Stem Cells Age-discriminate Organelles to Maintain Stemness

Stem Cells Age-discriminate Organelles to Maintain Stemness

Thursday, 02 April 2015

Tissue stem cells, that continuously renew our tissues, can divide asymmetrically to produce two types of daughter cells. One will be the new stem cell, whereas the other will give rise to the differentiating cells of the tissue.

A study jointly leads by laboratories in the Institute of Biotechnology, University of Helsinki and Massachusetts Institute of Technology (MIT) investigated whether stem cells may also use asymmetric cell division to reduce accumulation of cellular damage. Damage build-up can cause stem cell exhaustion that result in reduced tissue renewal and aging.

Human mammary stem-like cell apportions

aged mitochondria asymmetrically between

daughter cells. Mitochondria were labelled age-

selectively red 51 hours prior to imaging, leaving

mitochondria that are younger unlabelled. The

daughter cell that will become the new stem cell

(bottom left) receives only few old mitochondria.

Credit: Julia Döhla. 

Researchers developed a novel approach to follow cellular components, such as organelles, age-selectively during cell division. Scientists in David Sabatini's lab studied stem-like cells (SLCs) from cultures of immortalized human mammary epithelial cells. These SLCs were chosen because they express genes associated with the stem-cell state (referred to as stemness), are able to form structures known as mammospheres in culture. To track the destinations of subcellular components during cell division, the researchers, led by former postdoctoral scientist Pekka Katajisto, tagged the components – including lysosomes, mitochondria, Golgi apparatus, ribosomes, and chromatin – with a fluorescent protein that glows when hit by a pulse of ultraviolet light.

"We found that stem cells segregate their old mitochondria to the daughter cell that will differentiate, whereas the new stem cell will receive only young mitochondria" says Pekka Katajisto, a Group leader and Academy research fellow at BI.

By tracing the movements of the glowing organelles, the researchers were able to demonstrate that while the normal epithelial cells distributed all of the tagged components symmetrically to daughter cells, the SLCs localized their older mitochondria distinctly and passed on the lion's share of them to the daughter cells headed for differentiation. The researchers ultimately found that the number of older mitochondria in those cells was roughly six times that in daughter cells whose fate was to remain as stem cells.

Mitochondria appear to be particularly important for stem cells, as other analysed organelles were not similarly age-discriminated, and since inhibition of normal mitochondrial quality control pathways stopped their age-selective segregation.

"There is a fitness advantage to renewing your mitochondria," says David Sabatini, Professor at MIT and Whitehead Institute.

"Stem cells know this and have figured out a way to discard their older components."

“While the mechanism used by stem cells to recognize the age of their mitochondria remains unknown, forced symmetric apportioning of aged mitochondria resulted in loss of stemness in all of the daughter cells," says Katajisto.

"This suggests that the age-selective apportioning of old and potentially damaged organelles may be a way to fight stem cell exhaustion and aging," says Katajisto who now runs a lab at the Institute of Biotechnology at University of Helsinki.

Katajisto laboratory is now exploring how old mitochondria differ from old, and whether this phenomenon occurs in other cell types beyond the human mammary stem-like cells examined here as well as in in vivo.

Source: University of Helsinki

Contact: Pekka Katajisto

Reference:

Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness 

Pekka Katajisto, Julia Döhla, Christine Chaffer, Nalle Pentinmikko, Nemanja Marjanovic, Sharif Iqbal, Roberto Zoncu, Walter Chen, Robert A. Weinberg, David M. Sabatini

Science April 2, .2015, DOI:10.1126/science.1260384

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Researchers Find Protein 'Switch' Central to Heart Cell Division

Discovery advances efforts to replace damaged heart muscle

Tuesday, 04 March 2014

In a study that began in a pair of infant siblings with a rare heart defect, Johns Hopkins researchers say they have identified a key molecular switch that regulates heart cell division and normally turns the process off around the time of birth. Their research, they report, could advance efforts to turn the process back on and regenerate heart tissue damaged by heart attacks or disease.

In the heart muscle cell above, the arrows 

show an early sign of replication. Credit: 

Johns Hopkins Medicine. 

"This study offers hope that we can someday find a way to restore the ability of heart cells to divide in response to injury and to help patients recover from many kinds of cardiac dysfunction," says cardiologist Daniel P. Judge, M.D., director of the Johns Hopkins Heart and Vascular Institute's Center for Inherited Heart Diseases.

"Things usually heal up well in many parts of the body through cell division, except in the heart and the brain. Although other work has generated a lot of excitement about the possibility of treatment with stem cells, our research offers an entirely different direction to pursue in finding ways to repair a damaged heart."

Unlike most other cells in the body that regularly die off and regenerate, heart cells rarely divide after birth. When those cells are damaged by heart attack, infection or other means, the injury is irreparable.

Judge's new findings, reported online March 4 in the journal Nature Communications, emerged from insights into a genetic mutation that appears responsible for allowing cells to continue replicating in the heart in very rare cases.

The discovery, Judge says, began with the tale of two infants, siblings born years apart but each diagnosed in their earliest weeks with heart failure. One underwent a heart transplant at three months of age; the other at five months. When pathologists examined their damaged hearts after they were removed, they were intrigued to find that the babies' heart cells continued to divide — a process that wasn't supposed to happen at their ages.

The researchers then hunted for genetic abnormalities that might account for the phenomenon by scanning the small percent of their entire genome responsible for coding proteins. One stood out: ALMS1, in which each of the affected children had two abnormal copies.

The Johns Hopkins researchers also contacted colleagues at The Hospital for Sick Children in Toronto, Canada, who had found the same heart cell proliferation in five of its infant patients, including two sets of siblings. Genetic analysis showed those children had mutations in the same ALMS1 gene, which appears to cause a deficiency in the Alström protein that impairs the ability of heart cells to stop dividing on schedule. The runaway division may be responsible for the devastating heart damage in all of the infants, Judge says.

