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Archive for the ‘Cytoskeleton’ Category


Lesson 8 Cell Signaling and Motility: Lesson and Supplemental Information on Cell Junctions and ECM: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

Please click on the following link for the PowerPoint Presentation for Lecture 8 on Cell Junctions and the  Extracellular Matrix: (this is same lesson from 2018 so don’t worry that file says 2018)

cell signaling 8 lesson 2018

 

Some other reading on this lesson on this Open Access Journal Include:

On Cell Junctions:

Translational Research on the Mechanism of Water and Electrolyte Movements into the Cell     

(pay particular attention to article by Fischbarg on importance of tight junctions for proper water and electrolyte movement)

The Role of Tight Junction Proteins in Water and Electrolyte Transport

(pay attention to article of role of tight junction in kidney in the Loop of Henle and the collecting tubule)

EpCAM [7.4]

(a tight junction protein)

Signaling and Signaling Pathways

(for this lesson pay attention to the part that shows how Receptor Tyrosine Kinase activation (RTK) can lead to signaling to an integrin and also how the thrombin receptor leads to cellular signals both to GPCR (G-protein coupled receptors like the thrombin receptor, the ADP receptor; but also the signaling cascades that lead to integrin activation of integrins leading to adhesion to insoluble fibrin mesh of the newly formed clot and subsequent adhesion of platelets, forming the platelet plug during thrombosis.)

On the Extracellular Matrix

Three-Dimensional Fibroblast Matrix Improves Left Ventricular Function Post MI

Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel

 

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Lesson 5 Cell Signaling And Motility: Cytoskeleton & Actin: Curations and Articles of reference as supplemental information: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

Cell motility or migration is an essential cellular process for a variety of biological events. In embryonic development, cells migrate to appropriate locations for the morphogenesis of tissues and organs. Cells need to migrate to heal the wound in repairing damaged tissue. Vascular endothelial cells (ECs) migrate to form new capillaries during angiogenesis. White blood cells migrate to the sites of inflammation to kill bacteria. Cancer cell metastasis involves their migration through the blood vessel wall to invade surrounding tissues.

Please Click on the Following Powerpoint Presentation for Lesson 4 on the Cytoskeleton, Actin, and Filaments

CLICK ON LINK BELOW

cell signaling 5 lesson

This post will be updated with further information when we get into Lesson 6 and complete our discussion on the Cytoskeleton

Please see the following articles on Actin and the Cytoskeleton in Cellular Signaling

Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

This article, constitutes a broad, but not complete review of the emerging discoveries of the critical role of calcium signaling on cell motility and, by extension, embryonic development, cancer metastasis, changes in vascular compliance at the junction between the endothelium and the underlying interstitial layer.  The effect of calcium signaling on the heart in arrhtmogenesis and heart failure will be a third in this series, while the binding of calcium to troponin C in the synchronous contraction of the myocardium had been discussed by Dr. Lev-Ari in Part I.

Universal MOTIFs essential to skeletal muscle, smooth muscle, cardiac syncytial muscle, endothelium, neovascularization, atherosclerosis and hypertension, cell division, embryogenesis, and cancer metastasis. The discussion will be presented in several parts:
1.  Biochemical and signaling cascades in cell motility
2.  Extracellular matrix and cell-ECM adhesions
3.  Actin dynamics in cell-cell adhesion
4.  Effect of intracellular Ca++ action on cell motility
5.  Regulation of the cytoskeleton
6.  Role of thymosin in actin-sequestration
7.  T-lymphocyte signaling and the actin cytoskeleton

 

Identification of Biomarkers that are Related to the Actin Cytoskeleton

In this article the Dr. Larry Bernstein covers two types of biomarker on the function of actin in cytoskeleton mobility in situ.

  • First, is an application in developing the actin or other component, for a biotarget and then, to be able to follow it as

(a) a biomarker either for diagnosis, or

(b) for the potential treatment prediction of disease free survival.

  • Second, is mostly in the context of MI, for which there is an abundance of work to reference, and a substantial body of knowledge about

(a) treatment and long term effects of diet, exercise, and

(b) underlying effects of therapeutic drugs.

Microtubule-Associated Protein Assembled on Polymerized Microtubules

(This article has a great 3D visualization of a microtuble structure as well as description of genetic diseases which result from mutations in tubulin and effects on intracellular trafficking of proteins.

A latticework of tiny tubes called microtubules gives your cells their shape and also acts like a railroad track that essential proteins travel on. But if there is a glitch in the connection between train and track, diseases can occur. In the November 24, 2015 issue of PNAS, Tatyana Polenova, Ph.D., Professor of Chemistry and Biochemistry, and her team at the University of Delaware (UD), together with John C. Williams, Ph.D., Associate Professor at the Beckman Research Institute of City of Hope in Duarte, California, reveal for the first time — atom by atom — the structure of a protein bound to a microtubule. The protein of focus, CAP-Gly, short for “cytoskeleton-associated protein-glycine-rich domains,” is a component of dynactin, which binds with the motor protein dynein to move cargoes of essential proteins along the microtubule tracks. Mutations in CAP-Gly have been linked to such neurological diseases and disorders as Perry syndrome and distal spinal bulbar muscular dystrophy.

