Saturday, September 28, 2013

Intracellular Junctions

An intracellular junction is a highly specialized region of the plasma membrane that can be found within the tissues of multicellular organisms and serves to maintain cell and tissue polarity and integrity. These junctions consist of multiprotein complexes that provide contact both between neighboring cells and between cells and the extracellular matrix. Intracellular junctions also build up the paracellular barrier of epithelia and control paracellular transport. Various types of cell adhesion molecules are involved in the construction of intracellular junctions including selectins, cadherins, integrins and the immunoglobulin superfamily. The three major types of cell junctions found in vertebrates are adherens junctions, gap junctions and tight junctions.

Adherens junctions, also referred to as zonula adherens or intermediate junctions, are defined as cell junctions in which the cytoplasmic face is linked to the actin cytoskeleton. They can appear as either bands encircling the cell or spots of attachment to the extracellular matrix. Adherens junctions are composed primarily of cadherins, p120, alpha catenin and beta catenin.

Gap junctions are specialized intercellular connections that are found between multiple types of cells. They are composed of two connexons that connect across the intracellular space. These junctions directly connect the cytoplasm of two cells and allow various molecules and ions to pass freely for direct electrical communication between cells.

Tight junctions, which are also referred to as zonula occludens, are formed when the membranes of closely associated areas of two cells join together to form a virtually impermeable barrier to fluid. One of the functions of tight junctions involves the preservation of transcellular transport. Tight junctions maintain the polarity of cells by preventing the lateral diffusion of integral membrane proteins between the apical and lateral/basal surfaces. This allows the specialized functions of each surface to be preserved. Tight junctions are also involved in the prevention of the passage of molecules and ions through the spaces between cells.

The study of junction assembly and remodeling on the molecular level is a rapidly growing area of research. Studies that address the fundamental properties of junction dynamics will most likely further reveal the complexity of these specialized organelles.


Wednesday, September 25, 2013

Neural Stem Cell Marker

In the body, neural stem cells are found near blood vessels in the subventricular zone, the hippocampus, and the songbird higher vocal center. The cells in the ventricular zone release vascular endothelial growth factor (VEGF). Because blood vessel growth is attracted to VEGF, the vascular cells are near the central nervous system germinal zones. Scientists have suggested these cells provide a neural stem cell niche.

In order to investigate the relationship between the neural stem cells and vascular cells, researchers in the Center for Neuropharmacology and Neuroscience, the Center for Cardiovascular Sciences, and the Center for Cell Biology and Cancer Research at Albany Medical College cocultured vascular cells and neural stem cells. In transwell plates, mouse neural stem cells were seeded in the well bases and vascular type cells were seeded in the upper compartment of the wells. Various types of vascular cells were studied, including primary bovine pulmonary artery endothelial (BPAE) cells, vascular smooth muscle (VSM) cells, mouse brain endothelial (MbEND) cells, and NIH3T3 fibroblasts. High-density, age-matched cortical cells (CTX) were used as controls.

When BPAE and MbEND cells were used in the upper compartments, the endothelial cells were not found in the lower cells because they could not fit and migrate through the pores. Within one day of cell seeding, the neural stem cells cultured with the control, CTX, started creating neurons. The neuron growth was mostly in glial lineages. However, cells cultured with BPAE or MbEND grew into sheets of flattened progeny. Stem cells cultured with endothelial cells produce fewer neurons and larger stem cells, as compared to cells grown with CTX. Researchers concluded that endothelial cells aid in expansion of neural stem cells, but inhibit cell differentiation. VSM and NIH3T3 cells were also found to promote stem cell proliferation.

After researchers removed the transwell inserts, endothelial-expanded stem cells proliferated and differentiated. Approximately 31% of the progeny produced were neurons, while only 9% of stem cells cultured with CTX were neurons. Neural stem cells cocultured with BPAE contained as much as 64% neurons. However, neurogenesis was reduced in the stem cells cultured with VSM or NIH3T3 cells. Therefore, endothelial cells’ affect on neurogenesis in neural stem cells depends on the cell type and is not universal across all endothelial cells.

Scientists also found that endothelial cells activate proliferation and neurogenesis of neural stem cells in a variety of areas in the central nervous system (CNS). Another type of stem cells, neurosphere-expanded stem cells, was also influenced by endothelial cells. When exposed to the endothelial cells, neurosphere-expanded stem cells created 22% neurons, compared to 2% neuron production in control cells and CTX.