These mutations, it turned out, were also linked to a known rare recessive disorder called Alström syndrome, a condition associated with obesity, diabetes, blindness, hearing loss and heart disease.

In further experiments, the Johns Hopkins researchers cultured mouse heart cells, and then turned off the ALMS1 gene. Compared to those with normal ALMS1 genes, the number of heart cells in samples without this gene increased by an additional 10 percent. The researchers then contacted colleagues at Jackson Laboratory in Maine who had genetically engineered and bred mice with an ALMS1 mutation. They found that the animals with the mutation had increased proliferation of heart cells after two weeks of age, compared to mice with a normal version of the ALMS1 gene. The cell proliferation did eventually stop in the mice, says Judge, an associate professor at the Johns Hopkins University School of Medicine.

Judge says precise knowledge of the regulatory role played by the ALMS1 mutation should advance the search for ways to help regenerate heart muscle tissue in a controlled fashion. Much work in the field of regeneration has been focused on the use of stem cells, which have the remarkable potential to develop into many different cell types.

Judge cautions that efforts to manipulate ALMS1 to repair damage would be tricky, because uncontrolled proliferation may lead to serious and even lethal complications.

"The children who helped us recognize the importance of this gene were born with a rare condition that leads to heart failure and many other problems, such as diabetes, obesity, blindness and deafness," he says.

"Now we hope to apply these discoveries to help millions of others with heart disease."

Source: Johns Hopkins Medicine 

Contact: Stephanie Desmon

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How Stem Cells Produce Different Kinds of Cells

How Stem Cells Produce Different Kinds of Cells

Sunday, 05 May 2013

The human body contains trillions of cells, all derived from a single cell, or zygote, made by the fusion of an egg and a sperm. That single cell contains all the genetic information needed to develop into a human, and passes identical copies of that information to each new cell as it divides into the many diverse types of cells that make up a complex organism like a human being.

If each cell is genetically identical, however, how does it grow to be a skin, blood, nerve, bone or other type of cell? How do stem cells read the same genetic code but divide into very different types?

The apical tip of fruit fly testis containing germ line 

stem cells and differentiating germ cells. Copies of 

Y chromosome are marked with either red or blue. 

Using this method, the authors discovered that 

germ line stem cells inherit specific copies of Y (and 

X) chromosomes. Credit: Yukiko Yamashita. 

Researchers at the University of Michigan have found the first direct evidence that cells can distinguish between seemingly identical copies of chromosomes during stem cell division, pointing to the possibility that distinct information on the chromosome copies might underlie the diversification of cell types.

Scientists in the lab of Life Sciences Institute researcher Yukiko Yamashita explained how stem cells can distinguish between two identical copies of chromosomes and distribute them to the daughter cells in a process called non-random chromosome segregation. They also described the genes responsible. Their work is scheduled to be published online May 5 in Nature.

"If we can figure out how and why cells are dividing this way, we might be able to get a glimpse of how we develop into a complete human, starting from a single cell," Yamashita said.

"It is very basic science, but understanding fundamental biological processes always has wide-ranging implications that could be exploited in therapeutics and drug discovery."

During the cell division cycle, the mother cell duplicates its chromosomes, generating two identical sets. When the cell divides to become two cells, each cell inherits one set of chromosome copies. In many divisions, the daughter cells are identical to the mother — one skin cell becomes two, for instance.

But in a process called asymmetric division, a cell divides into two daughters that are not identical — a skin stem cell divides into another skin stem cell and a regular skin cell, for example. In that case, the genetic information within the chromosome copies remains the same, but the type of cell, or "cell fate," is different.

The Yamashita lab used stem cells from the testes of the fruit fly Drosophila to study the process of cell division.

"The Drosophila germ line stem cell can be identified at a single-cell resolution, so they are an ideal model," Yamashita said.

The stem cells cluster and are easy to identify; they divide to produce another germ line stem cell and a differentiating cell called a gonialblast, which goes on to eventually become a sperm cell.

The researchers marked the copies of each chromosome in the Drosophila stem cells as they divided. Using this method, they tracked the tendency of the X and the Y chromosome copies to move to the daughter germ line stem cell or to the gonialblast. They were able to demonstrate that copies of X and Y chromosomes (but not other chromosomes) are distinguished and delivered to the daughter cells with a striking bias.

This is the first direct evidence that cells indeed have an ability to distinguish identical copies of chromosomes and separate them in a regulated manner. This ability has been suspected and hypothesized, but never proven.

"We do not know yet why copies of X and Y chromosomes segregate non-randomly," Yamashita said.

"We think maybe specific epigenetic information is transmitted to the germ line stem cell and to the gonialblast."

The findings suggest that the information on the X and Y chromosomes that makes this division possible is primed during gametogenesis — the process of creating ovum or sperm cells — in the parents.

Many other cells throughout the body are able to divide into two different types, especially during embryonic development. Yamashita's next steps are to explore whether the non-random chromosome segregation seen in Drosophila is a widespread phenomenon that is shared by mammals, including humans.

Source: University of Michigan 

Contact: Laura J. Williams

Reference:

Chromosome-specific nonrandom sister chromatid segregation during stem-cell division

Swathi Yadlapalli &  Yukiko M. Yamashita

Nature (2013), doi:10.1038/nature12106

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A Single Stem Cell Mutation Triggers Fibroid Tumours

Mutated stem cell 'goes wild' in frenzied tumour expansion

Saturday, 05 May 2012

Fibroid uterine tumours affect an estimated 15 million women in the United States, causing irregular bleeding, anaemia, pain and infertility. Despite the high prevalence of the tumours, which occur in 60 percent of women by age 45, the molecular cause has been unknown.