 

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

Researchers have classified a brand-new organ inside human body. Known as the mesentery, the new organ is found in our digestive systems, and was long thought to be made up of fragmented, separate structures. But recent research has shown that it’s actually one, continuous organ. The evidence for the organ’s reclassification is now published in The Lancet Gastroenterology & Hepatology. Although we now know about the structure of this new organ, its function is still poorly understood, and studying it could be the key to better understanding and treatment of abdominal and digestive disease.

mesentery

J Calvin Coffey, a researcher from the University Hospital Limerick in Ireland, who first discovered that the mesentery was an organ. In 2012, Coffey and his colleagues showed through detailed microscopic examinations that the mesentery is actually a continuous structure. Over the past four years, they’ve gathered further evidence that the mesentery should actually be classified as its own distinct organ, and the latest paper makes it official. Mesentery is a double fold of peritoneum – the lining of the abdominal cavity – that holds our intestine to the wall of our abdomen. It was described by the Italian polymath Leanardo da Vinci in 1508, but it has been ignored throughout the centuries, until now. Although there are generally considered to be five organs in the human body, there are in fact now 79, including the mesentery. The heart, brain, liver, lungs and kidneys are the vital organs, but there are another 74 that play a role in keeping us healthy. The distinctive anatomical and functional features of mesentery have been revealed that justify designation of the mesentery as an organ. Accordingly, the mesentery should be subjected to the same investigatory focus that is applied to other organs and systems. This provides a platform from which to direct future scientific investigation of the human mesentery in health and disease.

References:

http://www.thelancet.com/journals/langas/article/PIIS2468-1253(16)30026-7/abstract

http://www.sciencealert.com/it-s-official-a-brand-new-human-organ-has-been-classified

http://www.bbc.com/news/health-38506708

http://www.independent.co.uk/news/science/new-organ-mesentery-found-human-body-digestive-system-classified-abdominal-grays-anatomy-a7507396.html

https://in.news.yahoo.com/scientists-discover-human-organ-064207997.html

https://en.wikipedia.org/wiki/Mesentery

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Signaling through the T Cell Receptor (TCR) Complex and the Co-stimulatory Receptor CD28

Curator: Larry H. Bernstein, MD, FCAP

 

 

New connections: T cell actin dynamics

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.

RELATED CONTENT

 

Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

 

RELATED CONTENT

T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex at the site of the T cell receptors (TCRs). Srikanth et al. found that the lymphocyte-specific large guanosine triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human CD4+ T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T cell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium and kinase signaling.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

 

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Insight into Blood Brain Barrier

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Gateway to The Brain

This image shows the structural model of critical transporter, Mfsd2a. Source: Duke-NUS Medical School
This image shows the structural model of critical transporter, Mfsd2a. Source: Duke-NUS Medical School.  http://www.dddmag.com/sites/dddmag.com/files/rd1604_brain.jpg

Scientists from Duke-NUS Medical School (Duke-NUS) have derived a structural model of a transporter at the blood-brain barrier called Mfsd2a. This is the first molecular model of this critical transporter, and could prove important for the development of therapeutic agents that need to be delivered to the brain — across the blood-brain barrier. In future, this could help treat neurological disorders such as glioblastoma.

Currently, there are limitations to drug delivery to the brain as it is tightly protected by the blood-brain barrier. The blood-brain barrier is a protective barrier that separates the circulating blood from the central nervous system which can prevent the entry of certain toxins and drugs to the brain. This restricts the treatment of many brain diseases. However, as a transporter at the blood-brain barrier, Mfsd2a is a potential conduit for drug delivery directly to the brain, thus bypassing the barrier.

In this study, recently published in the Journal of Biological Chemistry, first author Duke-NUS MD/PhD student Debra Quek and senior author Professor David Silver used molecular modeling and biochemical analyses of altered Mfsd2a transporters to derive a structural model of human Mfsd2a. Importantly, the work identifies new binding features of the transporter, providing insight into the transport mechanism of Mfsd2a.

“Our study provides the first glimpse into what Mfsd2a looks like and how it might transport essential lipids across the blood-brain barrier,” said Ms Quek. “It also facilitates a structure-guided search and design of scaffolds for drug delivery to the brain via Mfsd2a, or of drugs that can be directly transported by Mfsd2a.”

Currently this information is being used by Duke-NUS researchers to design novel therapeutic agents for direct drug delivery across the blood brain barrier for the treatment of neurological diseases. This initiative by the Centre for Technology and Development (CTeD) at Duke-NUS, is one of many collaborative research efforts aimed at translating Duke-NUS’ research findings into tangible commercial and therapeutic applications for patients.

