The striking conservation between genes involved in cytokinesis and axonal elongation, as well as the morphological similarity between growth cones and migrating cells, raises the question of how axonal elongation evolved. Based on the observation that axonal elongation, like cell migration, involves retrograde actin flow at the leading edge and bulk forward advance of microtubules, we have developed and applied an active fluid biophysical model to cell division, mesenchymal cell migration, ameboid migration, neuronal migration, and axonal elongation. We find patterns of motion can be simulated in all cases by modestly adjusting sub-cellular gradients in cellular adhesion, force generation, cytoskeletal viscosity, and cellular topology. Considering this problem in an evolutionary context suggests a hypothetical path linking them together, a means for testing whether neurons evolved once or twice during evolution, and new directions for developing therapies designed to treat neurological injury and disease.

Learning Objectives: 
1. Classify the core mechanism underlying cell motility and how to interpret kymographs.
2. Identify the differences and commonalities between the internal flow patterns and motion that occur during cytokinesis, amoeboid migration, mesenchymal migration, neuronal migration, and axonal elongation.
3. Approve that cell biological and biophysical analysis of axonal elongation may help us to better understand how neurons evolved.

Get to know Luminex’s xMAP® Technology all over again. For 27 years, xMAP Technology has become known as the worldwide leader in bead-based multiplexing. With the new xMAP INTELLIFLEX®, we have re-energized the xMAP platform with a modern, fast, flexible, and intuitive flow-based analysis system that can deliver twice the data in a single well as compared to previous instruments.  

Join us to learn about the exciting applications of the xMAP INTELLIFLEX and its new dual reporter capability. We’ll describe some recently published studies that used this system, focusing on serological analyses. xMAP Technology enabled the multiplexed analysis of antigen-specific antibodies and the xMAP INTELLIFLEX’s simultaneous measuring of two antibody isotypes within a single well.

For Research Use Only. Not for use in diagnostic procedures. 

Learning Objectives:

1. Refresh your knowledge of Luminex and xMAP bead-based multiplexing technology and how it has been used in life sciences research for the past 27 years.

2. Learn about the enhanced features and the novel second reporter channel of the innovative xMAP INTELLIFLEX, Luminex's newest instrument for xMAP Technology.

3. Learn about recent work that demonstrates the dual reporter functionality of the xMAP INTELLIFLEX for multiplexed serological analysis with antibody isotyping.

α-synuclein (α-syn) is a small presynaptic protein linked to the pathogenesis of Parkinson’s disease (PD) and related disorders, collectively called synucleinopathies. Phosphorylation of α-syn at Ser-129 is a pathologic hallmark of synucleinopathies and is widely considered to be a therapeutic target. Using pHluorin assays, we and others have shown that modest levels of α-syn overexpression attenuates synaptic vesicle (SV) recycling, suggesting that α-syn is a physiologic attenuator of neurotransmitter release. Furthermore, we have shown that binding of α-syn to VAMP2 is necessary for α-syn-induced synaptic attenuation. In this study, we evaluated the presynaptic targeting of phospho-incompetent (Ser-129A) and phospho-mimic (Ser-129D) α-syn mutants, and also evaluated effects of Ser-129 phosphorylation on SV-recycling using pHluorins. While mimicking Ser129 phosphorylation augmented synaptic α-syn accumulation and further attenuated SV-recycling as expected, surprisingly, preventing Ser129-P completely blocked the ability of α-syn to attenuate SV-recycling; suggesting a physiologic role for this post-translational modification at synapses. Mechanistically, we found that phosphorylation at the Ser-129 site was required for α-syn binding to VAMP2 and subsequent synaptic attenuation, explaining the inability of Ser129 phospho-incompetent α-syn to attenuate SV-recycling. Finally, AlphaFold2-based modeling and membrane-binding simulations support a scenario where Ser-129 phosphorylation triggers a conformational change in α-syn that exposes its C-terminus, allowing VAMP2 binding and consequent functionality. Collectively, the data suggest a physiologic role for α-syn S129 phosphorylation and warrant caution in developing therapeutic agents that inhibit this phosphorylation site.

