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Posts Tagged ‘Cell Biology’


Biologists Wondered—How Old are Cells in an Organism?

Reporter: Irina Robu, PhD

Scientists form Salk Institute discovered that the mouse brain, live and pancreas contain populations of cells and proteins with extremely long lifespans with some as old as neurons. The research was published in Cell Metabolism on June 6, 2019. The general idea is that most neurons in the brain do not divide during adulthood and experience a long lifespan and age-related decline. Yet, due to limitations the lifespan of cells outside of the brain was difficult to determine.

However, the researchers knew very well that neurons are not replaced during the lifespan, they used them as control to compare other non-dividing cells. The team used an electron isotope labeling with hybrid imaging method to visualize and quantify cell and protein age and turnover in the brain, pancreas and liver in the young and old rodent models.

To confirm that their method is correct, the scientist determined first the age of the neurons and then realized that the cells that line blood vessels, endothelial cells were as old as neurons. According to this research, it means that some non-neuronal cells do not replicate themselves throughout the lifespan. The pancreas, the organ responsible for maintaining blood sugar levels and secreting digestive enzymes showed cells of all ages. Still, some beta cells, replicate during the lifetime and are relatively young, while others do not divide and were long lived. Yet, delta cells found in stomach do not divide at all.
Unlike other type of cells, the liver cells have the capacity to regenerate during adulthood. The researchers expected to observe young liver cells, however the majority of liver cells were found to be as old as the animal, while the cells that line blood vessels and stellate like cells, another liver type cell were short lived.

But on the molecular level, a selection of long-lived cells contains protein complexes displaying age mosaicism. Due to the modern visualizing technologies, scientists were able to pinpoint the age of the cells and their supra-molecular complexes precisely. The ultimate goal to determining the age of the cells and sub-cellular structures is to provide insights into cell maintenance and repair mechanism and utilize these mechanisms to prevent or delay old age-linked decline of organs with limited cell regeneration.

SOURCE

https://www.salk.edu/news-release/how-old-are-your-organs-to-scientists-surprise-organs-are-a-mix-of-young-and-old-cells/

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First Haploid Human Stem Cells

Reported: Irina Robu, PhD

Most of the cells in our body are diploid, which indicate they carry two sets of chromosomes—one from each parent. So far, scientists have only succeeded in generating haploid embryonic stem cells—which comprise a single set of chromosomes in non-human mammals such as mice, rats and monkeys. Nevertheless, scientists have tried to isolate and duplicate these haploid ESCs in humans, which would allow them to work with one set of human chromosomes as opposed to a mixture from both parents.

Scientists from Hebrew from The Hebrew University of Jerusalem, Columbia University Medical Center (CUMC) and The New York Stem Cell Foundation Research Institute (NYSCF) were successful in generating a new type of embryonic stem cells that has a single copy of the human genome, instead of two copies which is typically found in normal stem cells.

This landmark was finally obtained by Ido Sagi, working as a PhD student at the Hebrew University of Jerusalem which was successful in isolating and maintaining haploid embryonic stem cells in humans. Unlike in mice, these haploid stem cells were capable to differentiate into various cell types such as brain, heart and pancreas, although holding a single set of chromosomes. Sagi and his advisor, Prof. Nissim Benvenisty showed that this new human stem cell type will play an important role in human genetic and medical research.  This new human cell type cell type will aid in understanding human development and it will make genetic screening simpler and more precise, by examining a single set of chromosomes.

Based on this research, the Technology Transfer arm of the Hebrew University, started a new company New Stem, which is developing a diagnostic kit for predicting resistance to chemotherapy treatments. By gathering a broad library of human pluripotent stem cells with various genetic makeups and mutations. The company is planning to use this kit for personalized medication and future therapeutic and reproductive products.

SOURCE

https://medicalxpress.com/news/2017-06-haploid-human-stem-cells-medical.html#jCp

Other related articles published in this Open Access Online Scientific Journal include the following:

Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

Reporter: Aviva Lev-Ari, PhD, RN

Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

 

 

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Reporter: Irina Robu, PhD

Using the century-old cutting method, it would take a researcher five hours to cut 100 cells, and by the time they were done, the cells they cut first would be well on their way to healing.

In an effort to comprehend how a single cell heal, mechanical engineer Sing Tand developed a microscopic guillotine that proficiently cuts cells into two.

