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New avenues for research in membrane biology reveals the mobility of protein at work
Curator and Reporter: Dr. Premalata Pati, Ph.D., Postdoc
Membrane proteins(MPs) are proteins that exist in the plasma membrane and conduct a variety of biological functions such as ion transport, substrate transport, and signal transduction. MPs undergo function-related conformational changes on time intervals spanning from nanoseconds to seconds. Many MP structures have been solved thanks to recent developments in structural biology, particularly in single-particle cryo-Electron Microscopy (cryo-EM). Obtaining time-resolved dynamic information on MPs in their membrane surroundings, on the other hand, remains a significant difficulty.
OmpG (Open state) in a fully hydrated dimyristoylphosphatidylcholine (DMPC) bilayer. The protein is shown in light green cartoon. Lipids units are depicted in yellow, while their phosphate and choline groups are illustrated as orange and green van der Waals spheres, respectively. Potassium and chloride counterions are shown in green and purple, respectively. A continuous and semi-transparent cyan representation is used for water. https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-021-24660-1/MediaObjects/41467_2021_24660_MOESM1_ESM.pdf
Weill Cornell Medicine (WCM) researchers have found that they can record high-speed protein movements while linking them to function. The accomplishment should allow scientists to examine proteins in more depth than ever before, and in theory, it should allow for the development of drugs that work better by hitting their protein targets much more effectively.
The researchers utilized High-Speed Atomic Force Microscopy (HS-AFM) to record the rapid motions of a channel protein and published in a report in Nature Communications on July 16. Such proteins generally create channel or tube-like structures in cell membranes, which open to allow molecules to flow under particular conditions. The researchers were able to record the channel protein’s rapid openings and closings with the same temporal resolution as single channel recordings, a typical technique for recording the intermittent passage of charged molecules through the channel.
Senior author Simon Scheuring, professor of physiology and biophysics in anesthesiology at WCM, said,
There has been a significant need for a tool like this that achieves such a high bandwidth that it can ‘see’ the structural variations of molecules as they work.
Researchers can now produce incredibly detailed photographs of molecules using techniques like X-ray crystallography and electron microscopy, showing their structures down to the atomic scale. The average or dominant structural positionings, or conformations, of the molecules, are depicted in these “images,” which are often calculated from thousands of individual photos. In that way, they’re similar to the long-exposure still photos from the dawn of photography.
Many molecules, on the other hand, are flexible and always-moving machinery rather than fixed structures. Scientists need to generate videos, not still photos, to reveal how such molecules move as they work, to see how their motion translates to function to catch their critical functional conformations, which may only exist for a brief moment. Current techniques for dynamic structural imaging, on the other hand, have several drawbacks, one of which being the requirement for fluorescent tags to be inserted on the molecules being photographed in many cases.
Scheuring and his lab were early adopters of the tag-free HS-AFM approach for studying molecular dynamics. The technology, which can photograph molecules in a liquid solution similar to a genuine cellular environment, employs an extremely sensitive probe, similar to a record player’s stylus, to feel its way over a molecule and therefore build up a picture of its structure. Standard HS-AFM isn’t quick enough to capture the high-speed dynamics of many proteins, but Scheuring and colleagues have developed a modified version, HS-AFM height spectroscopy(HS-AFM-HS), that works much faster by collecting dynamic changes in only one dimension: height.
The researchers used HS-AFM-HS to record the opening and closing of a relatively simple channel protein, OmpG, found in bacteria and widely studied as a model channel protein in the new study, led by the first author Raghavendar Reddy Sanganna Gari, a postdoctoral research associate in Scheuring’s laboratory. They were able to monitor OmpG gating at an effective rate of roughly 20,000 data points per second, seeing how it transitioned from open to closed states or vice versa as the acidity of the surrounding fluid varied.
More significantly, they were able to correlate structural dynamics with functional dynamics in a membrane protein of this size for the first time in a partnership with Crina Nimigean, professor of physiology and biophysics in anesthesiology, and her group at WCM.
The demonstration opens the door for a wider application of this method in basic biology and drug development.
Sanganna Gari stated,
We’re now in an exciting period of HS-AFM technology, for example using this technique to study how some drugs modulate the structural dynamics of the channel proteins they target.
Main Source
Technique reveals proteins moving as they work. By Jim Schnabel in Cornell Chronicle, August 16, 2021.