Learning Objectives:

1. Elucidate the importance of S129 phosphorylation of α-synuclein to physiology, in addition to its pathological association. 
 
2. Explain the mechanism by which α-synuclein S129site phosphorylation triggers synaptic vesicle recycling. 
 
3. Describe the current approaches to measure synaptic recycling as a result of neuronal activity. 

Heterotetrameric adaptor protein (AP) complexes play key roles in protein sorting and transport vesicle formation in the endomembrane system of eukaryotic cells. One of these complexes, AP-4, was identified over 20 years ago but, until recently, its function remained unclear. AP-4 associates with the trans-Golgi network (TGN) through interaction with small GTPases of the ARF family. Recent studies identified the autophagy protein ATG9A as a specific AP-4 cargo. Defective export of ATG9A from the TGN in AP-4-deficient cells was shown to reduce ATG9A delivery to pre-autophagosomal structures, impairing autophagosome maturation. In addition, mutations in AP-4-subunit genes were found to cause neurological dysfunction in mice and a form of complicated hereditary spastic paraplegia referred to as 'AP-4-deficiency syndrome' in humans. These findings demonstrated that mammalian AP-4 is required for the development and function of the central nervous system, likely through its role in the sorting of ATG9A for the maintenance of autophagic homeostasis. In my talk, I will review the properties and functions of AP-4, and discuss how they might explain the clinical features of AP-4 deficiency.

Learning Objectives:

1. Classify how adaptor protein (AP) complexes mediate protein sorting to different intracellular compartments.
 
2. Analyze the connection of the AP-4 complex to autophagy, development of the central nervous system, and neurodevelopmental disease.
 
3. Express how to go from basic research discoveries to the elucidation of pathogenetic mechanisms and potential therapies.
 

Cell migration is an essential process for many biological phenomena. It plays an important role in disease processes such as wound healing and cancer metastasis. The immune system, which protects us from diseases, also relies on the migration of various cell types with immensely diverse and highly specific receptors to recognize potential threats. To accomplish the processes that are involved in cell migration, including chemoattraction, rolling adhesion, tight adhesion collective cell migration, and/or extravasation and transmigration, cells need to successfully navigate the complex 3D environment of living tissues.

To study the detailed molecular and biophysical mechanisms related to T cell migration mouse models are often used. However, their biology does not always translate to humans and on top of that mouse models cost a lot of time and money and are ethically debatable. In vitro cell culture models do not recapitulate complex human biology, due to the lack of vasculature, extracellular matrix, and neighboring cells. Today, you will learn how microfluidic organ-on-a-chip models can be developed to study 3D cell dynamics and about the development of an assay which allows T cell extravasation under flow in real-time, using a high-throughput, artificial membrane-free microfluidic platform as an example.

Microphysiological systems (MPS) have proven to be a powerful tool for recreating human tissue- and organ-like functions. However, establishing complex human in vitro ADME models involving co-culture of key organs to mimic physiologically based pharmacokinetic (PBPK) distribution behavior still present a challenge. In our recent study, we developed a PBPK compliant ADME 4-Organ-Chip (Chip4) with a downscale factor of 1:100.000 of the human body. The integration of an intestinal barrier model for absorption and first-pass metabolism, liver microtissues for main metabolism, a kidney model with proximal tubular-like cells and podocytes for excretion, and neuronal spheroids as a potential target organ were optimized in the chip and co-cultured for 14 days. The setup was repeatedly exposed to Haloperidol, an antipsychotic medication and to Carbamazepine, a tricyclic compound with anticonvulsant properties through different routes. Results on direct as well as metabolite induced effects on organ-specific levels will be presented. Subsequently this data formed the basis for the development of an in silico PBPK model for compound prediction.