Tang, who is an assistant professor of mechanical engineering at Stanford University knew that finding a way to competently slice the cell in two could lead to engineering self-healing materials and machines. In order, to efficiently slice a cell in two he developed a tool that could cut 150 cells in just over 2 minutes, and the cuts were much more standardized and synchronized in the stage of their repair process. They attained this rate by creating a scaled-up version of their tool with eight identical parallel channels that run simultaneously. Being able to efficiently study cell healing could eventually help scientists study and treat a variety of human diseases such as cancer and neurodegenerative diseases. Prior to Tang’s cellular guillotine, scientists used to slice cells by hand under a microscope using a glass needle which is a method that can lead to errors.

Tang’s method can be the Holy Grail of engineering self-healing materials and machines.

SOURCE

http://news.stanford.edu/2017/06/26/stanford-scientists-create-cellular-guillotine-studying-single-cell-wound-repair/

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Nanostraws Developed at Stanford Sample a Cell’s Contents without Damage

Reporter: Irina Robu, PhD

Cells within our bodies change over time and divide, with thousands of chemical reactions happening within cell daily. Nicholas Melosh, Associate Professor of Materials Science and Engineering, developed a new, non-destructive system for sampling cells with nanoscale straws which could help uncover mysteries about how cells function.

Currently, cells are sampled via lysing which ruptures the cell membrane which means that it can’t ever be sampled again. The sample system that Dr. Melosh invented banks on, on tiny tubes 600 times smaller than a strand of hair that allow researchers to sample a single cell at a time. The nanostraws penetrate a cell’s outer membrane, without damaging it, and draw out proteins and genetic material from the cell’s salty interior.

The Nanostraw sampling technique, according to Melosh, will knowingly impact our understanding of cell development and could result to much safer and operational medical therapies because the technique allows for long term, non-destructive monitoring. The sampling technique could also inform cancer treatments and answer questions about why some cancer cells are resistant to chemotherapy while others are not. The sampling platform on which the nanostraws are grown is tiny, similar to the size of a gumball. It’s called the Nanostraw Extraction (NEX) sampling system, and it was designed to mimic biology itself.

The goal of developing this technology was to make an impact in medical biology by providing a platform that any lab could build.

SOURCE

http://news.stanford.edu/2017/02/20/minuscule-nanostraws-sample-cells-contents-without-damage

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Spermatogenic Defects in Sex Reversed Mice

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

“Sex reversed” (Sxr) is an inherited form of sex reversal that causes XX and XO mice to develop as phenotypically normal males. Adult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis reports when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one-half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases.

 

A minority of XYSxra mice are sterile, and these may have testes as small as those from XOSxra mice. Adult XOSxra mice have consistently small testes and are invariably sterile. The reported results document the testicular defects in XYSxra and XOSxra testes as they first arise during puberty. The only other quantitative data on XYSxra and XOSxra spermatogenesis are for adult mice. A previous report described XYSxra testes as being a “mosaic” of normal and defective spermatogenesis. Recently a more extensive analysis was carried out of adult XYSxra and XOSxra testes. Once again there is good agreement with the present results in that the spermatogenic failure in XYSxra testes was predominantly between pachytene and diplotene, while in XOSxra testes the block was predominantly during the meiotic metaphases. To explain the spermatogenic anomalies in XYSxra and XOSxra testes, Burgoyne and Baker (1984) invoked the “meiotic pairing site” hypothesis of Miklos (1974). The other notable feature of the present study was the demonstration that the testicular deficiency is manifested earlier (with respect to age and spermatogenic stage) in XYSxra testes than in XOSxra testes. Krzanowska (1989) recently reported increased levels of X-Y univalence in pubertal XY males. So, it is suggested that this reduced efficiency of X-Y pairing at puberty that leads to the increased incidence of diploid spermatids in pubertal XYSxra males and to the presence of diploid spermatids in pubertal XY males. The other feature of pubertal XYSxra testes that deserves mention is the increase in the number of differentiating spermatogonia.

