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Like most microorganisms, Bacillus subtilis bacteria are single-celled creatures with one goal: to reproduce by making copies of themselves. But survival isn't always that simple. For example, when food gets scarce, B. subtilis must decide between two possible paths: shut down, form a dormant spore -- a process called "sporulation" -- and wait for better times or split into two cells and gamble that there is enough food for at least one more generation.
"The decision about whether to form a spore and when is a very important one for B. subtilis," said Oleg Igoshin, associate professor of bioengineering at Rice and one of the lead researchers on the new study. "If the organism waits too long, it can starve before it finishes transforming into a spore. If it acts too early and forms a spore too soon, it can be overwhelmed and out-reproduced by competitors."
Igoshin's lab specializes in describing the workings of the complex genetic regulatory networks that cells use to make such decisions. He said dozens of studies over the past 25 years have identified a network of more than 30 genes that B. subtilis uses to bring about sporulation. When food is plentiful, this network is largely silent. But during times of starvation the genes work in concert to form a spore.
B. subtilis is harmless to humans, but some dangerous bacteria like Bacillus anthracis, the organism that causes anthrax, also form spores by a similar mechanism. Scientists are keen to better understand the process, both to protect public health and to explore the evolution of complex genetic processes.
The exact workings of the sporulation network are complex. In 2012, Igoshin and graduate student Jatin Narula analyzed a genetic circuit downstream of the protein known as Spo0A, the "sporulation master regulator," to explain how the network filters out noisy fluctuations in Spo0A activity. By filtering out noise, cells are able to accurately determine if Spo0A activity is above the threshold that triggers sporulation.
In the new study, Narula, Igoshin and collaborators set out to explain how B. subtilis times its sporulation decision with its cell-division cycle, a programmed series of events that cells normally follow to reproduce.
"Successful sporulation requires two complete copies of the bacterial chromosome, so coordination between the sporulation decision and the completion of DNA replication is very important," Narula said. "A good analogy might be a semester-long course in biology. Lessons are presented in a particular order, and students are tested after they learn. If the final exam were given in the first week, students would almost certainly fail."
Igoshin said that when the researchers set out to find how sporulation decisions were timed to the cell cycle, several studies including prior work by team members, provided a significant clue: Under starvation conditions, the activity of the master regulator gene had been shown to spike once per cell cycle.
In investigating how this spike occurred, Narula pored through dozens of published studies and noticed a discrepancy between some experimental results and the widely accepted view of the interactions between two key players in the sporulation network, a protein called Spo0F and a kinase called KinA. To resolve this discrepancy, Narula built a mathematical model in which excess Spo0F inhibits KinA activity. The new model showed that changes in the ratio of KinA to Spo0F could produce the pulse similar to those seen in experiments.
"The inhibition of KinA by 0F results in a 'negative feedback loop,' which means the circuit output works to counteract the input that triggers it," said Narula, co-lead author of the study. "Such loops are common in engineered and biological systems and usually work to keep things relatively constant despite external perturbations. A simple example of negative feedback would be the thermostat on your house. When temperature drops it will keep your heater on until the temperature is back to normal. If there is a delay in the feedback loop, the system may overreact and produce a surge. With the thermostat, for example, if the heating unit continues to run for some time after the desired temperature is reached, the temperature can transiently spike before settling back to the desired level."
Igoshin and Narula said similar spikes appear to be a consequence of the delayed negative feedback loop in the network that controls the amount of the active Spo0A. Furthermore, these spikes were timed based upon the positions of the KinA and Spo0F genes on the bacterial genome.
To divide and reproduce, bacteria must make a duplicate copy of their DNA. Because replication of circular bacterial DNA always initiates at one particular point, Narula surmised that the location of the KinA and Spo0F genes could be crucial. If one were located near the point where DNA replication began, the cell would contain two copies of that gene -- doubling the rate of production of that protein -- throughout the DNA replication period. If the other gene were located on the part of the circle that was copied last, the ratio of KinA to Spo0F would be one-to-one only when DNA replication was nearly completed.
Igoshin and Narula used a mathematical model of the network to show that this type of gene arrangement could account for spikes in Spo0A activity after each round of DNA replication. To verify their idea, they teamed with experimental biologists Anna Kuchina, co-lead author of the study, and Gürol Süel, co-lead investigator, both of the University of California at San Diego.
Experiments showed that the spikes of Spo0A activity always followed completion of DNA replication as the model predicted. In addition, Kuchina and colleagues used biotechnology to engineer mutant forms of B. subtilis in which the two critical genes were located near one another. The Spo0A spike from the delayed negative feedback loop was not observed in the mutants, and they failed to produce spores. In another engineered strain, the feedback loop between Spo0A and Spo0F was eliminated. This led to a gradual increase in Spo0A activity as opposed to a spike, and such cells were several times more likely to fail or die during sporulation.
"We found that the relative location of sporulation genes on the DNA circle were similar in more than 30 species of spore-forming bacteria, including Bacillus anthracis," Igoshin said. "This evidence suggests that the DNA timing mechanism is highly conserved, and it is possible that other time-critical functions related to the cell cycle may be regulated in a similar way.

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The above post is reprinted from materials provided by Rice University. The original item was written by Jade Boyd. Note: Materials may be edited for content and length.