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
New Georgia Tech research shows that cell stiffness could be a valuable clue for doctors as they search for and treat cancerous cells before they’re able to spread. The findings, which are published in the journal PLoS One, found that highly metastatic ovarian cancer cells are several times softer than less metastatic ovarian cancer cells. This study used atomic force microscopy (AFM) to study the mechanical properties of various ovarian cell lines. A soft mechanical probe “tapped” healthy, malignant and metastatic ovarian cells to measure their stiffness. In order to spread, metastatic cells must push themselves into the bloodstream. As a result, they must be highly deformable and softer. This study results indicate that cell stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.
Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. The results suggest either of two approaches. Atomic Force Microscopy is not normally used by pathologists in diagnostics. Electron microscopy requires space for making and cutting the embedded specimen, and a separate room for the instrument. The instrument is large and the technique was not suitable for anything other than research initially until EM gained importance in Renal Pathology. It has not otherwise been used. This new method looks like it might be more justified over a spectrum of cases.
A. Atomic Force Microscopy
So the first point related to microscopy is whether AFM has feasibility for routine clinical use in the pathologists’ hands. This requires:
suitable size of equipment
suitable manipulation of the specimen
The question of whether you are using overnight fixed specimen, or whether the material is used unfixed
Nothing is said about staining of cells for identification.
Then there is the question about whether this will increase the number of Pathologist Assistants used across the country, which I am not against. This would be the end of “house” trained PAs, and gives more credence to the too few PA programs across the country. The PA programs have to be reviewed and accredited by NAACLS (I served 8 years on the Board). A PA is represented on the Board, and programs are inspected by qualified peers. There is no academic recognition given to this for tenure and promotion in Pathology Departments, and a pathologist is selected for a medical advisory role by the ASCP, and must be a Medical Advisor to a MLS accredited Program. The fact is that PAs do gross anatomic dictation of selected specimens, and they do autopsies under the guidance of a pathologist. This is the reality of the profession today. The pathologist has to be in attendance at a variety of quality review conferences, for surgical morbidity and mortality to obstetrics review, and the Cancer Review. Cytopathology and cytogenetics are in the pathology domain. In the case of tumors of the throat, cervix, and accessible orifices, it seems plausible to receive a swab for preparation. However, sampling error is greater than for a biopsy. A directed needle biopsy or a MIS specimen is needed for the ovary.
B. identification of biomarkers that are related to the actin cytoskeleton
The alternative to the first approach is the identification of biomarkers that are related to the actin cytoskeleton, perhaps in the nature of the lipid or apoprotein isoform that gives the cell membrane deformability. The method measuring by Atomic Force Microscopy is shown with the current method of cytological screening, and I call attention to cells clustered together that have a scant cytoplasm surrounding nuclei occupying 1/2 to 3/4 of the cell radius. The cells are not anaplastic, but the clumps are suggestive of glnad forming epithelium.
The cell membrane, also called the plasma membrane or plasmalemma, is a semipermeable lipid bilayer common to all living cells. It contains a variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of cellular processes. It also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall. (Photo credit: Wikipedia)
AFM non contact mode (Photo credit: Wikipedia)
C. The diagnosis of ovarian cancer can be problematic because it can have a long period of growth undetected.
On the other hand, it is easily accessible once there is reason to suspect it. They are terrible to deal with because they metastasize along the abdominal peritoneum and form a solid cake. It is a problem of location and silence until it is late. Once they do announce a presence on the abdominal wall, there is probably a serous effusion. It was not possible to rely on a single marker, but when CA125 was introduced, Dr. Marguerite Pinto, Chief of Cytology at Bridgeport Hospital-Yale New Haven Health came to the immnunochemistry lab and we worked out a method for analyzing effusions, as we had already done with carcinoembryonic antigen. The use of CEA and CA125 was published by Pinto and Bernstein as a first that had an impact. This was followed by a study with the Chief of Oncology, Dr. Martin Rosman, that showed that the 30 month survival of patients post treatment is predicted by the half-life of disappearance of CA125 in serum. At the time of this writing, I am not sure of the extent of its use 20 years later. History has taught us that adoption can be slow, depending very much on dissemination from major academic medical centers. On the other hand, concepts can also be stuck at academic medical centers because of a rigid and unprepared mindset in the professional community. The best example of this is the story of Ignaz Semmelweis, the best student of Rokitansky in Vienna for discovering the cause and prevention of childbirth fever at a time that nursemaids had far better results at obstetrical delivery than physicians. Contrary to this, Edward Jenner, the best student of John Hunter (anatomist, surgeon, and physician to James Hume), discovered vaccination from the observation that milkmaids did not get smallpox (cowpox was a better alternative).