In the body, most neurons are produced in the early embryonic stage, but interneurons and glia arrive later, typically in adult stem cells. Stem cells were cocultured with endothelial cells and stained for glutamic acid decarboxylase, an interneuron marker, or Tbr1, an early neuron marker that labels projection neurons. Endothelial cell coculture produced more projection neuron and interneuron markers than the controls. Additional signaling experiments also demonstrated that endothelial cell and neural stem cell coculture activated the signaling molecules, Notch and Hes1, which promote neural stem cell self-renewal.

Through this research, scientists demonstrated that endothelial cells release some type of factors that maintain central nervous system stem self renewal and neurogenesis. In the presence of these endothelial cells, neural stem cells undergo proliferation to produce sheets of undifferentiated stem cells that can generate neurons, astrocytes, and oligodendroctyes. The growth of stem cells in the presence of endothelial cells may be important for neuron replacement therapies. If researchers can isolate the secreted factors from endothelial cells that cause neurogenesis and capture it in a drug, patients that have lost nerve function may be able to regrow their own neurons through neural stem cell self-renewal and neurogenesis.

Other neural stem cell marker: MAP2, Brn2 ,PAX6


Tuesday, September 24, 2013


The CD44 antigen is a glycoprotein that is found on the surface of many cell types, including resting T cells, and is involved in cell-cell interactions and cell adhesion. CD44 acts as a receptor for hyaluronic acid and plays an important role in cell migration, including the recruitment of effector T cells and other leukocytes to infection sites. CD44 also participates in lymphocyte activation, recirculation and homing, as well as hematopoiesis and tumor metastasis. The function of CD44 is controlled by posttranslational modifications including proteolytic cleavage, N- and O-glycosylation and phosphorylation.

The function of CD44 on T cells

Due to an increase in its expression following the activation of B-cells and T-cells, CD44 can serve as a valuable marker for memory cells. Furthermore, the upregulation of CD44 expression is sustained on effector cells and memory cells after the immune response has subsided, so CD44 expression can also be used as an indicator of prior exposure to an antigen. However, little is currently known about its function of T cells. Recent studies have suggested that the CD44 signaling pathway may be involved in ensuring proper T cell effector responses by providing contextual signals at various anatomical sites. CD44 ligation may also promote T cell survival through the augmentation of T cell activation in response to an antigen. Studies also suggest that CD44 may contribute to both the regulation of the contraction phase of an immune response and the maintenance of immune tolerance. Furthermore, CD44 may play a role in ensuring the functional fitness of memory T cells once the memory stage has been established.

The therapeutic potential of CD44 inhibitors

In addition to its function on T cells, current research is also focusing on CD44 as a potential therapeutic target for controlling inflammation. Early studies have shown that the blocking of CD44 results in a reduction in skin contact sensitivity responses due to a decrease in lymphocyte and leukocyte infiltration. Other studies have shown that pulmonary injury and allergic responses are reduced following treatment with CD44 antibodies. Additionally, interfering with CD44 function has shown therapeutic potential for the treatment of arthritis, multiple sclerosis, colitis and type 1 diabetes. However, a better understanding of the multiple, distinct roles of CD44 in various cell types will be required to fully realize the therapeutic potential of CD44 inhibitors.


Saturday, September 21, 2013

Identifying Stem Cell Markers

Stem cells and anything that has to do with them are certain subjects of fascination in today’s world. These tiny components of our bodies seem to promise indefinite health and well-being, as well as the reversal or cure of many modern and ancient maladies. Since stem cells are not yet specialized and can turn into virtually any kind of cell that the human body needs, doctors believe they can replace ailing organs and otherwise rejuvenate flesh with them. Stem cell markers are another facet of this medical phenomenon that stirs up great interest in every part of society.

What Are Stem Cell Markers?

Each cell in your body is covered in proteins known as receptors. These receptors help the cell to survive by allowing various molecule sot bind to them and work out necessary interactions that provide the cell with energy. The numbers and kinds of receptors make a long list. Each type of cell has receptors or sets of receptors that are particular to it. For instance, the cells that make up your kidney have some receptors that are specific to them and are not found on liver cells or skin cells.

Stem cells have their own distinct receptors on their exterior surfaces. This distinction allows scientists to easily identify and tag such cells. Were it not for the existence of these markers, scientists would not have made so many recent advancements in their study of stem cells. Each marker has a name based on the molecules to which it binds.

How Do Scientists Identify Stem Cell Markers?

There are many different ways to use stem cell markers in scientific study. Two approaches are outlined here. One involves the use of a technique known as fluorescence-activated cell sorting (FACS) and the other method uses the fluorescent tags on these markers to identify and assess the condition of stem cells present in tissues.

• Scientists frequently use FACS to distinguish stem cells from the millions of cells present in any view of tissues or organs. The process begins with a nozzle that is so narrow that it will only allow the exit of one cell at a time. As the cells exit the nozzle, they pass through a beam of light. Stem cells, which have been previously treated with fluorescent markers, become negatively charged as they pass through the light. Other cells take on a positive charge.