Learning Objectives:

1. Demonstrate ADME-on-a-chip.
2. Correlate selective drug transport.
3. Examine organ-specific toxicity.  

Human Allogeneic Liver-derived Progenitor Cells (HALPC; HepaStemTM) are an advanced therapy medicinal product composed of medicinal signaling cells derived and expanded from healthy human liver. Their mesenchymal and hepatocytic features make them promising cell therapy candidates for the treatment of life-threatening fibro-inflammatory liver diseases.

Safety, tolerability, and preliminary efficacy of HALPC infusion have been shown in 3 different trial:

1. A phase IIa prospective, open label, multi-center, randomized clinical trial in paediatric patients suffering from metabolic disorders, such as urea cycle disorders or Crigler-Najjar syndrome (HEP001 – NCT01765283; Smets et al., 2019).
2. In adults with Non Alcoholic Steato Hepatitis (NASH)
3 In adult with Acute decompensation and Acute on chronic Liver Failure (ACLF). HALPC act as a cargo inducing a hit-and-run decreasing of systemic and local inflammation and deactivation of stellate cells through paracrine activity. In vitro studies have shown a significant secretion of anti-inflammatory and anti-fibrotic cytokines including prostaglandin E2 (PGE2), indoleamine 2,3- dioxygenase (IDO), and hepatocyte growth factor (HGF), upon incubation of HALPC with an inflammatory cocktail.
ACLF is a life-threatening complication of cirrhosis, associated with deterioration of liver function followed by multiple organ failures. ACLF is linked to multiple cell type dysfunction, immunological imbalance and increased local and systemic inflammation. It is expected that HALPC exert immunomodulatory effects by interacting with immune cells of the patient and by paracrine effects through the various factors they secrete.

The phase IIa clinical trial (HEP101 – NCT02946554) assessed the safety and preliminary efficacy of repeated infusions and intermediate doses in cirrhotic patients with ACLF or AD. (Nevens et al., 2018). An ongoing phase IIb, proof of concept trial, is currently recruiting patients across multiple centers in Europe (HEP102 – NCT04229901). This is an interventional, double blind, randomized, and placebo- controlled study of 1 dose regimen of HALPC intravenously infused in patients newly diagnosed with ACLF grade 1 or 2 on top of Standard of Care (SoC).

Pre-clinical data coupled with clinical data support a continued investigation in the context of a clinical development platform program, targeting ACLF, NASH, Acute Alcoholic Hepatitis, Acute decompensation of cirrhosis and other fibro inflammatory liver diseases.
 

Learning Objectives:

1. Classify the principle of paracrine mechanism of action.

2. Describe the process of bench to bedside clinical development.

3. Identify the design of a clinical trial.

The use of primary human hepatocytes in basic research or other clinical applications (e.g., liver assist devices, ex vivo gene therapy, and hepatocyte transplantation) is limited by a shortage in the number of non-transplantable donor livers of sufficient quality for isolation of cells. As hepatocytes are unable to be expanded in culture, efforts have been undertaken to find ways to produce cells using in vivo mammalian bioreactors. One such bioreactor is a rodent model that combines deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme critical to the catabolism of tyrosine, together with knockouts in recombination activation gene 2 (RAG2-/-) and interleukin-2 receptor gamma (IL2rg-/y) to generate severe combined immunodeficiency. These three mutations, referred to as an ‘FRG’ model, allow for the implantation of human hepatocytes into the livers of the rodent and selective expansion of healthy, FAH+ hepatocytes over time. This model has been extensively validated and is in wide use; however, it is limited by the numbers of cells that can be produced due to the small sizes of mice and rats. To address this, and other logistical issues with the use of rodent-expanded hepatocytes for clinical applications, we have generated a large animal FRG model that can be used for safely producing a theoretically unlimited quantity of high-quality human hepatocytes for research and therapeutic purposes.