New Northwestern Medicine preclinical research has for the first time identified the molecular trigger of the tumour – a single stem cell that develops a mutation, starts to grow uncontrollably and activates other cells to join its frenzied expansion.

"It loses its way and goes wild," said Serdar Bulun, M.D., the chair of obstetrics and gynaecology at Northwestern University Feinberg School of Medicine and Northwestern Memorial Hospital.

"No one knew how these came about before. The stem cells make up only 1 ½ percent of the cells in the tumour, yet they are the essential drivers of its growth."

The paper is published in the journal PLoS ONE. Masanori Ono, M.D., a post-doctoral student in Bulun's lab, is the lead author.

The stem cell initiating the tumour carries a mutation called MED12. Recently, mutations in the MED12 gene have been reported in the majority of uterine fibroid tissues. Once the mutation kicks off the abnormal expansion, the tumours grow in response to steroid hormones, particularly progesterone.

For the study, researchers examined the behaviour of human fibroid stem cells when grafted into a mouse, a novel model initiated by Northwestern scientist Takeshi Kurita, a research associate professor of obstetrics and gynaecology. The most important characteristic of fibroid stem cells is their ability to generate tumours. Tumours originating from the fibroid stem cell population grew 10 times larger compared to tumours initiated with the main cell population, suggesting a key role of these tumour stem cells is to initiate and sustain tumour growth.

"Understanding how this mutation directs the tumour growth gives us a new direction to develop therapies," said Bulun, also the George H. Gardner Professor of Clinical Gynecology.

Source: Northwestern University

Contact: Marla Paul

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At The Crossroads of Chromosomes

Penn study reveals structure of cell division's key molecule
Thursday, 23 September 2010

On average, one hundred billion cells in the human body divide over the course of a day. Most of the time the body gets it right but sometimes, problems in cell replication can lead to abnormalities in chromosomes resulting in many types of disorders, from cancer to Down syndrome.



This is a human chromosome, with

conventional nucleosomes containing

the major form of the histones (green),

and localization of the centromere

histone H3 variant, CENP-A (red).

Credit: Ben E. Black, University of

Pennsylvania School of Medicine.

 Now, researchers at the University of Pennsylvania's School of Medicine have defined the structure of a key molecule that plays a central role in how DNA is duplicated and then moved correctly and equally into two daughter cells to produce two exact copies of the mother cell. Without this molecule, entire chromosomes could be lost during cell division.

Ben Black, PhD, assistant professor of Biochemistry and Biophysics, and Nikolina Sekulic, PhD, a postdoctoral fellow in the Black lab, report in the September 16 issue of Nature the structure of the CENP-A molecule, which defines a part of the chromosome called the centromere. Specialized molecules called spindle fibres attach that help pull daughter cells apart during cell division to this constricted area.

"Our work gives us the first high-resolution view of the molecules that control genetic inheritance at cell division," says Black.

"This is a big step forward in a puzzle that biologists have been chipping away at for over 150 years."

Investigators have known for the last 15 years that epigenetic processes, the series of actions that affect the protein spools around which DNA is tightly bound, rather than encoded in the DNA sequence itself, control part of cell division. Those spools are built of histone proteins, and chemical changes to these spool proteins can either loosen or tighten their interaction with DNA. Epigenetics alter the readout of the genetic code, in some cases ramping a gene's expression up or down. In the case of the centromere, it marks the site where spindle fibres attach independently of the underlying DNA sequence. CENP-A has been suspected to be the key epigenetic marker protein.

However, what hasn't been known is how CENP-A epigenetically marks the centromere to direct inheritance. The Black team found the structural features that confer CENP-A the ability to mark centromere location on each chromosome. This is important because without CENP-A or the centromere mark it creates, the entire chromosome — and all of the genes it houses — are lost at cell division.



Structure of the centromere histone complex
containing two chains of CENP-A (red) and
two copies of its close binding partner,
histone H4 (blue). Credit: Ben E. Black,

University of Pennsylvania School of
Medicine.

 In this study, Black solved CENP-A's structure to determine how it specifically marks the centromere on each chromosome and surmise from that how the epigenetic mark is copied correctly in each cell division. They found that CENP-A changes the shape of the nucleosome of which it's a part, also making it more rigid than other nucleosomes without CENP-A. The nucleosome is the combination of DNA wound around a histone protein core – the DNA thread wrapped around the histone spool. The CENP-A nucleosome is copied several times to create a unique epigenetic area, different from the rest of the chromosome. CENP-A replaces histone H3 in the nucleosomes located at the centromere.

This CENP-A centromere identifier attracts other proteins, and in cell division builds a massive structure, the kinetochore, for pulling the duplicated chromosomes apart during cell division.

Besides the major advance in the understanding of the molecules driving human inheritance, this work also brings about the exciting prospect that the key epigenetic components are now in hand to engineer clinically useful artificial chromosomes that will be inherited alongside our own natural chromosomes — and with the same high fidelity, says Black.

Source: University of Pennsylvania School of Medicine
Contact: Karen Kreeger
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Live Imaging Puts New Light on Stem Cell Division

University of Oregon team finds, in fruit flies, that the mitotic spindle does not act alone to divide a cell
Thursday, 02 September 2010

The asymmetrical division of a stem cell — in this case a neuroblast of a fruit fly — is shown, the result of research in the lab of Chris Doe at the University of Oregon. Credit: Courtesy of Clemens Cabernard.

A long-held assumption about asymmetrical division of stem cells has cracked. Researchers at the University of Oregon report that the mitotic spindle does not act alone — that cortical proteins help to position a cleavage furrow in the right location.

Their discovery, described in the Sept. 2 issue of the journal Nature, provides a new window on how stem cells divide to produce two unequal daughter cells: one that lives on as a new stem cell and other, smaller cell, which adopts a new function, in this case as a neuron.