 

The conclusion is that most of the spermatogenic deficiencies in XYSxra and XOSxra testes can be explained in terms of the “meiotic pairing site” hypothesis, which links spermatogenic failure with sex chromosome univalence during meiosis. In XYSxra testes a variable proportion of pachytene spermatocytes have the X and Y unpaired, and the elimination of these cells explains the variable reduction in testis size and fertility. In XOSxra testes all spermatocytes have a univalent sex chromosome, accounting for the almost total spermatogenic block in these mice. It is suggested that the affected spermatocytes are eliminated earlier in XYSxra testes than in XOSxra testes, because two univalent sex chromosomes have more unpaired sites than the univalent X alone.

 

References:

 

Sutcliffe M. J., Darling S. M., Burgoyne P. S. (1991) Spermatogenesis in XY, XYSxra and XOSxra Mice: A quantitative analysis of spermatogenesis throughout puberty. Molecular Reprod. Dev. 30(2), 81–89.

 

Burgoyne P. S., Baker T. G. (1984) Meiotic pairing and gametogenic failure. In CW Evans and HG Dickinson (eds): “Controlling Events in Meiosis (38th Symp SOC Exp Biol).” Cambridge Company of Biologists, pp 349-362.

 

Miklos G. L. G. (1974) Sex-chromosome pairing and male fertility. Cytogen. Cell Genet. 13, 558-577.

 

Krzanowska H (1989) X-Y chromosome dissociation in mouse strains differing in efficiency of spermatogenesis: Elevated frequency of univalents in pubertal males. Gamete. Res. 23, 357-365.

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3D “Squeeze” Helps Adult Cells Become Stem Cells

Reported by: Irina Robu, PhD

Scientists based at Ecole Polytechnique Fédérale de Lausanne led by Matthias Lutolf have been engineering 3D extracellular matrices—gels. These scientists report that they have developed a gel that boosts the ability of normal cells to revert into stem cells by simply “squeezing” them.

The detail of the scientists’ work appeared in Nature Materials, January 11, 2015 in an article entitled, “Defined three-dimensional microenvironments boost induction of pluripotency.” According to the authors they find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodeling. They concluded that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity.

The researchers discovered that they could reprogram the cells faster and more efficiently  by simply adjusting the composition, hence the stiffness and density of the surrounding gel. As a result, the gel exerts different forces on the cells, “squeezing” them.

The scientists propose that the 3D environment is key to this process, generating mechanical signals that work together with genetic factors to make the cell easier to transform into a stem cell. The technique can be applied to a large number of cells to produce stem cells on an industrial scale.

Source

http://www.genengnews.com/gen-news-highlights/3d-squeeze-helps-adult-cells-become-stem-cells/81252223/

 

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brown adipocyte protein CIDEA promotes lipid droplet fusion

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

 

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding

Parker, Nicholas T Ktistakis, Ann M Dixon, Judith Klein-Seetharaman, Susan Henry, Mark Christian Dirk Dormann, Gil-Soo Han, Stephen A Jesch, George M Carman, Valerian Kagan, et al.

eLife 2015;10.7554/eLife.07485     http://dx.doi.org/10.7554/eLife.07485

 

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

 

Evolutionary pressures for survival in fluctuating environments that expose organisms to times of both feast and famine have selected for the ability to efficiently store and release energy in the form of triacyclglycerol (TAG). However, excessive or defective lipid storage is a key feature of common diseases such as diabetes, atherosclerosis and the metabolic syndrome (1). The organelles that are essential for storing and mobilizing intracellular fat are lipid droplets (LDs) (2). They constitute a unique cellular structure where a core of neutral lipids is stabilized in the hydrophilic cytosol by a phospholipid monolayer embedding LD-proteins. While most mammalian 46 cells present small LDs (<1 Pm) (3), white (unilocular) adipocytes contain a single giant LD occupying most of their cell volume. In contrast, brown (multilocular) adipocytes hold multiple LDs of lesser size, increasing the LD surface/volume ratio which facilitates the rapid consumption of lipids for adaptive thermogenesis (4).

The exploration of new approaches for the treatment of metabolic disorders has been stimulated by the rediscovery of active brown adipose tissue (BAT) in adult humans (5, 6) and by the induction of multilocular brown-like cells in white adipose tissue (WAT) (7). The multilocular morphology of brown adipocytes is a defining characteristic of these cells along with expression of genes such as Ucp1. The acquisition of a unilocular or multilocular phenotype is likely to be controlled by the regulation of LD growth. Two related proteins, CIDEA and CIDEC promote LD enlargement in adipocytes (8-10), with CIDEA being specifically found in BAT. Together with CIDEB, they form the CIDE (cell death-inducing DFF45-like effector) family of LD-proteins, which have emerged as important metabolic regulators (11).

