RxPG News : Cytology

Ribosomes already showing medical importance

Wed, 07 Oct 2009 20:05:54 PST

( from http://www.rxpgnews.com ) Indian-born scientist Venkatraman Ramakrishnan, awarded the Nobel Prize for Chemistry for his work on ribosomes, Wednesday said his work has established the ribosome's 'medical importance', while researchers said his findings could help in the global fight against tuberculosis.

Praising the Medical Research Council's Laboratory of Molecular Biology in Cambridge and the University of Utah for supporting his work, Ramakrishnan said, 'The idea of supporting long term basic research like that at LMB does lead to breakthroughs; the ribosome is already starting to show its medical importance.'

Tamil Nadu-born Ramakrishnan, now a US citizen, won the Nobel along with American scientist Thomas A. Steitz and Israeli Ada E. Yonath for studies of the structure and function of the ribosome, an element which translates information contained in the DNA code into life.

The MRC said Ramakrishnan's research could lead to the development of better drugs to fight against extreme forms of tuberculosis - a disease that kills nearly 1.8 million people every year.

The MRC said Ramakrishnan's basic research on the arrangement of atoms in the ribosome could help researchers to design antibiotics to treat people who are infected with a bacterium that has developed antibiotic resistance, for example some of the strains of bacteria that cause tuberculosis.

'Better targeting of the bacterial ribosome should also help to avoid negative effects on human cells thereby reducing the side effects of taking antibiotics,' the MRC said in a statement.

Ramakrishnan added: 'I have to say that I am deeply indebted to all of the brilliant associates, students and post docs who worked in my lab as science is a highly collaborative enterprise.'

MRC chief executive Sir Leszek Borysiewicz said he was 'delighted' that Ramakrishnan had won the Nobel Prize. 'The MRC is committed to long-term support of the difficult areas of basic science as exemplified by Venki's success. It is only on the back of such discoveries that we can continue to drive translation into benefits for human health.'

According to the World Health Organisation, tuberculosis is spreading at the rate of one new infection every second. In 2007, there were 9.27 million new cases - 500,000 of them resistant to drugs and 50,000 'extensively drug resistant'.

How cells adhere so firmly to blood vessel walls

Sat, 04 Nov 2006 20:56:37 PST

( from http://www.rxpgnews.com ) Blood is the universal means with which different types of cells are transported in our bodies. Its movement is determined by hydrodynamic forces. The cells anchor themselves to the walls of the blood vessels in the target tissue with the aid of special adhesive molecules, which are also called receptors. In many cases these receptors are grouped in the cell surface in nanometer-sized patches. The adhesion process is based on the key and lock principle: as a rule, an adhesion molecule only bonds with specific partners. This guarantees that the cells are only brought to a halt where they are to fulfill their biological function.

These processes are of great relevance to medicine. For example, red blood corpuscles infected with malaria stick to blood vessel walls to escape destruction in the spleen and patrolling white blood corpuscles dock with the blood vessel walls in order to seek out foreign bodies in the adjacent tissue. These "wandering adhesive cells" also include stem cells, which move from the bone marrow to their target tissue, and cancer cells which metastasize in the body.

To understand these processes better, it is necessary to show and track the interplay of hydrodynamics and molecular adhesion patches in detail. To do this, scientists at the Max Planck Institute of Colloids and Interfaces in Potsdam and at the University of Heidelberg have developed a computer model which systematically examines how the density, size and number of the receptor groups affect the adhesion. In millions of computer experiments, the researchers established how much these parameters influenced the time it took for an adhesive patch to find a partner on the target tissue while a flow of liquid was moving the cell in accordance with the laws of hydrodynamics. These calculations are very complex because they have to take into account hundreds of patches for each cell.

The illustration shows the computer model for cell adhesion in the hydrodynamic flow. It consists of a sphere with randomly distributed adhesion patches and a substrate with the relevant complementary partners (Image: Max Planck Institute of Colloids and Interfaces)

The initial simulations investigating the influence of flow speed on adhesion revealed that the faster the flow of the liquid, the faster the cells find their adhesion partners as the cell can scan a larger area. The researchers then varied the density of the patches and established that beyond a threshold value of a few hundred receptor areas per cell, there was no further acceleration of adhesion rate because from that point the effective radii of the patches overlap due to their thermal random movement. Similar results were seen with the size of the adhesive areas, which obviously plays a less significant part in effective adhesion.

