Obscurin is a giant (720-900 kDa) modular cytoskeletal protein with multiple signaling domains. While it is most highly expressed in myocytes, obscurin is also the second most mutated protein in breast and colorectal cancers and is significantly downregulated in pancreatic cancer. Obscurin derives its antioncogenic properties, at least in part, through its ability to modulate cellular motility and migration; obscurin knockdown in cultured epithelial cells leads to increased migration and an epithelial-to-mesenchymal transition (EMT). Obscurin likely controls cell motility through the obscurin RhoGEF domain interaction with the RhoA/ROCK pathway and/or the obscurin PH domain interaction with a PI3K/PIP3 pathway. Here, we more fully describe which obscurin domains dictate subcellular localization and regulate cellular motility. The obscurin C-terminus adenovirally infected into MDCK and MCF-10A cells localizes to adhesion structures at the plasma membrane. This localization is driven by four regions in obscurin: the obscurin RhoGEF and PH domains, along with two regions in the unstructured C-terminus. Infected cells lack central stress fibers, and this morphology is linked to the RhoGEF domain, the PH domain, and the C-terminal 76 residues. These three obscurin regions also inhibit cell motility. Together these data demonstrate how both specific obscurin domains and specific cellular localization regulate cellular velocity.

Myotrem is an untreatable, early-onset, congenital myopathy characterized by hypotonia, muscle weakness, skeletal deformities, dysmorphia, respiratory insufficiency, and myogenic tremor (V. Shashi et al., Hum Mutat, 2019 and J. Stavusis et al., Ann. Neurol., 2019). It is associated with dominant variants in the pivotal M-domain of slow-skeletal Myosin Binding Protein-C (sMyBP-C) that modulates the dynamic binding to myosin and actin filaments and thereby crossbridge formation and kinetics. Herein, we report a nonmissense Myotrem variant, c.795_803dup p.(Leu266_Arg268dup), referred to as LKR-duplication. Our comprehensive studies, integrating clinical findings with biophysical, structural, and computational approaches, uncover the previously unreported structure and properties of the slow-skeletal M-domain, while elucidating the impact of the LKR-duplication. We show that the LKR-duplication stabilizes local helicity but alters global domain dynamics, leading to increased myosin binding, while impairing myosin-ATPase activity and crossbridge cycling. Critically, we pinpoint the specific amino acid residues facilitating the M-domain/myosin interaction and demonstrate that the LKR-duplicated residues not only directly contribute to myosin binding but also enhance the myosin interacting capability of neighboring and distant residues. Our multimodal approach sheds light on aspects of the pathobiology of the slow-skeletal M-domain-the Myotrem hotspot-by unveiling underlying pathogenic etiologies thereby paving the way for the development of targeted treatments.

Enteroaggregative Escherichia coli (EAEC) is a common cause of diarrhea worldwide and is associated with growth faltering in developing countries. EAEC are defined by a characteristic adherence pattern mediated by the aggregative adherence fimbriae (AAFs). Despite the critical role of AAF in the definition of the EAEC pathotype, it is not known what host molecules mediate adherence and EAEC pathogenesis during infection of the human gastrointestinal tract. Multiple receptor candidates have been proposed based on in vitro experimentation. We propose that AAFs interact with multiple receptors during colonization of the human gastrointestinal mucosa, and we hypothesize that structural features of the AafA protein (the major subunit of AAF variant II produced by EAEC strain 042) promote these diverse interactions. In this study, we utilize a panel of AafA variants encoding single amino acid substitutions to understand the role of individual residues in biofilm formation as well as adherence to mucin, fibronectin, and human intestinal cells. We identify both charged and uncharged residues that participate in these interactions, and these residues cluster in two regions of the protein that may define a binding pocket at the junction of polymerized subunits. Although both bovine submaxillary mucin and human fibronectin are sialylated molecules, adherence to mucin is diminished by the removal of sialic acid residues while adherence to fibronectin is not, suggesting that the mechanisms of adherence to these molecules are distinct. Overall, our data provide insight into the structural features that determine AAF/II binding to mucin, sialic acid, and human intestinal cells.