Different mechanisms have been proposed for LD enlargement, including in situ neutral lipid synthesis, lipid uptake and LD-LD coalescence (12-14). The study of CIDE 62 proteins has revealed a critical role in the LD fusion process in which a donor LD progressively transfers its content to an acceptor LD until it is completely absorbed (15). However, the underlying mechanism by which CIDEC and CIDEA facilitate the interchange of triacylglycerol (TAG) molecules between LDs is not understood. In the present study, we have obtained a detailed picture of the different steps driving this LD enlargement process, which involves the stabilization of LD pairs, phospholipid binding, and the permeabilization of the LD monolayer to allow the transference of lipids.

 

CIDEA expression mimics the LD dynamics observed during the differentiation of brown adipocytes

Phases of CIDEA activity: LD targeting, LD-LD docking and LD growth

A cationic amphipathic helix in C-term drives LD targeting

The amphipathic helix is essential for LD enlargement

LD-LD docking is induced by the formation of CIDEA complexes

CIDEC differs from CIDEA in its dependence on the N-term domain

CIDEA interacts with Phosphatidic Acid

PA is required for LD enlargement

 

The Cidea gene is highly expressed in BAT, induced in WAT following cold exposure (46), and is widely used by researchers as a defining marker to discriminate brown or brite adipocytes from white adipocytes (7, 28). As evidence indicated a key role in the LD biology (47) we have characterized the mechanism by which CIDEA promotes LD enlargement, which involves the targeting of LDs, the docking of LD pairs and the transference of lipids between them. The lipid transfer step requires the interaction of CIDEA and PA through a cationic amphipathic helix. Independently of PA-binding, this helix is also responsible for anchoring CIDEA in the LD membrane. Finally, we demonstrate that the docking of LD pairs is driven by the formation of CIDEA complexes involving the N-term domain and a C-term interaction site.

CIDE proteins appeared during vertebrate evolution by the combination of an ancestor N-term domain and a LD-binding C-term domain (35). In spite of this, the full process of LD enlargement can be induced in yeast by the sole exogenous expression of 395 CIDEA, indicating that in contrast to SNARE-triggered vesicle fusion, LD fusion by lipid transference does not require the coordination of multiple specific proteins (48). Whereas vesicle fusion implicates an intricate restructuring of the phospholipid bilayers, LD fusion is a spontaneous process that the cell has to prevent by tightly controlling their phospholipid composition (23). However, although phospholipid-modifying enzymes have been linked with the biogenesis of LDs (49, 50), the implication of phospholipids in physiologic LD fusion processes has not been previously described.

Complete LD fusion by lipid transfer can last several hours, during which the participating LDs remain in contact. Our results indicate that both the N-term domain and a C-term dimerization site (aa 126-155) independently participate in the docking of LD pairs by forming trans interactions (Fig. 7). Certain mutations in the dimerization sites that do not eliminate the interaction result in a decrease on the TAG transference efficiency, reflected on the presence of small LDs docked to enlarged LDs. This suggests that in addition to stabilizing the LD-LD interaction, the correct conformation of the 409 CIDEA complexes is necessary for optimal TAG transfer. Furthermore, the formation of stable LD pairs is not sufficient to trigger LD fusion by lipid transfer. In fact, although LDs can be tightly packed in cultured adipocytes, no TAG transference across neighbour LDs is observed in the absence of CIDE proteins (15), showing that the phospholipid monolayer acts as a barrier impermeable to TAG. Our CG-MD simulations indicate that certain TAG molecules can escape the neutral lipid core of the LD and be integrated within the aliphatic chains of the phospholipid monolayer. This could be a transition state 416 prior to the TAG transference and our data indicates that the docking of the amphipathic helix in the LD membrane could facilitate this process. However, the infiltrated TAGs in LD membranes in the presence of mutant helices, or even in the absence of docking, suggests that this is not enough to complete the TAG transference.