• When scientists wish to see how stem cells act in tissues under a microscope and cannot use the FACS device, they remove a thin slice of the desired tissue. Stem cell markers are tagged with an injection of signaling molecules with fluorescent tags. Observers activate these tags with light or chemical energy. The fluorescent light emitted by stem cell markers is visible in the microscope.

Scientists have also begun to study the possibility of identifying and studying stem cells without having to rely on the identification of their markers. However, this method had been useful for a considerable period, given the short amount of time in which stem cells have been identifiable. Stem cell markers will certainly remain an important method of identification in research.


Wednesday, September 18, 2013

Endoderm Stem Cell Markers

The endoderm is one of the three vital germ layers in triploblastic animals. It is formed from the epiblast during embryogenesis from cells that migrate toward the center of the developing gastrula. The endoderm provides the epithelial lining of both the digestive and respiratory tracts, and the cells that form the endoderm eventually differentiate to form the stomach, colon, intestines, liver, pancreas and gall bladder, as well as the lining of the trachea, bronchi and alveoli of the lungs.

Although endoderm development is currently less understood than ectoderm and mesoderm development, researchers have successfully generated endodermal cells from ES (embryonic stem) cells. Human PSCs (pluripotent stem cells) can also be differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells. While cells from these monolayer cultures have shown some therapeutic efficacy in animal models, the generation of three-dimensional organs that contain multiple cell types remains a challenge. In a recent publication by Spence et al. (Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro, Nature 2011; 470(7332): 105–109), the authors described how they were able to generate intestinal “organoids” from human PSCs by using a series of growth factor manipulations to induce definitive endoderm formation, posterior endoderm patterning, hindgut specification and morphogenesis. The resulting three dimensional organoids contained villus-like structures and crypt-like proliferative zones. These organoid cultures also comprised a variety of cell types including intestinal stem cell markers, functional enterocytes, goblet cells, Paneth cells and enteroendocrine cells. These recent advances should pave the way for more studies using PSCs to study development and disease.

The future is stem cell research is highly dependent on the availability of reliable markers for endodermal stem cells. Antibodies against proteins expressed in various cell types including pancreatic islets, hepatocytes, intestinal cells and lung cells can be used as markers for the endodermal cell lineage to confirm the identities of various differentiated cell cultures and thereby further stem cell research.

Endoderm Stem Cell Markers: GATA4beta CateninCytokeratin 19FOXA1


Sunday, September 15, 2013

Vimentin is an invaluable diagnostic protein with an important functional role

Vimentin is a type III intermediate filament (IF) protein present in many eumetazoan cell types of mesodermic origin, such as the mesenchyme and fibroblasts, as well as some ectodermal cells, including neural stem cells and some cancers. Vimentin is a 53.5 kDa protein that contains a long central α-helix that dimerizes with another vimentin molecule to form a coiled coil. The α-helix and the resultant coils are remarkably stable due to the regular spacing of charged and hydrophobic residues along the helix. The C- and N- terminals of the molecule contain globular domains capable of interacting with organelles, actin and tubulin filaments, cytoplasmic proteins, and other vimentin dimers.

In cells that express vimentin, it forms an integral part of the cytoskeleton. Unlike actin and tubulin filaments, which are relatively rigid in structure due to their regulated assembly into highly structured polymers, vimentin has a malleable structure due to its aggregation via terminal domain interactions. Consequently, cells with high vimentin expression, such as mesenchymal cells, tend to be elastic and structurally robust but lose this elasticity when the protein is knocked out or functionally inactivated. Vimentin anchors membrane-bound organelles such as the mitochondria into relatively stable positions within the cell and is responsible for facilitating transport toward these organelles.

The mechanisms and importance of vimentin-driven and -facilitated transport is a subject of ongoing research. During mitosis, vimentin filaments are disassembled upon S55 phosphorylation to allow for cytokinesis. The dissociation of vimentin filaments via phosphorylation in nondividing cells results in a cytoskeletal contraction (via the myosin light chain) that directs LDL droplets to the mitochondria where cholesterol is exposed to its side-chain cleavage enzyme, thus increasing steroid biosynthesis. This has implications for both endocrinology, where the synthesis of steroid hormones is tightly regulated by the endocrine cells, and cancer biology, where some tumor types secrete excessive levels of these hormones.

Immunohistochemical staining of vimentin is useful for a number of purposes; given that it is cell-type specific, especially outside of mesodermal tissues and that the intracellular expression profile varies somewhat based upon the functional role of vimentin in a given cell. Immunohistochemical assay for vitamin is useful for diagnosing cancer types where the protein is overexpressed, such as sarcomas, and some melanomas and lymphomas. This is especially true of malignant epithelial cells that are becoming undifferentiated, which often express high levels of the typically mesodermal protein.

