TF target binding sites, green triangles: TF protein. B) Cell after WGD. The cell volume has doubled and the concentrations of bound sites in the tetraploid (specifically or non-specifically) are the same as in the original cell. C) Cell after WGD+DNA deletions. Duplicated ‘superfluous’ DNA is removed leading to a volume shrinkage. This leads to doubling the concentration of TF-sDNA (specific interactions) with respect to the original autopolyploid or tetraploid. D) WGD+generation of junk/selfish DNA that replaces deleted DNA (red lines). Duplicated chromosomes are differentiated (diploidization) and cell volume is similar to that of the original tetraploid and the concentrations TF-sDNA and TF-nsDNA are respected.</p

Burst transmission in asymmetrical networks (DIV15).

<p>A—Explanatory diagrams for graphs presented on the left and right column respectively, with the same color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120680#pone.0120680.g004" target="_blank">Fig 4</a>. B—Typical example of a device showing unidirectional transmission. C—Results from a device where bursts also propagate in the reverse direction, albeit with a greater delay. Addition of 5 μM CNQX to the latter device yields the typical unidirectional transmission (D). Delays obtained by normalized cross correlation.</p

Compatibility of ChR2 and Calcium Orange.

<p>A- Quantification of background stimulation in ChR2(ET/TC) transduced cultures during calcium imaging with Calcium Orange at DIV12. The abscissa indicates the intensity of excitation light (549 nm) used during imaging relative to the recording conditions used in the rest of the experiments (1x); 2x is twice that level and 4x is 4 times that level. The different bursting rates at 2x and 4x were normalized by the bursting rates recorded at 1x for each culture so as to remove inter-culture variability for initial bursting rates. For information purpose, average absolute bursting rate at 1x was 5.9 ± 2.2 bursts per minute (n = 9 cultures). B- Absorption spectra of two ChR2 variants, including the ChR2(ET/TC) used in this study, as well as those of the commonly used Fluo-4 and its red-shifted counterpart Calcium Orange indicator. The figures of merit of each combination are indicated in % of maximal absorption.</p

Optogenetic induction of bursting events.

<p>A- Typical responses of a transduced culture (DIV 13) to different stimulation durations (1, 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 ms). Vertical blue bars represent stimulation times and durations. No fluorescence data was recorded during the stimulations as the photodiode, recording directly the stimulated spot, was saturated. B- Same experiment as in A after addition of 10 μM CNQX (AMPA receptor antagonist).</p

Design of a simple neuronal device.

<p>A- Scheme showing the neuronal device featuring two micro-wells seeded with hippocampal neurons (DIV 15) and connected by an array of axon diodes. The neurons colored in green correspond to neurons that are transduced by ChR2. Neurons colored in purple are not transduced. B- Image of a neuronal device obtained by confocal fluorescence microscopy showing neurons expressing ChR2-YFP (false color).</p

Local stimulation and burst propagation (DIV15).

<p>A- Picture of a symmetrical two-compartments device with only one side transduced (left). Green and red channels are for ChR2-YFP and Calcium Orange indicator, respectively. This dual marking reveals that 70% of cells are expressing ChR2 inside the transduced compartment. B and C- Averaged response after stimulation of the transduced (B) and non-transduced (C) compartments. The solid colour lines correspond to the peristimulus fluorescence signal averaged over 30 stimulations (0.1 Hz in alternation) while the filled surfaces indicate associated standard deviations. Vertical dashed lines indicate stimulations. Diagrams are presented for clarity, with matching line and population colors. Green neurons are transduced neurons.</p