Vesicles are responsible for transporting molecules between the different compartments within a cell and also for bringing material into cells from outside. There are several types of vesicle: each has a specific type of coat which is made up of different proteins and assembles onto a membrane surrounding the vesicle.
The EMBL team has been taking a close look at a coat called COPI. This surrounds vesicles that move material around within the Golgi apparatus and to the endoplasmic reticulum (ER) -- these are regions of the cell where proteins are made and modified in preparation for transport to the cell surface.
Using a technique called cryo-electron tomography, in which samples can be frozen at very low temperatures to avoid fixing or staining them chemically, the researchers combined data from hundreds of vesicles to build up a 3D image of the COPI coat. This enabled them to produce the most detailed pictures yet obtained of a vesicle coat assembled on the vesicle membrane.
This imaging technique is still in its infancy and although scientists have been able to gather structural information about parts of the membrane coats in other types of vesicle, this is the first time a model of a complete assembled vesicle coat has been produced.
What the images revealed was surprising: unlike other types of vesicle, where the coat is thought to be made from proteins assembled in different layers -- each with a specific function -- around the vesicle membrane, the EMBL team observed that the proteins in the COPI coat all intertwine together in one big layer, which is curved to fit around the membrane. More precisely, the COPI coat is made of a repetition of building blocks, called "triads," that contain all the important functional elements organised in a precise 3D structure.
"Until now we could see different elements of the vesicle coat, but not get a complete and detailed picture of the coat assembled onto the vesicle membrane. This is an important step forward for our understanding of intracellular transportation," explains John Briggs who led the study.
"Our images showed us how the proteins that make up the coat are arranged and it was surprising to discover how different COPI is from, for example, clathrin or COPII coated vesicles," adds Svetlana Dodonova, co-author of the paper. "Our next step will be to try to find out how this coat forms and binds to the vesicle membrane and how it arranges itself into such complex shapes."

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The above post is reprinted from materials provided by European Molecular Biology Laboratory (EMBL). Note: Materials may be edited for content and length.

For the first time a structure called 'the mesh' has been identified which helps to hold together cells. This discovery, which has been published in the online journal eLife, changes our understanding of the cell's internal scaffolding.
This also has implications for researchers' understanding of cancer cells as the mesh is partly made of a protein which is found to change in certain cancers, such as those of the breast and bladder.
The finding was made by a team led by Dr Stephen Royle, associate professor and senior Cancer Research UK Fellow at the division of biomedical cell biology at Warwick Medical School. Dr Royle said: "As a cell biologist you dream of finding a new structure in cells but it's so unlikely. Scientists have been looking at cells since the 17th Century and so to find something that no-one has seen before is amazing."
Researchers at the University's Warwick Medical School made the discovery by accident while looking at gaps between microtubules which are part of the cells' 'internal skeleton'. In dividing cells, these gaps are incredibly small at just 25 nanometres wide -- 3,000 times thinner than a human hair.
One of Dr Royle's PhD students was examining structures called mitotic spindles in dividing cells using a technique called tomography which is like a hospital CAT scan but on a much smaller scale. This meant that they could see the structure which they later named the mesh.
Mitotic spindles are the cell's way of making sure that when they divide each new cell has a complete genome. Mitotic spindles are made of microtubules and the mesh holds the microtubules together, providing support. While "inter-microtubule bridges" in the mitotic spindle had been seen before, the researchers were the first to view the mesh.
The study received funding and support from Cancer Research UK and North West Cancer Research.
Dr Royle said: "We had been looking in 2D and this gave the impression that 'bridges' linked microtubules together. This had been known since the 1970s. All of a sudden, tilting the fibre in 3D showed us that the bridges were not single struts at all but a web-like structure linking all the microtubules together."
The discovery impacts on the research into cancerous cells. A cell needs to share chromosomes accurately when it divides otherwise the two new cells can end up with the wrong number of chromosomes. This is called aneuploidy and this has been linked to a range of tumours in different body organs.
The mitotic spindle is responsible for sharing the chromosomes and the researchers at the University believe that the mesh is needed to give structural support. Too little support from the mesh and the spindle will be too weak to work properly, however too much support will result in it being unable to correct mistakes. It was found that one of the proteins that make up the mesh, TACC3, is over-produced in certain cancers. When this situation was mimicked in the lab, the mesh and microtubules were altered and cells had trouble sharing chromosomes during division.
Dr Emma Smith, senior science communications officer at Cancer Research UK, said: "Problems in cell division are common in cancer -- cells frequently end up with the wrong number of chromosomes. This early research provides the first glimpse of a structure that helps share out a cell's chromosomes correctly when it divides, and it might be a crucial insight into why this process becomes faulty in cancer and whether drugs could be developed to stop it from happening."
North West Cancer Research (NWCR) has funded the research as part of a collaborative project between the University of Warwick and the University of Liverpool, where part of the research is being carried out.
Anne Jackson, CEO at NWCR, said: "Dr Royle and Professor Ian Prior at the University of Liverpool have made significant inroads into our understanding of the way in which cancer cells behave, which could potentially better inform future cancer therapies.
"As a charity we fund only the highest standard of research, as evidenced by Dr Royle's work.
"All our funded projects undergo a thorough peer review process, before they are considered by our scientific committee. Our specially selected scientific committee includes some of the UK's leading professors, award-winning scientists and pioneering professionals."

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The above post is reprinted from materials provided by University of Warwick. Note: Materials may be edited for content and length.