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase1 (SERCA1) is responsible for the clearance of cytosolic Ca2+ in skeletal muscle. Due to its vital importance in regulating Ca2+ homeostasis, the regulation of SERCA1 has been intensively studied. Small ankyrin 1 (sAnk1, Ank1.5), a 17 kDa muscle-specific isoform of ANK1, binds to SERCA1 directly via both its transmembrane and cytoplasmic domains and inhibits SERCA1’s ATPase activity. Here we characterize the interaction between the cytoplasmic domain of sAnk1 (sAnk1(29-155)) and SERCA1. The binding affinity for sAnk1 (29-155) to SERCA1 was 444 nM by blot overlay, about 7-fold weaker than the binding of sAnk1(29-155) to obscurin, a giant protein of the muscle cytoskeleton. Site-directed mutagenesis identified K38, H39, and H41, in the juxtamembrane region, as residues likely to mediate binding to SERCA1. These residues are not required for obscurin binding. Residues R64-K73, which do contribute to obscurin binding, are also required for binding to SERCA1, but only the hydrophobic residues in this sequence are required, not the positively charged residues necessary for obscurin binding. Circular dichroism analysis of sAnk1(29-155) indicates that most mutants show significant structural changes, with the exception of those containing alanines in place of K38, H39 and H41. Although the cytoplasmic domain of sAnk1 does not inhibit SERCA1’s Ca2+-ATPase activity, with or without mutations in the juxtamembrane sequence, the inhibitory activity of full-length sAnk1 requires the WT juxtamembrane sequence. We used these data to model sAnk1 and the sAnk1-SERCA1 complex. Our results suggest that, in addition to its transmembrane domain, sAnk1 uses its juxtamembrane sequence and perhaps part of its obscurin binding site to bind to SERCA1, and that this binding contributes to their robust association in situ, as well as regulation of SERCA1’s activity.

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, the subcellular structure that links the intermediate filament network of one cell to its neighbor. A mutation “hot-spot” within the NH2-terminal of the DSP protein (residues 299–515) is associated with arrhythmogenic cardiomyopathy. In a subset of DSP variants, disease is linked to calpain hypersensitivity. Previous studies show that calpain hypersensitivity can be corrected in vitro through the addition of a bulky residue neighboring the cleavage site, suggesting that physically blocking calpain accessibility is a viable strategy to restore DSP levels. Here, we aim to find drug-like molecules that also block calpain-dependent degradation of DSP. To do this, we screened ~2500 small molecules to identify compounds that specifically rescue DSP protein levels in the presence of proteases. We find that several molecules, including sodium dodecyl sulfate, palmitoylethanolamide, GW0742, salirasib, eprosarten mesylate, and GSK1838705A prevent wildtype and disease-variant-carrying DSP protein degradation in the presence of both trypsin and calpain without altering protease function. Computational screenings did not predict which molecules would protect DSP, likely due to a lack of specific DSP–drug interactions. Molecular dynamic simulations of DSP–drug complexes suggest that some long hydrophobic molecules can bind in a shallow hydrophobic groove that runs alongside the protease cleavage site. Identification of these compounds lays the groundwork for pharmacological treatment for individuals harboring these hypersensitive DSP variants.

Dominant missense variants in MYBPC1 encoding slow Myosin Binding Protein-C (sMyBP-C) have been increasingly linked to arthrogryposis syndromes and congenital myopathy with tremor. Herein, we describe novel compound heterozygous variants - NM_002465.4:[c.2486_2492del];[c.2663A > G] - present in fibronectin-III (Fn-III) C7 and immunoglobulin (Ig) C8 domains, respectively, manifesting as severe, early-onset distal arthrogryposis type-1, with the carrier requiring intensive care and several surgical interventions at an early age. Computational modeling predicts that the c.2486_2492del p.(Lys829IlefsTer7) variant destabilizes the structure of the Fn-III C7 domain, while the c.2663A > G p.(Asp888Gly) variant causes minimal structural alterations in the Ig C8 domain. Although the parents of the proband are heterozygous carriers for a single variant, they exhibit no musculoskeletal defects, suggesting a complex interplay between the two mutant alleles underlying this disorder. As emerging novel variants in MYBPC1 are shown to be causatively associated with musculoskeletal disease, it becomes clear that MYBPC1 should be included in relevant genetic screenings.