However, changing the height at which the adhesive patches protrude above the cell membrane has surprising results: even small increases give rise to much faster adhesion. White blood corpuscles use this effect by covering themselves with hundreds of protrusions called microvilli, which stand about 350 nanometers above the cell surface - almost four per cent of the cell diametre. Red blood corpuscles infected with malaria also use this "hedgehog spine" strategy. They have "knobs" that are 20 nanometers high on their surface.

The scientists suspect that their simulations have helped them to discover a general biological design principle which also occurs in other hydrodynamic contexts - in bacteria, for example, which collect in medical devices through which liquids flow, such as catheters or dialysis equipment. In the future, the software they have developed will allow these situations to be examined more closely than ever before and is another step on the way to "computational" biology.

New Insight into Cell Division

Sun, 29 Oct 2006 21:35:37 PST

( from http://www.rxpgnews.com ) The rod-shaped filaments of the microtubules are responsible for dividing the chromosomes in a cell. They form a connection between the starting point in the chromosomes, the kinetochors, and the centrosome. The centrosome organizes the spindle-shaped arrangement of the microtubule filaments in the cell by means of gamma tubulin ring complexes. Until now it was assumed that control of even distribution of chromosomes was only monitored by the checkpoint kinases in the kinetochors. When microtubules are correctly attached to the kinetochors, these kinases then inform the cell that a precise distribution of chromosomes can be carried out.

Scientists at the Max Planck Institute for Molecular Genetics in Berlin have now discovered that the checkpoint kinases are also associated with the gamma tubulin ring complex proteins. Accordingly, the researchers have proved that these checkpoint kinases also exist alongside the centrosome and perform their function there. This is a crucial new finding, since it shows that the correct organization of these filaments at both ends is important for correctly distributing the chromosomes during cell division.

Abnormal mitotic spindle, whose deformation is caused by defective cell division control. The chromosomes are dyed red, the microtubules green. (Image: Max Planck Institute for Molecular Genetics)

Another surprising finding is that these control mechanisms function independently of the integrity of the kinetochors or the centrosomes. This reveals that a cell has widely differing mechanisms for controlling cell division and monitors these directly at the level of protein complexes.

These findings are extremely important for the understanding of cell division regulation, which is often disrupted in cancer cells. The checkpoint kinases in cancer cells are frequently modified or present in incorrect quantity ratios. In their next step, the researchers plan to carry out targeted analysis of the molecular reactions between the different regulatory proteins and then investigate how these interactions differ in healthy cells and cancer cells. Long term, this could lead to the development of new diagnostic or therapeutic strategies.

New method for the controlled initiation of membrane fusion

Thu, 19 Oct 2006 23:38:37 PST

( from http://www.rxpgnews.com ) The process of membrane fusion is essential for the structure and dynamics of all cells in our bodies. Fusion is indispensable for intracellular vesicle traffic, which sustains the compartmental organisation of cells. Likewise, membrane fusion is the basic molecular process that controls the communication between cells via the secretion of hormones, neurotransmitters, and growth factors. Furthermore, fusion processes are also crucial for the interactions between our cells and various pathogens such as viruses and bacteria. However, in spite of the ubiquity of membrane fusion, many aspects of this process have remained rather controversial. This situation reflects the absence of well-defined protocols by which one can induce fusion in a controlled manner and subsequently study its dynamics with high temporal resolution.

In order to clarify the dynamics of the fusion process in more detail, scientists from the Max Planck Institute of Colloids and Interfaces developed two different protocols for the fusion of unilamellar vesicles, which had a diameter of tens of micrometers but consist of only a single lipid membrane with a thickness of about four nanometers. Even though such a membrane is much thinner than the optical resolution limit, one can observe its shape using different methods of optical microscopy such as phase contrast and confocal microscopy, see Figure 1. The two protocols provide two different methods of bringing a pair of unilamellar vesicles into close contact, to initiate the fusion of their membranes in a controlled manner and to study the subsequent fusion dynamics with high temporal resolution.

Confocal microscopy images of lipid vesicles containing two different fluorescent dyes: (a) Two vesicles before fusion (equatorial section); (b) Vesicles fused by applying a short electric pulse; and (c) Three-dimensional image of a two-domain vesicle produced by fusion of two membranes with different composition (Image: Max Planck Institute of Colloids and Interfaces).