A three-member team focused on drosophila (fruit flies) neural stem cells known as neuroblasts, long known for dividing asymmetrically. What is learned in these flies often applies to many other mitotic (dividing) cells in other organisms such as mammals, including humans.

Clemens Cabernard, a postdoctoral researcher
at the University of Oregon, provided new
insight on the asymmetrical division of
neuroblasts (stem cells) in fruit flies. Credit:
Photo by Jim Barlow.

"This addresses a fundamental question in cell biology, namely how a cell knows where to place a cleavage furrow and thus divide in a symmetrical or asymmetrical fashion," said Clemens Cabernard, a postdoctoral fellow in the lab of Chris Doe, a Howard Hughes Medical Institute investigator in the UO Institute of Molecular Biology and director of the UO Institute of Neuroscience. Also on the team was Kenneth E. Prehoda, a UO biochemist and member of the Institute of Molecular Biology.

What the UO team found is that neuroblasts have two distinct dividing pathways that appear to work redundantly: one that is polarity induced and one that is spindle induced, Cabernard said.

Theories on cleavage furrow positioning during cell division have centred on the mitotic spindle, a network of fibres called microtubules.

One idea is that microtubules from spindle poles reach to the cortex, which delivers a positive or negative signal to determine the position of the cleavage ring. Another idea is that microtubule fibres from the centre of the spindle reach out to the cortex resulting in the assembly of a cleavage ring (a complex consisting of several proteins, one of which is called myosin). A third model involves both. It was thought that asymmetrically dividing cells, such as drosophila neuroblasts, generate an asymmetric spindle and can position the cleavage ring in an asymmetric position, as opposed to symmetrically dividing cells that construct a symmetric spindle.

"We found a new mechanism in which a cleavage furrow can be placed at an asymmetric position," Cabernard said.

"First, by way of a couple of experiments, we ruled out that the cleavage furrow is solely dependent on the position and symmetry/asymmetry of the mitotic spindle."

First researchers used mutants that lack astral microtubules, the microtubule fibres reaching out from the spindle poles towards the cortex and watched with live imaging what happens to the cleavage furrow. A cleavage furrow still occurred in an asymmetric position. This has been seen before but not using the same markers, Cabernard said.

Next, researchers removed the entire spindle from the picture with targeted drugs. Usually cells stop dividing in this condition, but a genetic trick allows these cells to initiate cell division despite the lack of a mitotic spindle. Surprisingly, researchers found, the proteins involved in constructing a cleavage furrow became localized in an asymmetric fashion and positioned a cleavage furrow in an asymmetric position — pretty much like in wild-type neuroblasts.

"Although cell division could not be completed, the dividing point was correctly marked," Cabernard said.

"This told us that there must be a mechanism independent of the spindle."

In a third set of experiments, the research team rotated the mitotic spindle of neuroblasts using genetic mutants and thus changed the position of any spindle-derived signal. Interestingly, the team found that two cleavage furrows now formed, but only one coincided with the new position of the mitotic spindle, strongly supporting the hypothesis that a spindle independent signal also is used. Further experiments revealed that a cortical protein, required for proper neural stem cell divisions in mice and humans, is necessary for the asymmetric positioning of the cleavage furrow.

One of the marker proteins watched closely in the experiments was myosin. When a cell starts the division process, Cabernard said, the spindle is symmetrical but the myosin markers segregated toward the basal side — and is localized in an asymmetric fashion — which becomes smaller and transforms into a neuron upon cell division.

Although this research addresses a basic question in cell biology, the findings have important implications. Asymmetric cell division in fly or human stem cells is important to generate a number of differentiating cells while retaining a stem cell as a back up copy.

Previous work by Cabernard and Doe showed that if drosophila neuroblasts divide in a symmetric manner, which does not normally happen, two neuroblasts are generated. Thus, the researchers said, it is crucial for a stem cell to know where to place a cleavage furrow to produce all the required neurons. Similar results have been observed in neural stem cells in mice.

Source: University of Oregon
Contact: Jim Barlow

Reference:
A spindle-independent cleavage furrow positioning pathway
Clemens Cabernard, Kenneth E. Prehoda & Chris Q. Doe
Nature Vol.: 467, 91–94, (02 September 2010), doi:10.1038/nature09334
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Gene Expression in Single Embryonic Stem Cells Analysed

Singapore scientists make breakthrough findings on early embryonic development
Wednesday, 21 April 2010

Scientists at the Genome Institute of Singapore (GIS) have recently generated significant single cell expression data crucial for a detailed molecular understanding of mammalian development from fertilization to embryo implantation, a process known as the preimplantation period. The knowledge gained has a direct impact on clinical applications in the areas of regenerative medicine and assisted reproduction.

This study, published in Developmental Cell on April 20, 2010, is the first of its kind to apply single cell gene expression analysis of many genes to hundreds of cells in a developmental system.

Using the new BioMark microfluidic technology and the mouse preimplantation embryo as a model, the scientists were able to study the expression of 48 genes from individual cells and applied this to analyze over 600 individual cells from the 1-cell to the 64-cell stage of preimplantation development.

This high throughput single cell research methodology provides the scientists with the ability to detect dynamic patterns in cellular behaviour, which is unprecedented in the field. Significantly, the findings of the study resolves some of the arguments pertaining to cellular differentiation events and places fibroblast growth factor signalling as the primary event in the later cell fate decisions.

Executive Director at the GIS, a biomedical research institute of the Agency for Science, Technology and Research (A*STAR), Professor Edison Liu said:

"This remarkable work by Guoji Guo, Mikael Huss, Paul Robson and colleagues uses new microgenomic technologies to map, over time, how a single cell decides to permanently become different parts of an embryo. Within one division, cells commit to specific developmental lineages by expressing defined sets of genes. This research now opens the possibility of assessing the genetic triggers for fate determination of individual cells in developmental time. On another level, this work highlights the importance of new micro-technologies in advancing the understanding of early embryonic events."