Ovarian Cancer a plot of the CA125 elimination half-life vs the Kullback-Liebler distance
Troponin(s) T, I, C and the contractile apparatus (contributed by Aviva Lev-Ari, PhD, RN)
For 2012 – 2013 Frontier Contribution in Cardiology on Gene Therapy Solutions for Improving Myocardial Contractility, see
Lev-Ari, A. 8/1/2013 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
For explanation of Conduction prior to Myocardial Contractility, see
Lev-Ari, A. 4/28/2013 Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis
The contraction of skeletal muscle is triggered by nerve impulses, which stimulate the release of Ca2+ from the sarcoplasmic reticulum—a specialized network of internal membranes, similar to the endoplasmic reticulum, that stores high concentrations of Ca2+ ions. The release of Ca2+ from the sarcoplasmic reticulum increases the concentration of Ca2+ in the cytosol from approximately 10-7 to 10-5 M. The increased Ca2+ concentration signals muscle contraction via the action of two accessory proteins bound to the actin filaments: tropomyosin and troponin (Figure 11.25). Tropomyosin is a fibrous protein that binds lengthwise along the groove of actin filaments. In striated muscle, each tropomyosin molecule is bound to troponin, which is a complex of three polypeptides: troponin C (Ca2+-binding), troponin I (inhibitory), and troponin T (tropomyosin-binding).
When the concentration of Ca2+ is low, the complex of the troponins with tropomyosin blocks the interaction of actin and myosin, so the muscle does not contract.
At high concentrations, Ca2+ binding to troponin C shifts the position of the complex, relieving this inhibition and allowing contraction to proceed.
Figure 11.25
Association of tropomyosin and troponins with actin filaments. (A) Tropomyosin binds lengthwise along actin filaments and, in striated muscle, is associated with a complex of three troponins: troponin I (TnI), troponin C (TnC), and troponin T (TnT). In (more…)
Contractile Assemblies of Actin and Myosin in Nonmuscle Cells
Contractile assemblies of actin and myosin, resembling small-scale versions of muscle fibers, are present also in nonmuscle cells. As in muscle, the actin filaments in these contractile assemblies are interdigitated with bipolar filaments of myosin II, consisting of 15 to 20 myosin II molecules, which produce contraction by sliding the actin filaments relative to one another (Figure 11.26).The actin filaments in contractile bundles in nonmuscle cells are also associated withtropomyosin, which facilitates their interaction with myosin II, probably by competing with filamin for binding sites on actin.
Figure 11.26
Contractile assemblies in nonmuscle cells. Bipolar filaments of myosin II produce contraction by sliding actin filaments in opposite directions.
Two examples of contractile assemblies in nonmuscle cells, stress fibers and adhesion belts, were discussed earlier with respect to attachment of the actin cytoskeleton to regions of cell-substrate and cell-cell contacts (see Figures 11.13 and 11.14). The contraction of stress fibers produces tension across the cell, allowing the cell to pull on a substrate (e.g., the extracellular matrix) to which it is anchored. The contraction of adhesion belts alters the shape of epithelial cell sheets: a process that is particularly important during embryonic development, when sheets of epithelial cells fold into structures such as tubes.
The most dramatic example of actin-myosin contraction in nonmuscle cells, however, is provided by cytokinesis—the division of a cell into two following mitosis (Figure 11.27). Toward the end of mitosis in animal cells, a contractile ring consisting of actin filaments and myosin II assembles just underneath the plasma membrane. Its contraction pulls the plasma membrane progressively inward, constricting the center of the cell and pinching it in two. Interestingly, the thickness of the contractile ring remains constant as it contracts, implying that actin filaments disassemble as contraction proceeds. The ring then disperses completely following cell division.
Figure 11.27
Cytokinesis. Following completion of mitosis (nuclear division), a contractile ring consisting of actin filaments and myosin II divides the cell in two.