In the first protocol, artificial fusogenic molecules (a liposome whose outer wall contains molecules that cause cell fusion) or ligands, synthesized by the collaborators from Collège de France, were incorporated into the lipid membranes. Two unilamellar vesicles were aspirated by two glass micropipettes. Close proximity of the vesicle membranes was achieved by displacing these micropipettes. Membrane fusion was subsequently induced by the local addition of ions that form a complex between two fusogenic molecules embedded in the opposing membranes. In the second protocol, two lipid vesicles were brought into contact by alternating electric fields. Once close contact was established, membrane fusion was induced by exposing the vesicles to an additional electric pulse. Such a pulse leads to the formation of membrane pores in the opposing membranes, which subsequently fuse in order to dispose of the edges of the pores.

Both for ligand-mediated fusion and for electrofusion, the dynamics of fusion was observed using a fast digital camera with an acquisition rate of 20 000 frames per second, which corresponds to a temporal resolution of 50 microseconds. "Since previous direct imaging studies of membrane fusion were limited to time scales that exceed tens of milliseconds, the new experiments improved the temporal resolution by three orders of magnitude and revealed that the fusion process is surprisingly fast", says Rumiana Dimova, group leader in the Max Planck Institute of Colloids and Interfaces, and one of the participating scientists.

Indeed, only a few hundred microseconds after the initiation of the fusion process, the fusion neck connecting the two vesicles has already reached a diameter of a couple of micrometres as shown in Figure 1(b). This implies that the fusion neck has an average expansion velocity of centimetres per second and that the initial formation of the fusion neck can be completed within about 200 nanoseconds. This is in good agreement with recent computer simulations of tension-induced fusion. In this way, the Max Planck researchers have managed to bridge the gap between theoretical predictions and available experimental knowledge about the fusion process.

The experimental fusion protocols developed in the present study can be applied to other biomimetic systems and can be used to construct new ones. Particularly interesting systems, which can be studied in this way, are mixed membranes containing both lipids and fusogenic proteins such as SNAREs. One example for the construction of new biomimetic systems is provided by the formation of large vesicles with several intramembrane domains as shown in Figure 1(c). Another example consists of vesicles that contain different chemical reactants. The fusion of such vesicles initiates the corresponding chemical reactions in these rather small compartments and might be useful in order to synthesize new nanomaterials. In general, controlled membrane fusion has many potential applications in bioengineering, pharmacology, and medicine.

CPK3 and CPK6 function as ion channel regulators in guard cell signaling

Wed, 11 Oct 2006 05:20:37 PST

( from http://www.rxpgnews.com ) When water is scarce, plants synthesize a hormone that facilitates conservation by closing stomatal pores on their leaves. Each pore is surrounded by a pair of guard cells that control stomatal aperture in response to various stimuli, including the drought-triggered hormone called abscisic acid (ABA). ABA signaling increases calcium levels in guard cells; calcium in turn acts on a variety of channels that regulate the transport of ions across the cell membranes. As both positively and negatively charged ions (called anions) cross the membrane, turgor pressure drops and stomata close.

These observations support a model in which ABA signaling includes calcium signaling. But ABA signaling also affects parallel pathways and mechanismsincluding raising pH levelsand no mutations in calcium-sensing proteins have been previously reported that positively transduce an ABA response in plants. Thus, identifying the molecules that sense and transduce calcium signals in guard cells would provide valuable insights into the mechanics of ABA signaling. Calcium-dependent protein kinases (CDPKs) are calcium-sensor candidates, but with 34 CDPK genes in Arabidopsis alone, functional redundancy in this enzyme family has likely thwarted efforts to characterize their contributionthat is, when the function of one is disrupted, another can step in to fill its role.

Confocal image of an Arabidopsis stomate showing two guard cells exhibiting green fluorescent protein and native chloroplast (red) fluorescence. (Image: Alex Costa)

In a new study, Izumi Mori, Julian Schroeder, and colleagues describe two CDPK genescpk6 and cpk3with clear roles in calcium and ABA signaling in guard cells. (In previous guard-cell microarray experiments, Schroeder and colleagues had narrowed down the number of guard cellexpressed CDPK genes to a more manageable number.) Losing function of the cpk3 and cpk6 genes in guard cells impairs ABA- and calcium-induces activation of a class of anion channels (slow, or S-type) and stomatal closure. Elevated calcium levels activate S-type anion channels through phosphorylationa chemical reaction that regulates protein activity; kinases typically function by phosphorylating target proteins.