Professor Davor Solter, Senior Principal Investigator of the Institute of Medical Biology, A*STAR, added:

"This is a real technological tour de force. The authors investigated changes in expression of multiple genes on the single cell level during preimplantation mouse development. They clearly demonstrated gradual and stochastic lineage allocation and absence of predetermination. These results conclusively resolved one of the hotly debated issues in mammalian development and provided important new insight into the mechanism which regulates early development in mammals."

"These are important findings. The team at GIS provided a new look into the complex and little-understood process of early embryo development. It also demonstrates the power of single cell gene expression. It is clear that individual cells and small groups of cells behave differently than the aggregate population, and these differences are key to understanding the biology of the system as a whole." said Gajus Worthington, president and chief executive officer of Fluidigm.

"It always provides a special thrill when researchers use the capabilities of Fluidigm's technology to bring insight to the body of scientific knowledge."

The preimplantation period involves the first cellular differentiation events in mammalian development including the formation of pluripotent cells from where embryonic stem (ES) cells are derived. Being one of the simplest mammalian developmental systems to study, it can provide comprehensive understanding of the complex molecular control of reprogramming and cell fate decisions.

Reference:
Resolution of cell fate decisions revealed by single cell gene expression analysis from zygote to blastocyst
Guoji Guo, Mikael Huss, Guo Qing Tong, Chaoyang Wang, Li Li Sun, Neil D. Clarke, Paul Robson
Developmental Cell, Volume 18, Issue 4, 675-685, 10.1016/j.devcel.2010.02.012
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Artificial Light at Night Disrupts Cell Division

Just one 'pulse' of artificial light at night disrupts the circadian mode of cell division
Tuesday, 13 April 2010

Just one "pulse" of artificial light at night disrupts circadian cell division, reveals a new study carried out by Dr. Rachel Ben-Shlomo of the University of Haifa-Oranim Department of Environmental and Evolutionary Biology along with Prof. Charalambos P. Kyriacou of the University of Leicester.

"Damage to cell division is characteristic of cancer, and it is therefore important to understand the causes of this damage," notes Dr. Ben-Shlomo. The study has been published in the journal Cancer Genetics and Cytogenetics.

The current research was carried out by placing lab mice into an environment where they were exposed to light for 12 hours and dark for 12 hours. During the dark hours, one group of mice was given artificial light for one hour. Changes in the expression of genes in the rodents' brain cells were then examined.

Earlier studies that Dr. Ben-Shlomo carried out found that the cells' biological clock is affected, and in the present research, she revealed that the mode of cell division is also harmed and that the transcription of a large number of genes is affected. She states that it is important to note that those genes showing changes in their expression included genes that are connected to the formation of cancer as well as genes that assist in the fight against cancer.

"What is certain is that the natural division is affected," Dr. Ben-Shlomo clarifies.

This research joins earlier studies from the University of Haifa on the effects of exposure to artificial light at night.
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For Stem Cells, Practice Makes Perfect

For Stem Cells, Practice Makes Perfect
Monday, 05 April 2010

Multipotent stem cells have the capacity to develop into different types of cells by reprogramming their DNA to turn on different combinations of genes, a process called "differentiation." In a new study, researchers from the Carnegie Institution for Science have found that reprogramming is imperfect in the early stages of differentiation, with some genes turned on and off at random. As cell divisions continue, the stability of the differentiation process increases by a factor of 100. The finding will help scientists understand how stem cells reprogram their genes and why fully differentiated cells are very hard to reprogram, knowledge with potential impacts on aging, regenerative medicine, and cancer research.

Allan Spradling and Andrew Skora of the Carnegie Institution's Department of Embryology studied stem cells in the ovaries of the fruit fly Drosophila. The stem cells develop into specialized cells, called follicle cells, over a series of nine generations of cell divisions. Using a biochemical method known as a GAL4-UAS reporter gene, the researchers were able to keep track of genes located at many different sites on the chromosomes as the follicle cells developed. If the programming of a reporter gene was perfectly transmitted from parent to daughter cell, then the follicle cells would express the gene at the same level after each division. But the researchers found that in the first division alone random changes occurred 41% of the time. By the fifth division, changes took place about 8% of the time. By the ninth division, however, such changes happened only 0.37% of the time, a stability increase of more than 100-fold.

The instability of epigenetic information during the early differentiation of ovarian stem cells surprised the researchers. They speculate that stem cells may be deficient in epigenetic inheritance machinery in order to prevent them from differentiating prematurely, and thereby to help maintain the flexibility to give rise to many different cell types.

"Stem cells appear unable to faithfully pass on a particular genetic program to their daughter cells," says Spradling.

"Apparently, before one particular kind of cell can differentiate from a stem cell, its progenitors have to learn how to maintain and transmit epigenetic (programming) information."

Spradling explains that the mechanism by which the reprogramming and stabilization occurs is not well understood, but their research confirmed the expectation that at least some of the critical changes take place in the gene-bearing chromosomes themselves, rather than in external factors such as the cell's environment or signals from other cells. Most likely, the reprogramming alters proteins on the chromosome, which package the DNA and control which genes are expressed. Changes in chromosome structure, as opposed to changes in the genes themselves, that can be passed on from one generation to the next are called epigenetic changes. The researchers hope that their research will provide a way to learn more about the methods cells use to transmit epigenetic information faithfully during cell division.

"Epigenetic inheritance underlies the ability of multi-celled organisms to develop from single-celled zygotes to complex creatures with an array of specialized cells and tissues," says Spradling.

"But the amount of epigenetic information transmitted at different stages of cellular differentiation remains little known. Applying the GAL4-UAS system within a defined stem cell lineage allows us to measure the stability of epigenetic information quantitatively, and to follow how it changes during development. This will have an impact across a broad swath of stem cell research."