Ms Quek plans to further validate her findings by purifying the Mfsd2a protein in order to further dissect how it functions as a transporter.

 

J Biol Chem. 2016 Mar 4. pii: jbc.M116.721035. [Epub ahead of print]
Structural insights into the transport mechanism of the human sodium-dependent lysophosphatidylcholine transporter Mfsd2a.

Major Facilitator Superfamily Domain containing 2A (Mfsd2a) was recently characterized as a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood-brain barrier endothelium. It is the primary route for importation of docosohexaenoic acid and other long-chain fatty acids into foetal and adult brain, and is essential for mouse and human brain growth and function. Remarkably, Mfsd2a is the first identified MFS family member that uniquely transports lipids, implying that Mfsd2a harbours unique structural features and transport mechanism. Here, we present three 3D structural models of human Mfsd2a derived by homology modelling using MelB- and LacY-based crystal structures, and refined by biochemical analysis. All models revealed 12 transmembrane helices and connecting loops, and represented the partially outward-open, outward-partially occluded, and inward-open states of the transport cycle. In addition to a conserved sodium-binding site, three unique structural features were identified: A phosphate headgroup binding site, a hydrophobic cleft to accommodate a hydrophobic hydrocarbon tail, and three sets of ionic locks that stabilize the outward-open conformation. Ligand docking studies and biochemical assays identified Lys436 as a key residue for transport. It is seen forming a salt bridge with the negative charge on the phosphate headgroup. Importantly, Mfsd2a transported structurally related acylcarnitines but not a lysolipid without a negative charge, demonstrating the necessity of a negative charged headgroup interaction with Lys436 for transport. These findings support a novel transport mechanism by which LPCs are flipped within the transporter cavity by pivoting about Lys436 leading to net transport from the outer to the inner leaflet of the plasma membrane.

 

Brain and eye contain membrane phospholipids that are enriched in the omega-3 fatty acid docosohexaenoic acid (DHA). It is widely accepted that DHA is important for brain and eye function and brain development (1,2), although mechanisms for DHA function in these tissues are not well defined.   The mechanism by which DHA and other conditionally essential and essential fatty acids cross the blood-brain barrier (BBB) has been a long-standing mystery. Recently, we identified Major Facilitator Superfamily Domain containing 2a (Mfsd2a, aka NLS1) as the primary transporter by which the brain obtains DHA. Importantly, Mfsd2a does not transport unesterified DHA, but transports DHA in the chemical form of lysophosphatidylcholine (LPC) that are synthesized by the liver and circulate largely on albumin (3). This is consistent with biochemical evidence that the brain does not transport unesterified fatty acids (4) and that LPC is the preferred carrier of DHA to the brain (5,6).   Mfsd2a is a sodium-dependent transporter that is part of the Major Facilitator Superfamily (MFS) of proteins. Members of this family with elucidated structures have 12 transmembrane domains composed of two evolutionarily duplicated 6 transmembrane units (7). Transporting an LPC is a unique feature of Mfsd2a, since most members of this family transport water-soluble and minimally polar substrates such as sugars (GLUT, MelB, LacY), and amino acids (TAT1). Mfsd2a transport is not limited to LPCs containing DHA, as it can transport LPCs containing a variety of fatty acyl chains, with higher specificity for LPCs with unsaturated fatty acyl chains with a minimum chain length of 14 carbons (6,8). Crystal structures have been solved for more than a dozen members of the MFS family, with more than 19 structures, including that of Melibiose permease (MelB) of S. typhimurium (9), Lactose permease (LacY) of Escherichia coli (10), glycerol-3-phosphate transporter of E. coli (11) and the mammalian glucose transporters 1, 3, and 5 (GLUT1, GLUT3, GLUT5) (12-14). A common transport mechanism has emerged from both biochemical and structural analyses of MFSs, in which they transport via a rocker-switch, alternating access mechanism (7,15). In the rocker-switch model, rigid-body relative motion of the N- and C-termini domains renders the substrate-binding site alternatively accessible from either side of the membrane.

Mfsd2a is highly expressed at the bloodbrain barrier in both mouse and human (6,16). Mfsd2a deficient mice (KO) have significantly reduced brain DHA as a result of a 90% reduction in brain uptake of LPC containing DHA as well as other LPCs. The most prominent phenotype of Mfsd2a KO mice is microcephaly, and KO mice additionally exhibit motor dysfunction, and behavioral disorders including anxiety and memory and learning deficits (6). In line with the mouse KO phenotypes, human patients with partially or completely inactivating mutations in Mfsd2a presented with severe microcephaly, intellectual disability, and motor dysfunction (8,16). Plasma LPCs are significantly elevated in both KO mice and human patients with Mfsd2a mutations, consistent with reduced uptake at the blood-brain barrier. Taken together, these findings demonstrate that LPCs are essential for normal brain development and function in mouse and humans.