Plant biologists can investigate gene function by inserting DNA (called transferred DNA, or T-DNA) from the soil bacterium Agrobacterium tumefaciens into a plant's genome. When the inserted T-DNA disrupts a gene's function, researchers can infer gene function based on observed defects in plants carrying the mutant genes, or alleles. After confirming that the cpk3 and cpk6 alleles were in fact function-disrupting mutants in Arabidopsis plants, Mori et al. sequenced the alleles and identified two different insertion mutations for both alleles (cpk3-1 and cpk3-2, and cpk6-1 and cpk6-2). Then they isolated single mutant plants, with two copies of just one allele, and double mutants, with two copies of different combinations of the alleles (for example, two copies of both cpk3-1 and cpk6-1 or of cpk3-2 and cpk6-2), for further study.

All the mutant plants looked normal, though both double mutants grew a bit behind schedule compared to the nonmutant (wild-type) plants. ABA-induced stomatal closure, however, was partially impaired. The researchers examined the mutants' effect on calcium and ABA activation of anion channels. In wild-type guard cells, elevated calcium levels activated large S-type anion channel currents, but this activation was significantly reduced in both cpk3 single mutant cells and even more so in single cpk6 mutants. Reduced currents were also observed in both double mutants, though they did maintain a background anion current. Double mutants also exhibited reduced ABA activation of the S-type anion channels. Interestingly, Mori et al. also found that ABA activation of another class of ion channels, calcium-permeable channels, was impaired in the single and double cpk mutants, revealing the first genetic mutants that impair both ABA regulation of calcium channels and calcium activation of anion channels. Thus, CDPKs play an important role in calcium-mediated ABA regulation of S-type anion channels, calcium channels, and stomatal closing.

This study provides direct genetic evidence that calcium sensors function in stomatal ABA signaling and that CPK3 and CPK6 function as ion channel regulators in guard cell signaling. Because stomatal closing was partially preserved in cells lacking these kinases and another class of (rapid) R-type anion channels was less affected in the cpk mutants, the authors further conclude that parallel calcium-dependent and -independent signaling mechanisms are at play in a branched guard-cell signaling network. Using a cell-specific signaling and protein regulation approach, as described here, researchers can begin the tall task of characterizing responses of gene-disruption mutants in other members of the large CDPK family function throughout the plant kingdom.

Disrupted Intercellular Communication Causes a Disfiguring Birth Defect

Wed, 13 Sep 2006 09:49:37 PST

( from http://www.rxpgnews.com ) Before a fertilized egg begins the repeated rounds of cell division that turn the single cell into a proliferating, streaming, differentiating mass of cells, its fate may already be sealed. Inherited mutations in genes involved in segregating and sorting embryonic cells can result in serious abnormalities in body patterning and appear to underlie an inherited X-linked disorder (so-called because the mutated genes lie on the X chromosome) called craniofrontonasal syndrome (CFNS). X-linked disorders tend to affect males more severely than females, because boys inherit just one X chromosome while girls inherit two: if one gene is defective, the other can fill in. CFNS is a rare departure from this pattern, with females exhibiting the most severe symptoms. This disfiguring disorder is characterized by a range of skull aberrations, including facial asymmetry, widely spaced eyes, and abnormal head shape, as well as polydactyly and fused digits.

A class of receptor protein-tyrosine kinases called Ephs and their ephrin binding partners (called ligands) regulate tissue patterning by restricting cell interactions, ensuring proper cell sorting, and establishing developmental compartment boundaries. Mutations in one ephrin gene, ephrin-B1, have been identified in patients with CFNS and have been associated with aberrant skeletal patterning in mutant âheterozygousâ female mice, which carry one normal and one nonfunctional copy of the ephrin-B1 gene. Mutations in connexins, structural proteins that form gap junction pores, also lead to cranial and skeletal defects in both mice and humans.

Localization of ephrin-B1 (green) and connexin43 (red) in 3T3 cells.

In a new study, Alice Davy, Jeffrey Bush, and Philippe Soriano elucidate the mechanisms of ephrin-mediated cell sorting, and show how the breakdown of the process causes physical abnormalities. The researchers worked with ephrin-B1 heterozygous female mice, polydactyl mutants with abnormally developed frontal bones in the skull (called the calvarial phenotype, after the name of the bones). They show that Eph/ephrin signaling regulates gap junction communication, which in turn controls cell sorting. Their results indicate that flawed cell sorting, resulting from dysregulated communication at gap junctionsâintracellular membrane channels with pores that allow coupled cells to exchange small moleculesâunderlies the skeletal abnormalities observed in the mice.

