Arie Horowitz

The complexity of prokaryotic and, exceedingly, of eukaryotic cells, challenges the implemen-tation of methodologies to quantify intracellular dynamics. Although stochastic thermodynam-ics can be used to effectively quantify the energetics of single proteins or complexes, there is no statistical approach to determine the macrostate of a whole cell from its subcellular mi-crostates. The objective of this study is to test if Onsager reciprocity, a coupling between ther-modynamic flows, can be established between time-dependent intracellular processes from their measurements in previous studies. A prototypical thermodynamic profile of the cell is for-mulated as a model system to enable representation of its sub systems as force-driven ther-modynamic flows. Onsager reciprocity is validated on the bacterial flagellum and ATP syn-thase. It is then tested on the putative coupling between the contractile ring and treadmilling of the cytoskeleton in bacterial binary fission and eukaryotic cytokinesis, with opposite out-comes. All thermodynamic flows and forces are formulated to comply with their canonical defi-nitions.

Epithelial tight junctions define the paracellular permeability of the intestinal barrier. Molecules can cross the tight junctions via two distinct size-selective and charge-selective paracellular pathways: the pore pathway and the leak pathway. These can be distinguished by their selectivities and differential regulation by immune cells. However, permeability increases measured in most studies are secondary to epithelial damage, which allows non-selective flux via the unrestricted pathway. Restoration of increased unrestricted pathway permeability requires mucosal healing. By contrast, tight junction barrier loss can be reversed by targeted interventions. Specific approaches are needed to restore pore pathway or leak pathway permeability increases. Recent studies have used preclinical disease models to demonstrate the potential of pore pathway or leak pathway barrier restoration in disease. In this Review, we focus on the two paracellular flux pathways that are dependent on the tight junction. We discuss the latest evidence that highlights tight junction components, structures and regulatory mechanisms, their impact on gut health and disease, and opportunities for therapeutic intervention.

Though membrane trafficking of cell junction proteins has been studied extensively for more than two decades, the accumulated knowledge remains fragmentary. The goal of this review is to synthesize published studies on the membrane trafficking of the five major junction transmembrane proteins: claudins, occludin, and junction adhesion molecules (JAMs) in tight junctions; cadherins and nectins in adherens junctions; to identify underlying common mechanisms; to highlight their functional consequences on barrier function; and to identify knowledge gaps. Clathrin-mediated endocytosis appears to be the main, but not exclusive, mode of internalization. Caveolin-mediated endocytosis and macropinocytosis are employed less frequently. PDZ-domain binding is the predominant mode of interaction between junction protein cytoplasmic tails and scaffold proteins. It is shared by claudins, the largest family of junction integral proteins, by junction adhesion molecules A, B, and C, and by the three nectins. All eight proteins are destined to either recycling via Rab4/Rab11 GTPases or to degradation. The sorting mechanisms that underlie the specificity of their endocytic pathways and determine their fates are not fully known. New data is presented to introduce an emerging role of junction-associated scaffold proteins in claudin membrane trafficking.

Though membrane trafficking of cell junction proteins has been studied extensively for more than two decades, the accumulated knowledge remains fragmentary. The goal of this review is to synthesize the numerous and disparate observations on the membrane trafficking of the five major junction transmembrane proteins accumulated to date to comprehend the current state of the art, to highlight differences and similarities among the trafficking pathways, and to identify topics that are not fully understood. Clathrin-mediated endocytosis appears to be the main but not exclusive mode of internalization. Caveolin-mediated endocytosis and macropinocytosis are employed less frequently. PDZ-domain binding is the predominant mode of interaction between junction protein cytoplasmic tails and scaffold proteins. It is shared by claudins, the largest family of junction integral proteins, and by the three nectins. All five proteins are destined to either recycling via Rab4/Rab11 GTPases or to degradation. The sorting mechanisms that underlie the specificity of their endocytic pathways and determine their fates are not fully known. New data is presented to introduce an emerging role of junction-associated scaffold proteins in claudin membrane trafficking.

Inference of Diagnostic Markers and Therapeutic Targets From CSF Proteomics for the Treatment of Hydrocephalus

The purpose of this mini-review is to examine if publicly available cerebrospinal fluid (CSF) proteomics data sets can be exploited to provide insight into the etiology of hydrocephalus, into the character of the injury inflicted on the parenchyma by ventriculomegaly, and into the response of the brain to this condition. While this undertaking was instigated by reanalysis of recent comparative proteomics of CSF collected from the brain of healthy and Mpdz knockout (KO) mice (Yang et al., 2019), it is an opportunity to survey previously published CSF proteomics data sets to determine if they can be pooled together to that end. The overabundance of extracellular matrix (ECM) proteins, complement factors, and apolipoproteins in the CSF of Mpdz KO mice was taken to indicate that the hydrocephalic brain underwent ischemia, inflammation, and demyelination. The overabundance of five cytokine-binding proteins could be linked uniquely to insulin-like growth factor (IGF) secretion and signaling. The overabundance of two serpins, angiotensinogen and pigment epithelium-derived factor (PEDF) was considered as a biomarker of anti-angiogenic negative-feedback mechanisms to reduce CSF production. These findings raise the intriguing propositions that CSF proteomics can identify biomarkers of case-specific injuries, and that IGF signaling and angiogenesis pathways can serve as therapeutic targets. It appears, however, that the currently available proteomics data is not amenable to comparison of CSF from normal and hydrocephalic patients and cannot be used test the premise of those propositions.

The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer’s intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 μm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.

Comment on "Close loop peer review" by Michael Hill.

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ . Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss‐of‐function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53‐fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz ‐linked hydrocephalus.