The fact that Mfsd2a transports a lysolipid, a non-canonical substrate for an MFS protein, might indicate unique structure features and a novel transport mechanism. However, no structural information or mechanism of transport of Mfsd2a is known. Human Mfsd2a is composed of 530 amino acids, with two glycosylation sites at Asn217 and Asn227. Mfsd2a is evolutionarily conserved from teleost fish to humans. Although not a functional ortholog of bacterial MFS transporters, Mfsd2a shares 25% and 26% amino acid sequence identity with S. typhimurium MelB (9,17), and LacY from E. coli (10), respectively. Given the high conservation of the MFS fold, the use of homology modeling to gain insight into the structure of S. typhimurium MelB, for example, has proven to be highly accurate and largely consistent with subsequent X-ray crystal data (9,18). Here, we take advantage of two recently derived high resolution X-ray crystal structures of S. typhimurium MelB (9), and a high resolution X-ray crystal structure of LacY (10) to generate three predictive structural models of human Mfsd2a. These models reveal three unique regions critical for function – an LPC headgroup binding site, a hydrophobic cleft occupied by the LPC fatty acyl tail, and three sets of ionic locks. These structural features indicate a novel mechanism of transport for LPCs.

Mfsd2a is a sodium-dependent lysophosphatidylcholine transporter essential for human brain growth and function (40). Mfsd2a is the only known MFS member or secondary transporter that transports a lipid. In line with its unique function, the current study has identified three unique structural features based on a combination of homology structural modeling and biochemical analysis – (1) a unique headgroup binding site and (2) a hydrophobic cleft for acyl chain binding, and (4) 3 sets of ionic locks that stabilize the outward open conformation. Drawing together these findings with studies of the mechanism of transport of other MFS family members, we propose the following alternatingaccess mechanism for LPC transport (Fig. 6). In the first steps, LPC inserts itself into the outer leaflet of the membrane and diffuses laterally into the transporter’s hydrophobic cleft. As Mfsd2a undergoes conformational changes from the outward open to the inward open conformation, the zwitterionic headgroup is inverted from the outer membrane leaflet to the inner membrane leaflet along a translocation pathway within the transporter, interacting with specific polar and charged residues lining the path. Since LPCs are hydrophobic phospholipids, it is unlikely that they will partition out of the transporter into the aqueous environment of the cytoplasm. We propose that the “flipped” LPC exits the transporter laterally into the membrane environment of the inner leaflet. This model of LPC flipping requires further biochemical proof. Of particular interest is the visualization of the interaction of the negatively charged phosphate headgroup of LPC with Lys436 that is maintained in both outward and inward open conformations. The sidechain of Lys436 is seen to be pointing in the upward direction in the outward open conformation, but pointing downward into the translocation cleft in the inward open conformation. These findings suggest that the Lys436 acts as a tether to push or pivot the headgroup down into the translocation cavity while the N- and C-termini of Mfsd2a rock and switch from outward to inward open.

Interestingly, Lys436 is orthologous to the residue Lys377 in the melibiose transporter of S. typhimurium. Based on the S. typhimurium MelB crystal structure, Lys377 has been predicted to be involved in binding melibiose, and in forming a hydrogen bond with Tyr120, likely separating the sodium binding site from the central hydrophilic cavity (9). In a recent molecular dynamic simulation of E. coli MelB, Lys377 was noted to interact differently with residues involved in the sodium binding site (Asp55, Asp59, and Asp124) in the presence or absence of a sodium ion, and thought to be critical for the spatial organization of the sodium binding site (41). Similarly, in our refined models of Mfsd2a, Lys436 is localized in close proximity to the sodium-binding site residue, Asp93, and the central translocation pathway where it has been identified by docking studies to interact with the charged headgroup of LPC. We hypothesize that Lys436 may shuttle between the two binding sites, communicating and coordinating the occupancy status of the two sites. Interestingly, there is a distinct mobility shift in Mfsd2a bands on SDS-PAGE between wild-type Mfsd2a and the L-3 mutant (R498E, R499E, R500E, K503E, K504E) (Fig. 5I) that is not seen when each of the residues are mutated individually (Fig. S1). These findings are consistent with a conformational change in the L-3 mutant. Given that the L-3 ionic lock is visualized in the outward partially occluded model, we hypothesize that the loss of the L-3 ionic lock results in Mfsd2a being trapped in an energetically more favorable inward open conformation, resulting in the loss of transport function (Fig. 5H).