Previous studies established that ephrin-B1 heterozygous females exhibit polydactyly while males lacking their copy of ephrin-B1 and females lacking both copies do not. Polydactyly accompanied a random inactivation of X chromosomes in female cells (in which one X chromosome is silenced in some cells and the second is silenced in others) that created a mosaic pattern of ephrin-B1 expression, with ephrin-B1-expressing cells segregated from cells that didnât express the gene. Ephrin-B1 mutants also develop multiple defects in tissue derived from neural crest cellsâwhich give rise to cartilage, bone, connective tissue, and other specialized tissues.

In this study, Davy et al. show that the mosaic loss of ephrin-B1 blocked the differentiation of neural crest cells by disrupting the distribution of a connexin (Cx43) that regulates bone cell differentiation and forms gap junctional pores. Cx43 aggregated between wild-type (nonmutant) cells and between cells that lack ephrin-B1, but was rarely seen at the border between ephrin-B1-positive and -negative cells, suggesting that the mosaic cells restricted the number of junctional pores. Expression of the ephrin-B1 receptor, EphB2, is elevated in ephrin-B1-negative regions in ephrin-B1 heterozygous embryos, so the researchers suspected that interactions between the receptor and ligand reduced Cx43 levels and disrupted gap junction formationâwhich they confirmed by tracking gap junction communication in cell cultures. This defect might be mediated by a physical interaction between ephrin-B1 and Cx43.

The researchers propose that gap junction communication is inhibited by interactions between Eph-positive and ephrin-positive cells that cause Cx43 to be sequestered inside cells, where they canât form gap junction pores and establish cell-to-cell communicationâleading to skeletal abnormalities. This explains why the CFNS phenotype is more prevalent in females (who exhibit mosaic expression of ephrin-B1 through X inactivation). By contributing a mouse model with skull and digit defects that mimic those seen in humans, the researchers have provided a valuable platform for future investigations into the role of ephrins and gap junction communication in disfiguring skeletal disorders.

Sharing Responsibility for Clathrin Coat Assembly

Wed, 16 Aug 2006 08:57:37 PST

( from http://www.rxpgnews.com ) Membranes protect cells from extracellular insults, but in so doing also block entry to nutrients and other essential molecules. One way cells circumvent this problem is by selectively binding such molecules to receptors on the membrane, then pulling the whole lot into the cell and packaging them into vesicles. Clathrin moleculesthree-pronged pinwheel-shaped proteinsform an elaborate lattice coat around the vesicles, which ultimately bud off from the membrane and transport their cargo to their cellular destination.

This highly complex process, called clathrin-mediated endocytosis, requires a constellation of accessory proteins that interact with key protein hubs. Vesicle formation has traditionally been described as a linear process with the core proteins being clathrin and adaptor protein (AP) complexes. In a previous paper, Harvey McMahon and colleagues suggested that the process can be viewed as a network of protein interactions with clathrin and APs forming the two main hubs of the network. In a new study, Eva Schmid, Marijn Ford, McMahon, and colleagues use an impressive array of toolsbiophysical, biochemical, structural, and cell biologicalto shed light on the network dynamics of this endocytic interactome. APs orchestrate the process of cargo recruitment and assembly of the nascent vesicle and are the first hub of the endocytic network. They found that clathrin takes over from adaptors as a hub as clathrin assembles into a coat. This shift requires collaboration between the hubs, which operate within a dynamic network that performs multiple tasks simultaneously.

Of four AP complexes involved in cellular transport, AP2 figures mostly in plasma membrane endocytosis. The AP2 structure has long been likened to Mickey Mouse, with the four-subunit core representing Mickeys body and the two flanking appendages forming his ears, but mounting evidence suggests the British childrens book character Mr. Ticklea circular blob with gangly, elastic arms and little handsmay be a more apt comparison. Mr. Tickles body is the core, his arms are the two flexible hinge domains, and his hands are the two appendages, β-appendage and α-appendage. Whichever character you prefer, the core anchors the complex to the membrane and interacts with cargo molecules, and the appendages recruit accessory proteins for vesicle formation.