To be transferred to the adjacent LD, the TAGs integrated in the hydrophobic region of the LD membrane should cross the energy barrier defined by the phospholipid polar heads, and the interaction of CIDEA with PA could play a role in this process, as suggested by the disruption of LD enlargement by the mutations preventing PA-binding (K167E/R171E/R175E) and the inhibition of CIDEA after PA depletion. The minor effects observed with more conservative substitutions in the helix, suggests that the presence of positive charges is sufficient to induce TAG transference by attracting anionic phospholipids present in the LD membrane. PA, which requirement is indicated by our PA-depletion experiments, is a cone-shaped anionic phospholipid which could locally destabilize the LD monolayer by favoring a negative membrane curvature incompatible with the spherical LD morphology (51). Interestingly, while the zwitterion PC, the main component of the monolayer, stabilizes the LD structure (23), the negatively charged PA promote their coalescence (29). This is supported by our CD-MD results which resulted in a deformation of the LD shape by the addition of PA. We propose a model in which the C-term amphipathic helix positions itself in the LD monolayer and interacts with PA molecules in its vicinity, which might include trans interactions with PA in the adjacent LD. The interaction with PA disturbs the integrity of the phospholipid barrier at the LD-LD interface, allowing the LD to LD transference of TAG molecules integrated in the LD membrane (Fig. 7). Additional alterations in the LD composition could be facilitating TAG transference, as differentiating adipocytes experience a reduction in saturated fatty acids in the LD phospholipids (52), and in their PC/PE ratio (53) which could increase the permeability of the LD membranes, and we previously observed that a change in the molecular structures of TAG results in an altered migration pattern to the LD surface (32).

During LD fusion by lipid transfer, the pressure gradient experienced by LDs favors TAG flux from small to large LDs (15). However, the implication of PA, a minor component of the LD membrane, could represent a control mechanism, as it is plausible that the cell could actively influence the TAG flux direction by differently regulating the levels of PA in large and small LDs, which could be controlled by the activity of enzymes such as AGPAT3 and LIPIN-1J (13, 30). This is a remarkable possibility, as a switch in the favored TAG flux direction could promote the acquisition of a multilocular phenotype and facilitate the browning of WAT (24). Interestingly, Cidea mRNA is the LD protein- encoding transcript that experiences the greatest increase during the cold-induced process by which multilocular BAT-like cells appear in WAT (24). Furthermore, in BAT, cold exposure instigates a profound increase in CIDEA protein levels that is independent of transcriptional regulation (54). The profound increase in CIDEA is coincident with elevated lipolysis and de novo lipogenesis that occurs in both brown and white adipose tissues after E-adrenergic receptor activation (55). It is likely that CIDEA has a central role in coupling these processes to package newly synthesized TAG in LDs for subsequent lipolysis and fatty acid oxidation. Importantly, BAT displays high levels of glycerol kinase activity (56, 57) that facilitates glycerol recycling rather than release into the blood stream, following induction of lipolysis (58), which occurs in WAT. Hence, the reported elevated glycerol released from cells depleted of CIDEA (28) is likely to be a result of decoupling lipolysis from the ability to efficiently store the products of lipogenesis in LDs and therefore producing a net increase in detected extracellular glycerol. This important role of CIDEA is supported by the marked depletion of TAG in the BAT of Cidea null mice following overnight exposure to 4 °C (28) and our findings that CIDEA-dependent LD enlargement is maintained in a lipase negative yeast strain.

Cidea and the genes that are required to facilitate high rates of lipolysis and lipogenesis are associated with the “browning” of white fat either following cold exposure (46) or in genetic models such as RIP140 knockout WAT (59). The induction of a brown- like phenotype in WAT has potential benefits in the treatment and prevention of metabolic disorders (60). Differences in the activity and regulation of CIDEC and CIDEA could also be responsible for the adoption of unilocular or multilocular phenotypes. In addition to their differential interaction with PLIN1 and 5, we have observed that CIDEC is more resilient to the deletion of the N-term than CIDEA, indicating that it may be less sensitive to regulatory posttranslational modifications of this domain. This robustness of CIDEC activity together with its potentiation by PLIN1, could facilitate the continuity of the LD enlargement in white adipocytes until the unilocular phenotype is achieved. In contrast, in brown adipocytes expressing CIDEA the process would be stopped at the multilocular stage for example due to post-translational modifications that modulate the function or stability of the protein or alteration of the PA levels in LDs.

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