The PTCD3 gene product (protein PTCD3 or MRPS39) forms the entry channel of the mitochondrial small ribosomal subunit and binds to single-stranded mRNA. Here, we expand on the clinical manifestations of PTCD3 pathogenic variants by describing an early-onset patient with Leigh-like syndrome and two patients with milder form of disease, with combined oxidative phosphorylation deficiency. A 34-year-old male and his 33-year-old sister both have horizontal nystagmus, pronounced rough tremor, truncal ataxia, dysmetria, spasticity and hyperreflexia. The basal respiration rate decreased significantly for the male patient and his mother (p < 0.0001) compared to the controls. The whole genome sequencing analysis revealed two heterozygous variants in the PTCD3: c.1182T>A, p.(Tyr394Ter) and c.805C>T, p.(His269Tyr). Tyr394Ter variant ablates the C-terminal half of the protein, including a significant portion of the central fold. In silico modelling for the variant His269Tyr shows that the inclusion of the slightly larger tyrosine sidechain is well tolerated, with no significant change in either the position or the movement of the surrounding area. The third case is a 9-year-old boy, who has a global developmental delay, central hypotonia, hyperreflexia and abnormal MRI. PTCD3 pathogenic variant c.538+4A>G was identified by whole exome sequencing. To test the variant's effect on splicing, an RT-PCR experiment was performed, which revealed skipping of an out-of-frame exon 7.

The giant cytoskeletal protein obscurin contains multiple cell signaling domains that influence cell migration. Here, we follow each of these pathways, examine how these pathways modulate epithelial cell migration, and discuss the cross-talk between these pathways. Specifically, obscurin uses its PH domain to inhibit phosphoinositide-3-kinase (PI3K)-dependent migration and its RhoGEF domain to activate RhoA and slow cell migration. While obscurin's effect on the PI3K pathway agrees with the literature, obscurin's effect on the RhoA pathway runs counter to most other RhoA effectors, whose activation tends to lead to enhanced motility. Obscurin also phosphorylates cadherins, and this may also influence cell motility. When taken together, obscurin's ability to modulate three independent cell migration pathways is likely why obscurin knockout cells experience enhanced epithelial to mesenchymal transition, and why obscurin is a frequently mutated gene in several types of cancer.

Obscurin is a large cytoskeletal protein found in epithelial and muscle cells. It is the second most mutated protein in breast and colorectal cancers and is significantly downregulated in pancreatic cancer. Obscurin activates ROCK via RhoA, which in turn activates actomyosin contractility, F-actin polymerization, and modulates cellular motility and migration. Obscurin knockdown leads to an epithelial to mesenchymal transition (EMT), a hallmark of cancer progression. Here, we extend these findings to show that the C-terminus of obscurin A localizes near the cell membrane in MDCK cells.

Mutations in the desmosomal protein desmoplakin (DSP) underlie about 5% of arrhythmogenic cardiomyopathy (AC) cases. Recent data from our lab show that some disease-linked DSP mutations are hypersensitive to calpain, an endogenous calcium-dependent protease. The resulting loss of DSP destabilizes the desmosome and leads to weakened cell-cell adhesion, which is correlated with fibrofatty infiltration in AC. Our lab has probed the molecular mechanism of this DSP degradation, showing that DSP mutant hypersensitivity to calpain is dependent upon the exposure of a usually-occluded cleavage site on the DSP surface.

The shift of funding organizations to prioritize interdisciplinary work points to the need for workflow models that better accommodate interdisciplinary studies. Most scientists are trained in a specific field and are often unaware of the kind of insights that other disciplines could contribute to solving various problems. In this paper, we present a perspective on how we developed an experimental pipeline between a microscopy and image analysis/bioengineering lab. Specifically, we connected microscopy observations about a putative mechanosensing protein, obscurin, to image analysis techniques that quantify cell changes. While the individual methods used are well established (fluorescence microscopy; ImageJ WEKA and mTrack2 programs; MATLAB), there are no existing best practices for how to integrate these techniques into a cohesive, interdisciplinary narrative. Here, we describe a broadly applicable workflow of how microscopists can more easily quantify cell properties (e.g., perimeter, velocity) from microscopy videos of eukaryotic (MDCK) adherent cells. Additionally, we give examples of how these foundational measurements can create more complex, customizable cell mechanics tools and models.