Learning Objectives:

1. Explain the functions of hepatocytes and how these cells are being used for research and clinical purposes
2. Describe the rationale for use of fumarylacetoacetate hydrolase (FAH) as a useful selectable marker for the expansion of hepatocytes in vivo
3. Compare and contrast the benefits of rodent vs. large-animal FRG models for the expansion of healthy human hepatocytes

Mechanical forces are crucial to essentially every cellular activity including intracellular transportation, cell division and differentiation, cell-cell interaction and cell death. The quantification of mechanical forces at the cellular and molecular level, however, is difficult especially for proteins localized to the inside of the cell. The state-of-the-art genetically encoded in vivo force sensors rely on Förster resonance energy transfer (FRET), which requires the insertion of at least two fluorescent proteins and are limited in their readouts. We propose to use a series of engineered protein motifs called coiled-coils to construct new in vivo force sensors in order to reduce the footprint of in vivo force sensors and to diversify the readout. We demonstrate the versatile uses of coiled-coil force sensors by quantifying the forces during clathrin-mediated endocytosis (CME) in fission yeast Schizosacchromyces pombe. We will also discuss how future coiled-coil force sensors will make in vivo force measurements faster and more accessible. 

Learning Objectives

1) Examine the importance of mechanical forces in cellular activities. 
2) Evaluate current strategies for force measurement at the cellular and molecular level. 
3) Elucidate the production and transmission of mechanical forces during clathrin-mediated endocytosis, as revealed by different force sensors.
 

One of the primary limitations in drug discovery and toxicology research is the lack of good model systems between the single cell level and animal or human systems. This is especially true for neurodegenerative diseases such as ALS, Alzheimer’s, and spinal cord injury. In addition, with the banning of animals for toxicology testing in many industries body-on-a-chip systems to replace animals with human mimics is essential for product development and safety testing. Our research focus is on the establishment of functional in vitro systems to address this deficit where we seek to create organs and subsystems to model motor control, muscle function, myelination and cognitive function, as well as cardiac and other subsystems. The idea is to integrate microsystems fabrication technology and surface modifications with protein and cellular components, for initiating and maintaining self-assembly and growth into biologically, mechanically and electronically interactive functional multi-component systems. Our advances in culturing adult rat, mouse and human mammalian spinal cord, hippocampal neurons, muscle and cardiac cells in a defined serum-free medium, suggest outstanding potential for answering questions related to maturation, aging, neurodegeneration and injury. We are using this ability to manipulate the biological systems and integrate it with silicon-based systems to create functional organ mimics for high content drug discovery. We are also using what we learn for a more fundamental understanding of cellular development, protein adsorption and neuronal regeneration. Examples will be given of some of the more advanced human-on-a-chip systems being developed as well as the results of six workshops held at NIH to explore what is needed for validation and qualification of these systems.

​In the United States, prostate cancer (PCa) is the second-leading cause of cancer death in men. Multiple observations indicate the link between prostate carcinogenesis, stress of endoplasmic reticulum (ER stress), and impairment of Golgi morphology. We reported that PCa cells employ a self-activation mechanism of ATF6-mediated ER stress caused by Golgi disorganization and associated with enhanced androgen receptor transactivation. However, the role of ER stress and Golgi fragmentation in the development of castration-resistant prostate cancer (CRPC) is still unknown. CRPC is characterized by the enhanced activity and expression of integrins. Different classes of integrins, mostly alphav beta3, alphav beta5, and alphav beta6, form abnormal integrin repertoire at the plasma membrane (PM) due to the high affinity of their glycans (N-acetyllactosamine, LacNAc) to Galectin-3. This, however, is possible only when integrins are specifically glycosylated in Golgi by glycosyltransferases, MGAT5 or GalT/B3GN, which activation relies on the structural reorganization of Golgi matrix proteins golgins. In addition, underglycosylated integrins, carrying high mannose glycans, can also enhance prostate tumor cells' migratory and invasive capabilities. Here, we provide data indicating that: (a) Golgi disorganization in advanced PCa cells associates with the atypical activity of MGAT1 glycosyltransferase in Golgi, which is caused monomerization of the largest golgin giantin; (b) severity of PCa correlates with expression of MGAT5- and GalT/B3GNT-modified integrins; (c) appearance of high-mannose integrins at the cell surface is likely mediated by ER-PM junctions and triggered by ATF6-mediated ER stress; (d) treatment of advanced PCa cells with anti-malarial drug hydroxychloroquine (HCQ) restores compact Golgi, thus blocking the activity of MGAT5 and GalT/B3GNT enzymes. Our central hypothesis is that ER stress, Golgi disorganization, and remodeling of Golgi matrix proteins impair N-glycosylation of integrins, which results in their enhanced representation at the cell surface and more aggressive phenotype of PCa cells.