Patients with the partially inactivating mutation p.(S399L) exhibited significant increases specifically in plasma LPCs having monounsaturated (18:1 – 92%, p=0.004) and polyunsaturated LPCs (18:2, 20:4, 20:3 – 254%, p=0.002; 117%, p=0.007, and 238%, p=0.002), but not in the most abundant LPCs – saturated LPCs (C16:0, C18:0) (8). This is consistent with a greater specificity of Mfsd2a for LPCs with unsaturated fatty acyl chains (6)…A possible explanation for this acyl chain specificity is related to the mobility of the acyl tail in the membrane. It is known that phospholipids with unsaturated fatty acyl chains disrupt the packing of the bilayer, resulting in greater lateral membrane fluidity (42). Therefore, one possible mechanism for LPC specificity is that LPCs with unsaturated fatty acyl chains have greater lateral mobility in the membrane, increasing the Ka for interacting with the transport cleft of Mfsd2a.

Another important structural feature of the physiological ligand, LPC, is a minimum acyl chain length of 14 carbons is required for transport by Mfsd2a. A possible explanation for this requirement is that the hydrocarbon chain must extend beyond the cleft, protruding into the hydrophobic milieu of the phospholipid bilayer core. This interaction of the fatty acyl tail with the acyl chains of the membrane bilayer may provide a hydrophobic force strong enough to pull the molecule through and out of the transporter as the LPC headgroup partitions into the inner leaflet of the membrane. A similar scenario is seen in the Sec translocon where a hydrophobic transmembrane domain of a protein partitions laterally from the Sec61p complex channel into the lipid bilayer (43,44). This proposal that the omega carbon of the fatty acyl chain sticks out of the Mfsd2a pocket is consistent with the observation that Mfsd2a can transport nitrobenzoxadiazole (NBD) or Topfluor when these moieties are attached to the omega carbon of the LPC fatty acyl tail [1].

Other known transmembrane phospholipid transporters include flippases, floppases, and scramblases. Flippases and floppases utilize ATP to drive the uphill transport of aminophospholipids from the outer to the inner leaflet, and specific substrates from the inner to the outer leaflet, respectively (45-47). Scramblases are less well understood, facilitating transport of substrates in either direction down concentration gradients upon activation. While the substrates are similar, several differences make comparisons between Mfsd2a and phospholipid transporters of limited relevance. First, the shapes of the substrates differ in shape and size – lysophospholipids are smaller and conical while phospholipids are cylindrical. Second, unlike flippases and floppases, Mfsd2a is a secondary transporter, utilizing a sodium electrochemical gradient to drive the transport of lysophospholipids from one leaflet to the other. Third, the overall structure of MFS members is different from P4- ATPases and ABC transporters. Consequently, the mechanism of action between Mfsd2a and flippases such as P4-ATPases and ABC transporters, or floppases is expected to differ.

Being expressed at the blood-brain barrier, Mfsd2a is a potential conduit for drug delivery to the brain. The blood-brain barrier is highly impermeable, protecting the brain from bloodderived molecules, pathogens, and toxins. However, its impermeability poses a challenge for pharmacological treatment of brain diseases. It has been predicted that 98% of small molecule drugs are excluded from the brain by the blood-brain barrier (48). Currently, most drugs used to treat brain diseases are lipid soluble small molecules with a molecular weight of less than 400 Da (49). A small number of drugs traverse the blood-brain barrier by carrier-mediated transport. An example of this is Levodopa, a treatment for Parkinson’s Disease, which is a precursor of the neurotransmitter dopamine. Levodopa is transported across the blood-brain barrier by the large neutral amino acid transporter, LAT1 (50). Our findings here provide a further refinement of understanding of the structure-activity relationship of LPCs to their transport, and educates the search and design of drugs that can be transported by Mfsd2a. Candidates for transport, whether as a drug itself or as a LPC scaffold, must have a zwitterionic headgroup, but not necessarily a phosphate, and a minimal threshold of hydrophobic character. As the binding pocket is several times larger than LPC, it is sterically feasible to attach a small molecule drug onto LPC or LPC-like scaffolds for delivery across the blood-brain barrier.

In summary, these studies represent a first structural model of human Mfsd2a based on homology modeling and biochemical interrogation. We expect that this model will serve as a foundation for the future development of X-ray crystal structures of the protein, which would provide further insight into the structure and function of this physiologically important transporter required for human brain growth and function.

REFERENCES

1. Salem, N., Jr., Litman, B., Kim, H. Y., and Gawrisch, K. (2001) Mechanisms of action of docosahexaenoic acid in the nervous system. Lipids 36, 945-959

2. Bazan, N. G. (2009) Neuroprotectin D1-mediated anti-inflammatory and survival signaling in stroke, retinal degenerations, and Alzheimer’s disease. Journal of lipid research 50 Suppl, S400- 405

3. Baisted, D. J., Robinson, B. S., and Vance, D. E. (1988) Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes. The Biochemical journal 253, 693-701

4. Edmond, J., Higa, T. A., Korsak, R. A., Bergner, E. A., and Lee, W. N. (1998) Fatty acid transport and utilization for the developing brain. Journal of neurochemistry 70, 1227-1234