The results of this research are published in the Proceedings of the National Academy of Sciences.
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Movies for the Human Genome

EMBL scientists identify the genes involved in cell division in humans
Thursday, 01 April 2010

Name a human gene, and you'll find a movie online showing you what happens to cells when it is switched off. This is the resource that researchers at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, and their collaborators in the Mitocheck consortium are making freely available, as the result of a study in which they have identified the genes involved in mitosis – the most common form of cell division – in humans. Published today in Nature, their work begins to unravel the molecular workings of one of the most fundamental processes of life: how one cell becomes two.

 "Without mitosis, nothing happens in life, really", says Jan Ellenberg, who led the study at EMBL, "and when mitosis goes wrong, you get defects like cancer."

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Normal Cell Division. When no genes are silenced, cell division occurs normally, with each cell giving rise to two. This video was generated within the Mitocheck consortium, whose dataset containing more videos can be found online. Credit: Thomas Walter/EMBL.

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Of the 22,000 genes in each human cell, almost 600 play a part in mitosis, Ellenberg and colleagues found. To uncover which genes are involved in this process, the scientists developed a new method using high-throughput imaging of living cells. They silenced, or inactivated, each of the 22,000 human genes one by one in a different set of cells, and filmed those cells for 48 hours under a microscope. This generated almost 200,000 time-lapse movies of mitosis. Having a person – or even a group of people – process such vast amounts of information would be almost impossible, so the scientists created a new computer program that analyses the footage and automatically detects what characteristic defects cells display, and in what order. By grouping genes with similar effects – for instance, genes which when inactivated led to cells with 2 nuclei instead of one, after division – they were able to identify genes involved in mitosis, which they confirmed with further experimental assays.

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Silencing Gene Hinders Cell Division. When a gene called OGG1 is silenced, cell division starts normally, but then the daughter-cells are unable to separate from each other. The result: individual cells (green) with more than one nucleus (red). Credit: Thomas Walter & Jutta Bulkescher / EMBL.

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"The end result is that we now have a very rich resource for the scientific community, as we're making all the movies and all the analysis data freely available online," Ellenberg emphasises.

"Scientists can go to the website, type in the name of their favourite gene, and watch what happens when it is silenced; they can find out what other genes have similar effects – all in a few mouse clicks, instead of months or years of work in the lab!"

But mitosis is not solved yet, the scientists say. They have yet to uncover exactly how these genes act at the molecular level – a task that will be tackled by a follow-up project called Mitosys. All data from this follow-up work will also be made freely available online, creating what Ellenberg describes as a 'one-stop-shop' for mitosis research.

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This image of a dividing cell is composed of several microscopy images of human cells in which different individual genes were silenced. The images are placed according to genes’ effects: images for genes that affect chromosomes make up the chromosomes (red), while the mitotic spindle (green) is composed of images for genes that affect it. Credit: Thomas Walter & Jutta Bulkescher / EMBL.

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In the mean time, the new methodology the EMBL scientists developed to silence all of an organism's genes in a fast and systematic manner is itself proving a boon to the scientific community.

"A year after we developed these new siRNA microarrays," says Rainer Pepperkok, who led the method's development at EMBL, "they're already in use by over 10 research groups from across Europe."

The current study looked at HeLa cells, a widely studied line of cancer cells. Now that they have narrowed the search from a daunting 22,000 to a more manageable 600 genes, the scientists would like to investigate how these same genes act in other cancers and in healthy cells, as such comparisons could help to identify markers which could be used for diagnosis or to help make better-informed treatment decisions.

The study was carried out as part of the Mitocheck consortium, coordinated by Jan-Michael Peters at the Research Institute of Molecular Pathology in Vienna, Austria, and the data is available at http://www.mitocheck.org/.

References:
Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes
Beate Neumann, Thomas Walter, Jean-Karim Hériché, Jutta Bulkescher, Holger Erfle, Christian Conrad, Phill Rogers, Ina Poser, Michael Held, Urban Liebel, Cihan Cetin, Frank Sieckmann, Gregoire Pau, Rolf Kabbe, Annelie Wünsche, Venkata Satagopam, Michael H. A. Schmitz, Catherine Chapuis, Daniel W. Gerlich, Reinhard Schneider, Roland Eils, Wolfgang Huber, Jan-Michael Peters, Anthony A. Hyman, Richard Durbin, Rainer Pepperkok & Jan Ellenberg
Nature, 1 April 2010, doi:10.1038/nature08869

Systematic Localization and Purification of Human Protein Complexes Identifies Chromosome Segregation Proteins
Hutchins et al.
Science Express, published online 1 April 2010
.........

ZenMaster

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Regenerative Medicine: One Gene Lost Equals One Limb Regained