Learning Objectives:

1. Elucidate the mechanism of abnormal glycosylation of integrins by studying the impact of Golgi disorganization on differential distribution of golgins and N-glycosylation enzymes. 

2. Examine the effects of ATF6-mediated ER stress on the expression and trafficking of integrins to the cell surface. 

3. Discuss the outcome of ATF6 depletion/inhibition combined with hydroxychloroquine treatment on the progression of a prostate tumor.

Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein’sintracellular role in a spatially and temporally precise manner. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers.  Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28oC to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these “enhanced” Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

Affiliations:

1 Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA.

Learning Objectives:

1. Identify the variety of the tools available for inducible dimerization

2. Outline the features and unique characteristics that determine the appropriateness of a given dimerization tool for one’s desired application.

3. Investigate the most important features of optical dimerization tools that should guide in the choice of the most suitable to modulate intracellular reactions.

The gastrointestinal (GI) epithelium is the body’s first line of defense to luminal contents such as food, microbiota, pharmaceuticals, and toxins. A small pool of proliferative stem and progenitor cells are responsible for maintaining and repairing the entire GI epithelium. Oncology chemotherapeutics target these rapidly dividing cells, contributing to adverse event risks, with 50-80% of patients developing diarrhea. Historically, these compounds have the lowest overall success rate for regulatory approval. A preclinical model for rapid screening of a candidate compound’s GI toxicity profile would provide a better safety profile prior to entering clinical trials. Although animal models, immortalized cell lines, and other complex in vitro systems exist for GI tox testing, RepliGut Planar, a simpler approach utilizing a monolayer of primary human epithelial cells, can more accurately predict GI toxicity.

Utilizing human colonic monolayers isolated from multiple donors, we investigated how chemotherapeutics affect the epithelium during proliferation and differentiation. Using an industry-backed test set of compounds, which included known positive and negative compounds for GI toxicity, each compound was tested to provide a dose-response curve. For proliferative cell testing, compounds were given to actively dividing cells and read-outs included resistance measurements indicative of barrier formation potential, total cell counts indicative of cell viability, and total EdU-positive cell counts indicative of monolayer proliferative capacity. For differentiated cell testing, compounds were given to non-dividing, cells and read-outs included barrier resistance measurements. Overall, the toxic concentration in the epithelial cell model were well-aligned with clinically relevant concentrations that produce toxicity (as measured by Cmax).

Within the kidney, the glomerulus is the structure in charge of the renal ultrafiltration. Loss of glomerular function leads to renal damage and could end with irreversible damage. The major roadblocks to advancing new drugs and therapeutics designed specifically to preserve glomerular function stem from the inability to effectively develop multicellular in vitro models that can accurately mimic the architecture of the glomerular filtration barrier. We have recently developed a barrier-free, human based glomerulus-on-a-chip system that closely replicates the glomerular filtration barrier and its functions. In this session we will present our platform and discuss our newer insights on modeling of various diseases including membranous nephropathy, Alport Syndrome and diabetic nephropathy as well as describe potential applications ranging from mechanistic studies, biomarker discovery and personalized medicine. 

Learning Objectives:

1. Appreciate the complexity of the glomerular filtration barrier and the difficulty of establishing faithful and functional in vitro models

2. Classify the emerging role of microfluidic systems to study the pathophysiology of human renal diseases

3. Recognize the potential of the glomerulus-on-a-chip to enable studies on how different factors might affect response to injury in podocytes and glomerular endothelial cells.