In addition to the typical extension and dissociation of vimentin filaments, cells that are rapidly expressing vimentin form "fountains" where the vimentin filaments rapidly spread out toward the cell membrane and then spread out and circle back upon approach. Such assessments of vimentin mechanics generally use a GFP-tagged vimentin protein in a transgenic system such that the protein can be visualized in vivo. However, pathology and diagnostics work generally utilizes fluorescence or DAB immunohistochemistry in fixed tissue using an anti-vimentin antibody.

A quality anti-vimentin antibody can also be used as a neural stem cell marker. While the classical BrdU/NeuN double stain identifies recently-formed neurons, it cannot identify neural stem cells that are not actively dividing. This gives vimentin an important role in the functional assessment of neural stem cells. For instance, a vimentin stain coupled with BrdU can identify the fraction of neural stem cells that are proliferating during an experimental treatment or time window. A number of treatments and conditions are known to alter the rate of neurogenesis, including major depression and cortisol imbalance (such as in Cushing's Syndrome), where hippocampal neurogenesis is substantially inhibited, and low-level radiation exposure, which temporarily halts neural stem cell division.


Thursday, September 12, 2013

Beta-catenin is an integral component of the adherens junction and the Wnt signaling cascade

Beta-catenin is a structural protein that forms part of the extended cadherin complex and also functions as a signaling molecule in the Wnt pathway. It is an 88 kDa protein that is mostly composed of "armadillo repeat domain" copies. Each domain is approximately 42 amino acids long and contains two alpha helices separated by a hairpin. Beta-catenin contains 12 copies of this domain, forming an alpha helix "supersolenoid" structure that contains a large, positively-charged groove that is well-adapted for protein-protein binding. This binding is regulated via phosphorylation; for instance, phosphorylation at the S552 residue by AKT promotes binding to transcription factors.

Adherens junctions often appear as broad bands that completely encircle epithelial cells. In this form, they are called belt desmosomes and are commonly seen in tissues that closely regulate the flow of nutrient and waste molecules, such as the epithelial cells lining the small intestine. Beta-catenin forms an important part of these junctions, where it comprises part of the protein complex between E-cadherin and the actin cytoskeleton. The cell's cadherin proteins bind to cadherins in adjascent cells. Thus, beta-catenin is a crucial component in a juncture that physically links the cytoskeletons of neighboring cells, allowing for the close regulation of molecular transport across a thin tissue layer.

Beta-catenin also acts as a signaling molecule within the Wnt signaling pathway. This pathway is especially important during embryogenesis, where it is responsible for axial development and the development of the dorsal neural tube. Dysregulation of this pathway is associated with exposure to carcinogens. Through this pathway, beta-catenin acts as a transcription factor. Outside of the adherens junction, beta-catenin binds to a "destruction complex" that results in its ubiquitination and rapid degradation. Upon activation of the Wnt pathway, this complex is inhibited and beta-catenin translocates to the nucleus, where it binds with TCF/LEF family transcription factors to alter gene expression. Through this pathway, beta-catenin has the potential to substantially alter cellular function.

The visualization of this protein using a beta-catenin antibody is useful for both diagnostic and research purposes. Due to its role as a gene regulator, beta-catenin is considered to be an oncogene. It binds to the APC protein, a regulator of cell cycle. Mutations and dysregulations of the beta-catenin/APC interaction are often seen in individuals susceptible to colorectal and ovarian cancers. Its transcriptional activity is controlled by phosphorylation at S552 by AKT. This phosphorylation causes the release of beta-catenin from the adherens complex and leads to a buildup of the protein in both the cytosol and the nucleus. Beta-catenin is most commonly visualized via immunofluorescence using a beta-catenin antibody and an appropriate secondary antibody, though DAB staining is also common for diagnostic purposes.

In cells without Wnt pathway activity, beta-catenin is seen largely at the cell periphery, where it expresses at the adherens junction but not the interior of the cell, where it rapidly undergoes proteolysis. Activation of the Wnt pathway or pharmacological intervention, such as via GSK3 inhibition, results in the rapid dispersal of beta-catenin into the cytosol, with especially high concentrations seen in the nucleus if AKT is also present. However, if the S33 and S37 residues are phosphorylated, as occurs in the destruction complex, the protein is degraded and little expression is seen outside of whatever remains at the adherens. Visualization using a beta-catenin antibody can therefore determine the functional and phosphorylation states of the protein. This can be further confirmed via antibodies that are specific to the various beta-catenin phosphorylation states.