5. Lagarde, M., Bernoud, N., Brossard, N., Lemaitre-Delaunay, D., Thies, F., Croset, M., and Lecerf, J. (2001) Lysophosphatidylcholine as a preferred carrier form of docosahexaenoic acid to the brain. Journal of molecular neuroscience : MN 16, 201-204; discussion 215-221

6. Nguyen, L. N., Ma, D., Shui, G., Wong, P., Cazenave-Gassiot, A., Zhang, X., Wenk, M. R., Goh, E. L., and Silver, D. L. (2014) Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 509, 503-506

7. Law, C. J., Maloney, P. C., and Wang, D. N. (2008) Ins and outs of major facilitator superfamily antiporters. Annual review of microbiology 62, 289-305

8. Alakbarzade, V., Hameed, A., Quek, D. Q. Y., Chioza, B. A., Baple, E. L., Cazenave-Gassiot, A., Nguyen, L. N., Wenk, M. R., Ahmad, A. Q., Sreekantan-Nair, A., Weedon, M. N., Rich, P., Patton, M. A., Warner, T. T., Silver, D. L., and Crosby, A. H. (2015) A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome. Nat Genet 47, 814-817

9. Ethayathulla, A. S., Yousef, M. S., Amin, A., Leblanc, G., Kaback, H. R., and Guan, L. (2014) Structure-based mechanism for Na(+)/melibiose symport by MelB. Nature communications 5, 3009

10. Guan, L., Mirza, O., Verner, G., Iwata, S., and Kaback, H. R. (2007) Structural determination of wild-type lactose permease. Proceedings of the National Academy of Sciences of the United States of America 104, 15294-15298

…. more

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Glycobiology advances

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

The Evolution of the Glycobiology Space

The Nascent Stage of another Omics Field with Biomarker and Therapeutic Potential

Enal Razvi, Ph.D. , Gary Oosta, Ph.D

http://www.genengnews.com/insight-and-intelligence/the-evolution-of-the-glycobiology-space/77900638/

 

The Evolution of the Glycobiology Space

 

Glycobiology is an important field of study with medical applications because it is known that tumor cells alter their glycosylation pattern, which may contribute to their metastatic potential as well as potential immune evasion. [iStock/© vjanez]    http://www.genengnews.com/media/images/AnalysisAndInsight/Apr12_2016_iStock_41612310_PlasmaMembraneOfACell1211657142.jpg

There is growing interest in the field of glycobiology given the fact that epitopes with physiological and pathological relevance have glyco moieties.  We believe that another “omics” revolution is on the horizon—the study of the glyco modifications on the surface of cells and their potential as biomarkers and therapeutic targets in many disease classes. Not much industry tracking of this field has taken place. Thus, we sought to map this landscape by examining the entire ensemble of academic publications in this space and teasing apart the trends operative in this field from a qualitative and quantitative perspective. We believe that this methodology of en masse capture and publication and annotation provides an effective approach to evaluate this early-stage field.

Identifiation and Growth of Glycobiology Publications

http://www.genengnews.com/Media/images/AnalysisAndInsight/thumb_April12_2016_SelectBiosciences_Figure11935315421.jpg

For this article, we identified 7000 publications in the broader glycobiology space and analyzed them in detail.  It is important to frame glycobiology in the context of genomics and proteomics as a means to assess the scale of the field. Figure 1 presents the relative sizes of these fields as assessed by publications in from 1975 to 2015.

Note that the relative scale of genomics versus proteomics and glycobiology/glycomics in this graph strongly suggests that glycobiology is a nascent space, and thus a driver for us to map its landscape today and as it evolves over the coming years.

Figure 2. (A) Segmentation of the glycobiology landscape. (B) Glycobiology versus glycomics publication growth.

 

http://www.genengnews.com/Media/images/AnalysisAndInsight/thumb_April12_2016_SelectBiosciences_Figure2ab1917013624.jpg

To examine closely the various components of the glycobiology space, we segmented the publications database, presented in Figure 2A. Note the relative sizes and growth rates (slopes) of the various segments.

Clearly, glycoconjugates currently are the majority of this space and account for the bulk of the publications.  Glycobiology and glycomics are small but expanding and therefore can be characterized as “nascent market segments.”  These two spaces are characterized in more detail in Figure 2B, which presents their publication growth rates.

Note the very recent increased attention directed at these spaces and hence our drive to initiate industry coverage of these spaces. Figure 2B presents the overall growth and timeline of expansion of these fields—especially glycobiology—but it provides no information about the qualitative nature of these fields.

Focus of Glycobiology Publications

http://www.genengnews.com/Media/images/AnalysisAndInsight/April12_2016_SelectBiosciences_Figure2c1827601892.jpg

Figure 2C. Word cloud based on titles of publications in the glycobiology and glycomics spaces.