Obscurin is a large cytoskeletal protein found in epithelial and muscle cells. It is the second most mutated protein in breast and colorectal cancers and is significantly downregulated in pancreatic cancer. Obscurin activates ROCK via RhoA, which in turn activates myosin, stimulates F-actin polymerization, and modulates cellular motility and migration. Likewise, obscurin knockdown leads to an epithelial to mesenchymal transition (EMT), a hallmark of cancer progression. Obscurin is not known to be modulated in any way by any part of this downstream RhoA pathway, yet it seems likely that a cytoskeletal protein would be affected by cell motility and motion. Here we begin to describe the relationship between global obscurin structure and individual components of this downstream pathway, including cell architecture and cell motility. We show that our model system, MDCK cells, express either no or very little obscurin. Upon the introduction of mini-obscurin constructs containing a precalibrated FRET-based force sensor, we see that obscurin localizes around the cell membrane in a dose-dependent manner. In normal cells, all obscurin constructs under >5 pN of force. When either the actin or myosin cytoskeleton is disrupted, the cells show a characteristic round morphology and obscurin is under significantly less force. However, when ROCK is inhibited, obscurin tension remains unchanged yet cells show morphological differences. This suggests that obscurin tension is not directly modulated by the pathway which it controls. Instead some other external force, possibly physical or specifically directed at the actin/myosin cell architecture, modulates obscurin tension and thus influences global obscurin architecture. Experiments are underway to disambiguate this stimulus.

Cell membranes are normally thought of as viscoelastic materials because they exhibit both viscous and elastic properties when deforming, however, some researchers describe membranes as elastic only. Our project seeks to quantify the membrane deformations during a splitting event, and mathematically predict membrane tensions if it were an elastic or viscoelastic material. Knowing the mechanical description that is most appropriate can help future research interested in describing cell mechanics when cells split, change shape, or move.

Obscurin is a giant signaling protein believed to be responsible for resisting applied forces on a cell by localizing to areas of high tension. We are interested to know: (1) what is the effect of obscurin on cells’ area and perimeter, and (2) where does obscurin move within the cell over time. While cell size and location of a protein can be described “by eye,” the challenge is finding a way to quantify these properties consistently. To address the first question, we used a machine learning algorithm called Weka, a plugin within the FIJI image analysis software, to accurately segment many cells at once. Once segmented, area and perimeter statistics are further analyzed in a custom Python program that compares the mean area and perimeter between experimental conditions using t-tests. We found that the presence of Blebbistatin, a drug that inhibits myosin II, significantly decreases the overall size of the cell, while the presence of Cytochalasin D, a drug that inhibits actin polymerization, significantly increases cell size. Experimentation with these drugs, known to disrupt cell mechanics, allows us to better understand obscurin's possible role in cell tension. To determine where obscurin moves within the cell over time, we utilized kymographs and colocalization plots. These techniques can help determine how much obscurin is present within the cell, and its location relative to the cell membrane. The utilization of adenovirus to increase the amount of obscurin in epithelial cells led to a wide range of florescent obscurin expression. Our localization analysis suggests that obscurin colocalizes to the cell membrane when an optimal amount of fluorescent obscurin is present. Altogether, quantifying cell shape properties and obscurin movement can help determine if obscurin is localizing to areas of high tension.

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (similar to 870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent K-cat of approximately 5.05 min(-1). Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurinkin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

The intercalated disk is a cardiac specific structure composed of three main protein complexes-adherens junctions, desmosomes, and gap junctions-that work in concert to provide mechanical stability and electrical synchronization to the heart. Each substructure is regulated through a variety of mechanisms including proteolysis. Calpain proteases, a class of cysteine proteases dependent on calcium for activation, have recently emerged as important regulators of individual intercalated disk components. In this review, we will examine how calcium homeostasis regulates normal calpain function. We will also explore how calpains modulate gap junctions, desmosomes, and adherens junctions activity by targeting specific proteins, and describe the molecular mechanisms of how calpain dysregulation leads to structural and signaling defects within the heart. We will then examine how changes in calpain activity affects cardiomyocytes, and how such changes underlie various heart diseases.

In an effort to promote more STEM engagement with hard-to-reach rural school children, we designed and implemented a distinctive set of interactive biophysics-related displays for our local county fair. Due to the location and the audience, these demonstrations had a unique list of design criteria, including being attractive to an average fairgoer, being exceptionally rugged, transportable, and being able to fit into the fair's indoor booth. Through a combination of using pop culture to engage students, building fun and interactive displays, and emphasizing real-world applications in our lessons, we were able to engage with hundreds of local children.