In their previous study, McMahon and colleagues found that α-appendages have two distinct interaction sites, allowing for clustered adaptor proteins to interact with many accessory proteins simultaneously. The AP2 α-appendage becomes a hub for protein interactions only in the initial stages of assembly. In this study, they focused on the β-appendage.

First, Schmid et al. determined the interaction partners of both appendages by removing the bound partners from cell extracts then analyzing them with mass spectrometry. They found a number of previously unidentified interaction partners for the β-appendage (and a few more for the α-appendage). Some interact only with the β-appendage, but many also interact with the α-appendage.

To understand the molecular details of the interactions, the researchers mutated key regions of the β-appendage interaction sites (the β-appendage also has a top and side site) then assessed the impact on their binding partners. They found that the top site mediates most interactions for the α-appendage and the side site does the same for the β-appendage. With this setup, accessory proteins that bind to the α-appendages top site can also bind to the β-appendages side site, leaving the appendages other sites free to interact with still more proteins. Interactors can bind to multiple appendages, allowing APs to serve as scaffolds for protein assembly. These results do not fully explain why two appendages exist, the researchers acknowledge, but because the same proteins interact with the top and side sites, its likely that the appendages collaborate to mediate these interactions.

Clathrin coat formation, Schmid et al. propose, is an outgrowth of increasingly stable interactions among a shifting network of proteins. Rapidly shifting interactions between isolated proteins give rise to coordinated, dynamic interactions between a network of proteins centered around the membrane, then to increasingly stable interactions as the coat assembles. The presence of both activated cargo receptors and lipid signaling molecules (phosphoinositides) in the membrane trigger the accumulation of adaptor complexes, which rapidly stabilize with the help of accessory proteins with multiple sites for AP2 appendage interactions. The accessory proteins recruit clathrin, which interacts with β-appendages and displaces accessory proteins as it accumulates and self-assembles during coat formation. Accessory proteins that interact only with appendages are shunted to the side, where clathrin polymers have not yet formed, while accessory proteins that can interact with clathrin are maintained. Having demonstrated the power of using a multidisciplinary approach to study the endocytic interactome, the researchers believe that the principles uncovered will apply to other protein networks.

Understanding the process of AIF release following MOMP during apoptosis

Wed, 02 Aug 2006 13:04:37 PST

( from http://www.rxpgnews.com ) Scientists at St. Jude Children's Research Hospital have demonstrated that a key event during apoptosis (cell suicide) occurs as a single, quick event, rather than as a step-by-step process. Apoptosis eliminates extraneous cells from the developing body; and disposes of cells that sustain irreparable harm to their DNA or are infected with microorganisms. The researchers photographed individual cells undergoing that process, allowing investigators to observe the release of certain proteins from pores in the membranes of mitochondria. These cellular structures contain enzymes that extract energy from food molecules, and the space within the membrane surrounding them holds a variety of proteins that are released during apoptosis.

Results of the study indicate the formation of pores in the mitochondrial membranes is a rapid process that allows a nearly simultaneous rather than a sequential release of many apoptosis proteins, according to Douglas Green, Ph.D., chair of the St. Jude Department of Immunology. Green is senior author of a report on this work that appears in the August 1 issue of Proceedings of the National Academy of Sciences. The process of pore formation, called mitochondrial outer membrane permeabilization (MOMP), allows apoptosis proteins stored underneath the membrane to escape and orchestrate the cell's destruction.

MOMP is controlled by a family of proteins called Bcl-2; some of these support apoptosis and others interrupt the process. The pro- and anti-apoptotic Bcl-2 proteins cooperate to weigh and balance cell signals that promote survival or death, in this way determining the final outcome. During apoptosis, these proteins are either already on the mitochondrial membranes or migrate to the membranes, where they trigger MOMP.

"The slow, continuous release of one of the proteins, apoptosis-inducing factor (AIF), suggests that the pore formed during MOMP remains open for many hours," Green said. "Our finding of nearly simultaneous rather than sequential release of the mitochondrial membrane proteins helps to explain the timing of the movement of these apoptosis proteins following MOMP. The findings also suggest that release of these proteins is not controlled by multiple levels of regulators, but rather occurs as a single event."

The study also highlights the importance of the Bcl-2 family in regulating the formation of pores in the mitochondrial membrane and emphasizes how critical the formation of these pores is to the regulation of apoptosis, Green said.