To understand the focus of publications in this field, and indeed the nature of this field, we constructed a word cloud based on titles of the publications that comprise this space presented in Figure 2C.

There is a marked emphasis on terms such as oligosaccharides and an emphasis on cells (this is after all glycosylation on the surface of cells). Overall, a pictorial representation of the types and classes of modifications that comprise this field emerge in this word cloud, demonstrating the expansion of the glycobiology and to a lesser extent the glycomics spaces as well as the character of these nascent but expanding spaces.

Characterization of the Glycobiology Space in Journals

Figure 3A. Breakout of publications in the glycobiology/glycomics fields.   http://www.genengnews.com/Media/images/AnalysisAndInsight/April12_2016_SelectBiosciences_Figure3a_5002432117316.jpg
Having framed the overall growth of the glycobiology field, we wanted to understand its structure and the classes of researchers as well as publications that comprise this field. To do this, we segmented the publications that constitute this field into the various journals in which glycobiology research is published. Figure 3A presents the breakout of publications by journal to illustrate the “scope” of this field.

The distribution of glycobiology publications across the various journals suggests a very concentrated marketplace that is very technically focused. The majority of the publications segregate into specialized journals on this topic, a pattern very indicative of a field in the very early stages of development—a truly nascent marketplace.

http://www.genengnews.com/Media/images/AnalysisAndInsight/thumb_April12_2016_SelectBiosciences_Figure3b1012091061.jpg

Figure 3B. Origin of publications in the glycobiology/glycomics fields.
We also sought to understand the “origin” of these publications—the breakout between academic- versus industry-derived journals. Figure 3B presents this breakout and shows that these publications are overwhelmingly (92.3%) derived from the academic sector. This is again a testimonial to the early nascent nature of this marketplace without significant engagement by the commercial sector and therefore is an important field to characterize and track from the ground up.

Select Biosciences, Inc. further analyzed the growth trajectory of the glycobiology papers in Figure 3C as a means to examine closely the publications trajectory. Although there appears to be some wobble along the way, overall the trajectory is upward, and of late it is expanding significantly.

In Summary

Figure 3C. Trajectory of the glycobiology space.   http://www.genengnews.com/Media/images/AnalysisAndInsight/April12_2016_SelectBiosciences_Figure3c1236921793.jpg
Glycobiology is the study of what coats living cells—glycans, or carbohydrates, and glycoconjugates. This is an important field of study with medical applications because it is known that tumor cells alter their glycosylation pattern, which may contribute to their metastatic potential as well as potential immune evasion.

At this point, glycobiology is largely basic research and thus it pales in comparison with the field of genomics. But in 10 years, we predict the study of glycobiology and glycomics will be ubiquitous and in the mainstream.

We started our analysis of this space because we’ve been focusing on many other classes of analytes, such as microRNAs, long-coding RNAs, oncogenes, tumor suppressor genes, etc., whose potential as biomarkers is becoming established. Glycobiology, on the other hand, represents an entire new space—a whole new category of modifications that could be analyzed for diagnostic potential and perhaps also for therapeutic targeting.

Today, glycobiology and glycomics are where genomics was at the start of the Human Genome Project. They respresent a nascent space and with full headroom for growth. Select Biosciences will continue to track this exciting field for research developments as well as development of biomarkers based on glyco-epitopes.

Enal Razvi, Ph.D., conducted his doctoral work on viral immunology and subsequent to receiving his Ph.D. went on to the Rockefeller University in New York to serve as Aaron Diamond Post-doctoral fellow under Professor Ralph Steinman [Nobel Prize Winner in 2011 for his discovery of dendritic cells in the early-70s with Zanvil Cohn]. Subsequently, Dr. Razvi completed his research fellowship at Harvard Medical School. For the last two decades Dr. Razvi has worked with small and large companies and consulted for more than 100 clients worldwide. He currently serves as Biotechnology Analyst and Managing Director of SelectBio U.S. He can be reached at enal@selectbio.us. Gary M. Oosta holds a Ph.D. in Biophysics from Massachusetts Institute of Technology and a B.A. in Chemistry from E. Mich. Univ. He has 25 years of industrial research experience in various technology areas including medical diagnostics, thin-layer coating, bio-effects of electromagnetic radiation, and blood coagulation. Dr. Oosta has authored 20 technical publications and is an inventor on 77 patents worldwide. In addition, he has managed research groups that were responsible for many other patented innovations. Dr. Oosta has a long-standing interest in using patents and publications as strategic technology indicators for future technology selection and new product development. To enjoy more articles like this from GEN, click here to subscribe now!

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3D Imaging of Cancer Cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions

Dark Daily Apr 8th 2016        Jon Stone

https://www.linkedin.com/pulse/3d-imaging-cancer-cells-could-lead-improved-ability-plan-joseph-colao

 

3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions.

New technology from researchers at the University of Texas Southwestern Medical Center enables the ability to study cancer cells in their native microenvironments.