Wistar scientists demonstrate mammalian regeneration through a single gene deletion Tuesday, 16 March 2010 A quest that began over a decade ago with a chance observation has reached a milestone: the identification of a gene that may regulate regeneration in mammals. The absence of this single gene, called p21, confers a healing potential in mice long thought to have been lost through evolution and reserved for creatures like flatworms, sponges, and some species of salamander. In a report published today in the Proceedings of the National Academy of Sciences, researchers from The Wistar Institute demonstrate that mice that lack the p21 gene gain the ability to regenerate lost or damaged tissue. Unlike typical mammals, which heal wounds by forming a scar, these mice begin by forming a blastema, a structure associated with rapid cell growth and de-differentiation as seen in amphibians. According to the Wistar researchers, the loss of p21 causes the cells of these mice to behave more like embryonic stem cells than adult mammalian cells, and their findings provide solid evidence to link tissue regeneration to the control of cell division. "Much like a newt that has lost a limb, these mice will replace missing or damaged tissue with healthy tissue that lacks any sign of scarring," said the project's lead scientist Ellen Heber-Katz, Ph.D., a professor in Wistar's Molecular and Cellular Oncogenesis program. "While we are just beginning to understand the repercussions of these findings, perhaps, one day we'll be able to accelerate healing in humans by temporarily inactivating the p21 gene." Heber-Katz and her colleagues used a p21 knockout mouse to help solve a mystery first encountered in 1996 regarding another mouse strain in her laboratory. MRL mice, which were being tested in an autoimmunity experiment, had holes pierced in their ears to create a commonly used life-long identification marker. A few weeks later, investigators discovered that the ear-holes had closed without a trace. While the experiment was ruined, it left the researchers with a new question: Was the MRL mouse a window into mammalian regeneration? The discovery set the Heber-Katz laboratory off on two parallel paths. Working with geneticists Elizabeth Blankenhorn, Ph.D., at Drexel University, and James Cheverud, Ph.D., at Washington University, the laboratory focused on mapping the critical genes that turn MRL mice into healers. Meanwhile, cellular studies ongoing at Wistar revealed that MRL cells behaved very differently than cells from "non-healer" mouse strains in culture. Khamilia Bedebaeva, M.D., Ph.D., having studied genetic effects following the Chernobyl reactor radiation accident, noticed immediately that these cells were atypical, showing profound differences in cell cycle characteristics and DNA damage. This led Andrew Snyder, Ph.D., to explore the DNA damage pathway and its effects on cell cycle control. Snyder found that p21, a cell cycle regulator, was consistently inactive in cells from the MRL mouse ear. The tumour suppressor p53, another regulator of cell division and a known factor in many forms of cancer tightly control P21 expression. The ultimate experiment was to show that a mouse lacking p21 would demonstrate a regenerative response similar to that seen in the MRL mouse. And this indeed was the case. As it turned out, p21 knockout mice had already been created, were readily available, and widely used in many studies. What had not been noted was that these mice could heal their ears. "In normal cells, p21 acts like a brake to block cell cycle progression in the event of DNA damage, preventing the cells from dividing and potentially becoming cancerous," Heber-Katz said. "In these mice without p21, we do see the expected increase in DNA damage, but surprisingly no increase in cancer has been reported." In fact, the researchers saw an increase in apoptosis in MRL mice – also known as programmed cell death – the cell's self-destruct mechanism that is often switched on when DNA has been damaged. According to Heber-Katz, this is exactly the sort of behaviour seen in naturally regenerative creatures. "The combined effects of an increase in highly regenerative cells and apoptosis may allow the cells of these organisms to divide rapidly without going out of control and becoming cancerous," Heber-Katz said. "In fact, it is similar to what is seen in mammalian embryos, where p21 also happens to be inactive after DNA damage. The down regulation of p21 promotes the induced pluripotent state in mammalian cells, highlighting a correlation between stem cells, tissue regeneration, and the cell cycle." ......... ZenMaster

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Cells Send Dirty Laundry Home to Mom

How mother cells manage to make young daughter’s Monday, 01 February 2010 Understanding how aged and damaged mother cells manage to form new and undamaged daughter cells is one of the toughest riddles of ageing, but scientists now know how yeast cells do it. In a groundbreaking study researchers from the University of Gothenburg, Sweden, show how the daughter cell uses a mechanical "conveyor belt" to dump damaged proteins in the mother cell. "This ensures that the daughter cell is born without age-related damage," says Professor Thomas Nyström from the Department of Cell and Molecular Biology. Thomas Nyström is a professor of microbiology at the University of Gothenburg and one of Sweden's leading researchers in the field of cellular and molecular biology. His research group has published countless scientific discoveries about cell ageing which have provided a new understanding of aging and age-related diseases. Now he and his colleagues have identified a key piece in the ageing puzzle. Mechanic transport In a study published as a feature article in the scientific journal Cell, two collaborating research groups at the Department of Cell and Molecular Biology have been able to show how newly formed yeast cells transport damaged and aged proteins back to the mother cell, guaranteeing that the new cell is born young and healthy. Mother dustbin "Previously it was believed that these structures allowed only one-way traffic of proteins and organelles from mother cell to daughter cell," says Nyström. "We can now show that damaged proteins are transported in the opposite direction. In principle, this means that the daughter cell uses the mother cell as a dustbin for all the rubbish resulting from the ageing process, ensuring that the newly formed cell is born without age-related damage." Conveyor belt In the study, the researchers show that this transportation is mechanical, using conveyor-like structures called actin cables. A special gene, which controls the rate of ageing, called SIR2, is needed for these cables to form properly. Previous research has shown that changing the SIR2 gene can markedly extend the life span of an organism. Longer life "Increased SIR2 activity means a longer life, whereas a damaged SIR2 gene accelerates ageing," says Nyström. "This has been demonstrated in studies of yeast, worms, flies and fish, and may also be the case in mammals." Future treatment This knowledge of how age-damaged proteins are transported from daughter cell to mother cell could eventually be used in the treatment of age-related diseases caused by protein toxicity in humans, but Nyström says that it is too early to say how. The first step "The first step is to study whether this transportation of damaged proteins also occurs in the cells of mammals, including humans, for example in the formation of sex sells and stem cells." Reference: The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates Beidong Liu, Lisa Larsson, Antonio Caballero, Xinxin Hao, David Öling, Julie Grantham, Thomas Nyström Cell, Volume 140, Issue 2, 257-267, 22 January 2010, 10.1016/j.cell.2009.12.031 ......... ZenMaster

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Handle with Care: Telomeres Resemble DNA Fragile Sites