The team found that after cells were treated with a chemical that triggers apoptosis, it took three to 10 minutes for several proteins, cytochrome c, Smac, Omi and adenylate kinase-2 to escape together immediately following MOMP.

However, the AIF protein escaped from the mitochondrial membrane much more slowly and incompletely, starting with the release of cytochrome c but continuing during the next few hours. The St. Jude researchers concluded that while AIF is known to regulate other cellular processes, the protein itself is not involved in triggering apoptosis. The researchers made the movement of the proteins visible by attaching fluorescent tags to make them glow when observed under a special microscope equipped with a laser that scanned the cells.

Researchers discover new cell structures

Fri, 30 Jun 2006 01:31:37 PST

( from http://www.rxpgnews.com ) Carnegie Mellon University researchers Kris Noel Dahl and Mohammad F. Islam have made a new breakthrough for children suffering from an extremely rare disease that accelerates the aging process by about seven times the normal rate.

Dahl, an assistant professor of chemical and biomedical engineering at Carnegie Mellon, said her work with researchers at the National Cancer Institute of the National Institutes of Health (NIH), the John Hopkins University School of Medicine and the University of Pennsylvania reveals that children suffering from Hutchinson-Gilford Progeria Syndrome (HGPS) have an excessively stiff shell of proteins.

The nucleus in all three trillion cells of the human body contains the DNA genome, which is wrapped with a stiff protein shell called the nuclear lamina. Children with HGPS have a mutation in one of the proteins of the lamina shell. For years, experts have thought this mutation made their nuclei much softer and more likely to be ruptured when cells were under stress.

But in a Proceedings of the National Academy of Sciences (PNAS) Journal article to be published this month, Dahl and her colleagues show that the lamina shell in HGPS patients is stiffer than normal. However, stiffer isn't necessarily better. The stiffer lamina did protect the HGPS nucleus from some forces, but under excessive force the HGPS lamina was more brittle and eventually fractured.

"The mutant HGPS lamina is like an egg shell that cracks when excessive pressure or force is exerted against it," Dahl said. "By contrast, normal lamina resembles the rubbery outer shell of a racquetball, which does not break under stress or force but can assume its original shape even after hard play."

The researchers also think that the stiffer lamina in HGPS patients may be unable to communicate the proper biological signals to the DNA inside the nucleus to help the cell grow, which contributes to the disease.

Islam, an assistant professor of chemical engineering and materials science and engineering, says that the increased stiffness of the lamina may be caused by mutant proteins self-organizing into ordered structures within the HGPS lamina.

"This could make the lamina stiffer and cause fractures in the nuclei," Islam said. The healthy lamina remains disordered and therefore less rigid.

"Once we understand what causes the lamina to stiffen, we can try to reverse or stop the problem," Dahl said. "We think this stiffening mechanism happens over time with increased protein concentration, so we need to determine the tipping point that causes real problems."

When people grow old, the walls of the cell nuclei exhibit similar problems to the HGPS nuclei, like losing their round shape and perkiness. "Our NIH collaborators have also found that the normal aged nuclei show the same structural changes as HGPS," Dahl said.

Cilia also contribute to cellular response to external signals

Thu, 04 May 2006 23:12:37 PST

( from http://www.rxpgnews.com ) By studying microscopic hairs called cilia on algae, researchers at UT Southwestern Medical Center have found that an internal structure that helps build cilia is also responsible for a cell's response to external signals.

Cilia perform many functions on human cells; they propel egg and sperm cells to make fertilization possible, line the nose to pick up odors, and purify the blood, among other tasks.

With such a range of abilities, cilia serve as both motors and "cellular antennae," said Dr. William Snell, a professor of cell biology at UT Southwestern and senior author of new research on cilia published in the May 5 issue of Cell.

Genetic defects in cilia can cause people to develop debilitating kidney disease or to be born with learning disabilities, extra fingers or toes, or the inability to smell.

But no one really knows how cilia work, or, in some parts of the body, what their function is.

"There are cilia all over within our brain, and we don't have a clue about what they're doing," Dr. Snell said.

He and his team use the microscopic green alga, Chlamydomonas reinhardtii, which has two individual cilia. This alga allows researchers to manipulate genes and study the resulting effects on cilia in a way that would be impossible in animals such as mice.

"Chlamy is one of the few model organisms in which it's possible to do these kinds of studies," Dr. Snell said.
Normally, cilia also called flagella are built and maintained by an internal bidirectional, escalator-like system that ferries molecules to and from the tips by a process called intraflagellar transport, or IFT.