Imaging research is one step closer to giving clinicians a way to do high-resolution scans of malignant cells in order to diagnose cancer and help identify useful therapies. If this technology were to prove successful in clinical studies, it might change how anatomic pathologists and radiologists diagnose and treat cancer.

Researchers at the University of Texas Southwestern Medical Center developed a way to create near-isotropic, high-resolution scans of cells within their microenvironments. The process involves utilizing a combination of two-photonBessel beams and specialized filtering.

New Imaging Approach Could be Useful to Both Pathologists and Radiologists

In a recent press release, senior author Reto Fiolka, PhD, said “there is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned. Our microscope is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor.”

In a study in Developmental Cell, Erik S. Welf, PhD, et al, described the new microenvironmental selective plane illumination microscopy (meSPIM). When developing the technology, the team outlined three goals:

1. The microscope design must not prohibitively constrain microenvironmental properties.

2. Spatial and temporal resolution must match the cellular features of interest.

3. Spatial resolution must be isotropic to avoid spatial bias in quantitative measurements.

This new technology offers pathologists and medical laboratory scientists a new look at cancer cells and other diseases. The study notes that meSPIM eliminates the influence of stiff barriers, such as glass slide covers, while also allowing a level of control over both mechanical and chemical influences that was previously impossible.

Early meSPIM Research Reveals New Cell Behaviors

Early use of meSPIM in observing melanoma cells is already offering new insights into the relationship between the cell behavior of cellular- and subcellular-scale mechanisms and the microenvironment in which these cells exist. The study notes, “The ability to image fine cellular details in controllable microenvironments revealed morphodynamic features not commonly observed in the narrow range of mechanical environments usually studied in vitro.”

One such difference is the appearance of blebbing. Created by melanoma cells and lines, these small protrusions are thought to aid in cell mobility and survival. Using meSPIM, observers could follow the blebbing process in real-time. Formation of blebs on slides and within an extracellular matrix (ECM) showed significant differences in both formation and manipulation of the surrounding microenvironment.

The team is also using meSPIM to take a look at membrane-associated biosensor and cytosolic biosensor signals in 3D. They hope that investigation of proteins such as phosphatidylinositol 3-kinase (PI3K) and protein kinase C will help to further clarify the roles these signals play in reorientation of fibroblasts.

meSPIM combined with computer vision enables imaging, visualization, and quantification of how cells alter collagen fibers over large distances within an image volume measuring 100 mm on each side. (Photo Copyright: Welf and Driscoll et al.)

The research team believes this opens new possibilities for studying diseases at a subcellular level, saying, “Cell biology is necessarily restricted to studying what we can measure. Accordingly, while the last hundred years have yielded incredible insight into cellular processes, unfortunately most of these studies have involved cells plated onto flat, stiff surfaces that are drastically different from the in vivo microenvironment …

“Here, we introduce an imaging platform that enables detailed subcellular observations without compromising microenvironmental control and thus should open a window for addressing these fundamental questions of cell biology.”

Limitations of meSPIM

One significant issue associated with the use of meSPIM is the need to process the large quantity of data into useful information. Algorithms currently allow for automatic bleb detection. However, manual marking, while time consuming, still provides increased accuracy. Researchers believe the next step in improving the quality of meSPIM scans lie in computer platforms designed to extract and process the scan data.

Until this process is automated, user bias, sample mounting, and data handling will remain risks for introducing errors into the collected data. Yet, even in its early stages, meSPIM offers new options for assessing the state of cancer cells and may eventually provide pathologists and radiologists with additional information when creating treatment plans or assessments.

 

Seeing cancer cells in 3-D (w/ Video)

http://phys.org/news/2016-02-cancer-cells-d-video.html

 

Cancer in 3-D

http://cdn.phys.org/newman/csz/news/800/2016/cancerin3d.png

Extracted surfaces of two cancer cells. (Left) A lung cancer cell colored by actin intensity near the cell surface. Actin is a structural molecule that is integral to cell movement. (Right) A melanoma cell colored by PI3-kinase activity near the cell surface. PI3K is a signaling molecule that is key to many cell processes. Credit: Welf and Driscoll et al.

Cancer cells don’t live on glass slides, yet the vast majority of images related to cancer biology come from the cells being photographed on flat, two-dimensional surfaces—images that are sometimes used to make conclusions about the behaviour of cells that normally reside in a more complex environment. But a new high-resolution microscope, presented February 22 in Developmental Cell, now makes it possible to visualize cancer cells in 3D and record how they are signaling to other parts of their environment, revealing previously unappreciated biology of how cancer cells survive and disperse within living things.

“There is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned,” says senior author Reto Fiolka, an optical scientist at the University of Texas Southwestern Medical Center. “Our is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor environments.”

Read more at: http://phys.org/news/2016-02-cancer-cells-d-video.html#jCp

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