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Trying to understand cells' interior design



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IMAGE: This is a fluorescent microscopy image of liquid-like droplets (yellow) formed or poly-L-lysine, DNA (dark spots), and adenosine triphosphate.
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Credit: IBS

How do you imagine the interior of our cells? Often compared to tiny factories, cells found smart and sophisticated ways to organize their 'interior'. De meeste biologische processen vereisen cellen om samen hun 'werknemers', zoals eiwitten en nucleïnezuren (zoals DNA), op de juiste tijd. Scientists at the Center for Soft and Living Matter, within the Institute for Basic Science (IBS, South Korea) have explained how liquid-like droplets are made of proteins and DNA form in vitro. For tiden er der stor interesse for at forstå molekylære mekanismer bag oprettelsen af ​​sådanne droplets, da det er forbundet med nogle humane sygdomme, såsom amyotrophic lateral sclerosis (ALS). The results, published as a featured article in Biophysical Journal showed how much the sequence of DNA matters in the formation of such droplets.

In the same way as walls divide a factory into departments, the cell has lipid membranes to divide its space into organelles. Men i de sidste 1

0 årene har forskere påvist at celler som ikke er omgivet av membraner, også kjent som membran-mindre organeller, oppfører like tette væskedråpler. A bit like a team of people meeting up in an open space office to carry out a job, these are dynamic assemblies with specific tasks. Men, hvordan er disse membranfrie organeller samlet, og de er påvirket af deres indhold er stadig uklare.

Til at svare på nogle af disse spørgsmål, IBS-forskere har testet, hvordan forskellige sekvenser af DNA-form dråber med et enkelt protein lavet af en enkelt repeating amino acid; lysine (poly-L-lysine). De to har motsatte gebyrer, så de tiltrækker hverandre, men er fortsatt i stand til å forbli i løsning.

The IBS team compared double- and single-stranded DNA. Double-stranded DNA is twisted into a helix like a spiral staircase. Each step of the staircase is made by two bonded nucleotides: adenosine with thymidine (A-T) and guanine with cytosine (G-C). Because of its helix structure, double-stranded DNA is quite stiff, and is often modeled as a rigid rod. In contrast, single stranded DNA – half of the staircase in the vertical direction, with unpaired nucleotides – is more flexible.

"It was a frustrating time about two years ago, when we wanted to form a model droplet system containing double-stranded DNA and poly-L-lysine," recalls Anisha Shakya, the key contributor to the study. "The two were kept on aggregating and getting precipitated. On the other hand, single-stranded DNA formed droplets easily."

The two IBS researchers involved in the study found that even when the overall electrical charge between two DNA molecules is the same, the DNA sequence ultimately determines the stability and appearance of the liquid-like droplets. "As the rigidity of DNA molecules can be slightly tuned, depending on its nucleotide sequence, we compared DNA molecules with the same change density, but different sequence," explains John T. King. For example, single stranded DNA with only T's was able to form droplets more readily than single stranded DNA with only A's. De reden dat poly (T) is meer flexibel dan poly (A). In concert, double stranded DNA rich in A's and T's is known to be more rigid than a poly (GC) and required the addition of more salts to obtain droplets.

The team also demonstrated that adenosine triphosphate (ATP), which typically acts as a fuel source in cells, facilitates the formation of liquid-like droplets. Mixtures of poly-L-lysine and double stranded DNA, which would typically precipitate at low salt concentrations, readily formed stable liquid-like droplets in presence of ATP.

This is a perfect platform to investigate how the flexibility of nucleic acids affects liquid-liquid phase separation. "The most fascinating part is to imagine how cells may take advantage of this sequence-dependent information to guide and regulate liquid-liquid phase separation in vivo," concludes Shakya.

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