The UT Southwestern researchers used a mutant temperature-sensitive strain of the alga that behaved normally at lower temperatures. At higher temperatures, however, the IFT process stopped, and its components disappeared from the cilia. The cilia themselves were still able to beat, or move back and forth, for about 40 minutes before they began to shorten.

The team focused on fertilization of the alga, a process that requires a cilium to bind to a molecule on a cilium from a cell of the opposite mating type. They found that when the external molecule binds to a cilium, it activates an enzyme that signals the start of a chain of chemical reactions.

Although the cilia could move without IFT and bind to the molecules of the cilia of the opposite type, those cells were unable to respond to the signaling molecules. The failure to activate the chain of chemical reactions indicated that IFT was necessary for this function.

Analysis showed that the cilia signaling process was similar to that found in human cells, such as those in the nose involved in the sense of smell and those in the developing nervous system that sculpt our brains.

Uncovering this series of reactions will make it possible to test, for instance, drugs that can affect cilia, in the hope of finding substances that would also be effective in higher animals, Dr. Snell said.

"This is another example of how basic science research can have big results," he said. "Studies on Chlamydomonas will help us understand the unique qualities of cilia that have led to their use in chemosensory pathways in humans."

A riboswitch might sense magnesium levels in the cell

Wed, 12 Apr 2006 13:06:37 PST

( from http://www.rxpgnews.com ) Magnesium, essential for energy-production and structural integrity, is critical to cell survival. Researchers have now found that cells use specialized segments of RNA called riboswitches to ensure that there is an adequate supply of the mineral. The newly described riboswitch can both sense magnesium levels and respond directly by regulating production of a magnesium transport protein.

Riboswitches are a recently discovered class of gene expression regulators. They control gene expression through a segment of messenger RNA (mRNA)the copy of a gene that is used to produce a proteinthat interacts with a target molecule to regulate its own translation into protein. Usually, the protein regulated by the riboswitch is part of the cellular machinery that regulates the levels of the target molecule.

In this case, the riboswitch lies on an mRNA that the cell uses to produce a transporter protein that carries magnesium into the cell. When the switch detects that magnesium has dropped to too low a level, it can boost the translation of the RNAmeaning the cell produces more of the transporter protein, thereby correcting the magnesium deficiency.

The discovery, which was described in an article published in the April 7, 2006, issue of the journal Cell, is important for two reasons, said Howard Hughes Medical Institute investigator Eduardo A. Groisman. First, the finding solves a biological puzzle about one of the cell's most importantalbeit underappreciatedsubstances, he said.

Every energy-producing reaction in the cell depends on magnesium as an accompanying cofactor for the cell's main energy molecule, ATP. Magnesium is also essential for the stability of the cell's membranes and its protein-producing ribosomes. Nevertheless, almost nothing was known about how the cell senses low magnesium levels, said Groisman, who is at the Washington University School of Medicine.

The finding also helps advance understanding of how riboswitches regulate gene expression, which is quite different from the more familiar regulation by proteins called transcription factors. The proteins that transport magnesium into the cellMgtA and MgtBhad been know for decades, Groisman said. And he and his colleagues discovered a decade ago that a regulatory system they called PhoP/PhoQ switches the genes for the transporters on or off in response to changing magnesium levels. But before this work, it wasn't suspected at all that a riboswitch might sense magnesium levels in the cell, Groisman said.

That mutation in the PhoQ protein should have rendered the cell unable to respond to low magnesium levels, but the transporter genes remained sensitive to fluctuations in the mineral, said Groisman.

So, the researchers decided to analyze in detail how the mRNA molecule for mgtA responded to magnesium, in hopes of discovering a basis for magnesium-sensing. To do so, they dissected the function of the components of the Salmonella bacterium's mRNA for mgtA by systematically altering those parts' function and observing the results.

Their studies revealed that a region at one end of the mRNA moleculewhich is not translated into the MgtA proteinresponded to levels of magnesium. A specific structure in this untranslated region, they showed, adopted different shapes depending on the level of magnesium in the bacterium.

In further studies, Groisman and his colleagues hope to understand in greater structural detail how the riboswitch senses magnesium levelspinpointing the particular part of the molecule influenced by magnesium. Also, he said, the researchers will seek to understand how this magnesium sensor applies the brakes on translation of the mRNA into the magnesium transporter protein, MgtA.