Troponins T and I are released into the circulation at the time of an acute coronary syndrome (ACS). Troponin T was first introduced by Roche (developed in Germany) for the Roche platform as a superior biomarker for identifying acute myocardial infarction (AMI), because of a monoclonal specificity to the cardiac troponin T. It could not be measured on any other platform (limited license patent), so the Washington University Clinical Chemistry group developed a myocardiocyte specific troponin I that quickly became widely available to Beckman, and was adapted to other instruments. This was intended to replace the CK isoenzyme MB, that is highly elevated in rhabdomyolysis associated with sepsis or with anesthesia in special cases.
The troponins I and T had a tenfold scale difference, and the Receiver Operator Curve Generated cutoff was accurate for AMI, but had significant elevation with end-stage renal disease. The industry worked in concert to develop a high sensitivity assay for each because there were some missed AMIs just below the ROC cutoff, which could be interpreted as Plaque Rupture. However, the concept of plaque rupture had to be reconsidered, and we are left with type1 and type 2 AMI (disregarding the case of post PCI or CABG related). This led to the current establishment of 3 standard deviations above the lowest measureable level at 10% coefficient of variation. This has been discussed sufficiently elsewhere. It did introduce a problem in the use of the test as a “silver bullet” once the finer distinctions aqnd the interest in using the test for prognosis as well as diagnosis. This is where the use of another protein associated with heart failure came into play – either the B type natriuretic peptide, or its propeptide, N-terminal pro BNP. The prognostic value of using these markers, secreted by the HEART and acting on the kidneys (sodium reabsorption) has proved useful. But there has not been a multivariate refinement of the use of a two biomarker approach. In the following part D, I illustrate what can be done with an algorithmic approach to multiple markers.
Software Agent for Diagnosis of AMI
Isaac E. Mayzlin, Ph.D., David Mayzlin, Larry H. Bernstein, M.D. The so called gold standard of proof of a method is considered the Receiver-Operating Characteristic Curve, developed for detecting “enemy planes or missiles”, and adopted first by radiologists in medicine. This matches the correct “hits” to the actual calssification and it is generally taught as a plot of sensitivity vs (1 – specifity). But what if you had no “training” variable? Work inspired by Eugene Rypka’s bacterial classification led to Rosser Rudolph’s application of the Entropy of Shannon and Weaver to identify meaningful information, referring to what was Kullback-Liebler distance as “effective information”. This allowed Rudolph and Bernstein to classify using disease biomarkers obtaing the same results as the ROC curve using an apl program. The same data set was used by Bernstein, Adan et al. previously, and was again used by Izaak Mayzlin from University of Moscow with a new wrinkle. Dr. Mayzlin created a neural network (Maynet), and then did a traditional NN with training on the data, and also clustered the data using geometric distance clustering and trained on the clusters. It was interesting to see that the optimum cluster separation was closely related to the number of classes and the accuracy of classification. An earlier simpler model using the slope of the MB isoenzyme increase and percent of total CK activity was perhaps related to Burton Sobel’s work on CK-MB disappearance rate for infarct size. The main process consists of three successive steps: (1) clustering performed on training data set, (2) neural network’s training on clusters from previous step, and (3) classifier’s accuracy evaluation on testing data. The classifier in this research will be the ANN, created on step 2, with output in the range [0,1], that provides binary result (1 – AMI, 0 – not AMI), using decision point 0.5. Table 1. Effect of selection of maximum distance on the number of classes formed and on the accuracy of recognition by ANN
Clustering Distance Factor F(D = F * R)
Number ofClasses
Number of Nodes in The Hidden Layers
Number of Misrecognized Patterns inThe TestingSet of 43
Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
refer to: Cardiac ischemia in patients with septic shock randomized to vasopressin or norepinephrine
Mehta S, Granton J, Gordon AC, Cook DJ, et al.
Critical Care 2013, 17:R117 http://dx.doi.org/10.1186/cc12789
Troponin and CK levels, and rates of ischemic ECG changes were similar in the VP and NE groups. In multivariable analysis
only APACHE II was associated with 28-day mortality (OR 1.07, 95% CI 1.01-1.14, p=0.033).
Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs
Up-regulation of sarcoplasmic reticulum Ca2+ uptake leads to cardiac hypertrophy, contractile dysfunction and early mortality in mice deficient in CASQ2Cardiovasc Res. 2013;98:297-306,
Gene and cytokine therapy for heart failure: molecular mechanisms in the improvement of cardiac functionAm. J. Physiol. Heart Circ. Physiol.. 2012;303:H501-H512,
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