A protein at the ends of chromosomes, helps prevent DNA replication from stalling at telomeres Friday, 10 July 2009 Telomeres, the repetitive sequences of DNA at the ends of linear chromosomes, have an important function: They protect vulnerable chromosome ends from molecular attack. Researchers at Rockefeller University now show that telomeres have their own weakness. They resemble unstable parts of the genome called fragile sites where DNA replication can stall and go awry. But what keeps our fragile telomeres from falling apart is a protein that ensures the smooth progression of DNA replication to the end of a chromosome. The research, led by Titia de Lange, head of the Laboratory of Cell Biology and Genetics, and first author Agnel Sfeir, a postdoctoral associate in the lab, suggests a striking similarity between telomeres and common fragile sites, parts of the genome where breaks tend to occur, albeit infrequently. (Humans have 80 common fragile sites, many of which have been linked to cancer.) De Lange and Sfeir found that these newly discovered fragile sites make it difficult for DNA replication to proceed, a discovery that unveils a new replication problem posed by telomeres. At the centre of the discovery is a protein known as TRF1, which de Lange, in an effort to understand how telomeres protect chromosome ends, discovered in 1995. Using a conditional mouse knockout, de Lange and Sfeir have now revealed that TRF1, which is part of a six-protein complex called shelterin, enables DNA replication to drive smoothly through telomeres with the aid of two other proteins.

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Fragile telomeres. This is a series of images showing chromosomes with fragile telomeres (green). Without the protein TRF1, telomeres resemble common fragile sites, unstable regions on chromosomes that break into segments or stretch due to faulty DNA replication. Credit: Cell.

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“Telomeric DNA has a repetitive sequence that can form unusual DNA structures when the DNA is unwound during DNA replication,” says de Lange. “Our data suggest that TRF1 brings in two proteins that can take out these structures in the telomeric DNA. In other words, TRF1 and its helpers remove the bumps in the road so that the replication fork can drive through.” The work, published in the July 10 issue of Cell, began when Sfeir deleted TRF1 and saw that the telomeres resembled common fragile sites, suggesting that TRF1 protects telomeres from becoming fragile. Instead of a continuous string of DNA, the telomeres were broken into fragments of twos and threes. To see if the replication fork stalls at telomeres, de Lange and Sfeir joined forces with

Carl L. Schildkraut, a researcher at Albert Einstein College of Medicine in New York City. Using a technique called SMARD, the researchers observed the dynamics of replication across individual DNA molecules — the first time this technique has been used to study telomeres. In the absence of TRF1, the fork often stalled for a considerable amount of time. The only other known replication problem posed by telomeres was solved in 1985 when it was shown that the enzyme telomerase elongates telomeres, which shorten during every cell division. The second problem posed by telomeres, the so-called end-protection problem, was solved by de Lange and her colleagues when they found that shelterin protects the ends of linear chromosomes, which look like damaged DNA, from unnecessary repair. Working with TRF1, the very first shelterin protein ever to be identified, de Lange and Sfeir have not only unveiled a completely unanticipated replication problem at telomeres, they have also shown how it is solved. The research lays new groundwork for the study of common fragile sites throughout the genome, explains de Lange. “Fragile sites have always been hard to study because no specific DNA sequence precedes or follows them,” she says. “In contrast, telomeres represent fragile sites with a known sequence, which may help us understand how common fragile sites break throughout the genome — and why.” Reference: Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient Replication Agnel Sfeir, Settapong T. Kosiyatrakul, Dirk Hockemeyer, Sheila L. MacRae, Jan Karlseder, Carl L. Schildkraut and Titia de Lange Cell, July 10, 2009, 138(1): 90-103 ......... ZenMaster

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How an Enzyme Tells Stem Cells Which Way to Divide

University of Oregon biochemists report a mechanism dictating cell division is not a long cascade of events Thursday, 14 May 2009 Driving Miranda, a protein in fruit flies crucial to switch a stem cell's fate, is not as complex as biologists thought, according to University of Oregon biochemists. They've found that one enzyme (aPKC or atypical protein kinase C) stands alone and acts as a traffic cop that directs which roads daughter cells will take. "Wherever aPKC is at or on a cell's cortex or membrane, Miranda isn't," says Kenneth E. Prehoda, a professor in the chemistry department and member of the UO's Institute of Molecular Biology. When a stem cell duplicates into daughter cells, the side, or cortical domain, containing aPKC continues as a stem cell, while the other domain with Miranda becomes a differentiated cell such as a neuron that forms the central nervous system. Prehoda and co-author Scott X. Atwood, who studied in Prehoda's lab and recently earned his doctorate, describe how the mechanism works in the May 12 issue of the journal Current Biology. Instead of a complex cascade of protein deactivation steps that many biologists have theorized, Prehoda said, aPKC strips phosphate off an energy-transfer nucleotide known as ATP and then attaches it to Miranda. This process forces Miranda away from aPKC and helps determine the fates of subsequent daughter cells. "This process is pretty simple," he said, when viewed from a biochemical perspective. "What happens is that Miranda gets phosphorylated by aPKC, turning it into an inactivated substrate and pushing it into another location in the cell."

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Dividing Drosophila neuroblast. aPKC is shown in green at the top half of a fruit fly neuroblast. Miranda, in blue, has been driven away to the opposite side. Upon division, the top half will remain a stem cell, while the bottom will become a differentiated cell. Credit: Courtesy of Kenneth Prehoda.

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Much of the paper in Current Biology is devoted to discuss why the more complex scenarios are not accurate. "There have been a lot of ideas on how this works, and most seemed to be really complicated and difficult to explain. We have found it's a much simpler mechanism," Prehoda said, adding that the mechanism likely is similar in many other types of cells, not just stem cells. "It's a basic-research question. How does this polarity occur? In order to develop stem cell-specific therapeutics based on a rational methodology you have to understand the mechanism," he said. If Miranda is improperly isolated into other regions by aPKC, the stem cell divides symmetrically, with both daughter cells adopting the same fate, In turn, Prehoda said, these cells can become tumours as they continue to rapidly divide without proper polarization. ......... ZenMaster

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