bims-bicyki 2020-10-25 papers

bims-bicyki

Biomed News

on Bicaudal-C1 and interactors in cystic kidney disease

Issue of 2020–10–25
twenty-one papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne

Recognizing and treating autosomal dominant polycystic kidney disease.
Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects.
Non-ciliary Roles of IFT Proteins in Cell Division and Polycystic Kidney Diseases.
The Kidney Score Platform for Patient and Clinician Awareness, Communication, and Management of Kidney Disease: Protocol for a Mixed Methods Study.
CystAnalyser: A new software tool for the automatic detection and quantification of cysts in Polycystic Kidney and Liver Disease, and other cystic disorders.
My CRISPR Book.
Acute Kidney Injury: A Bona Fide Complication of Diabetes.
A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy.
A Regulation Loop between YAP and NR4A1 Balances Cell Proliferation and Apoptosis.
Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination.
Live Visualization of ERK Activity in the Mouse Blastocyst Reveals Lineage-Specific Signaling Dynamics.
Near-Single-Cell Proteomics Profiling of the Proximal Tubular and Glomerulus of the Normal Human Kidney.
Advances in Understanding the Role of Adiponectin in Renal Fibrosis.
A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.
ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-β1.
Polymer Stiffness Regulates Multivalent Binding and Liquid-Liquid Phase Separation.
Roles for 53BP1 in the repair of radiation-induced DNA double strand breaks.
Siah2 integrates mitogenic and extracellular matrix signals linking neuronal progenitor ciliogenesis with germinal zone occupancy.
Polycystin-1 Downregulation Induced Vascular Smooth Muscle Cells Phenotypic Alteration and Extracellular Matrix Remodeling in Thoracic Aortic Dissection.
Assessment of fibrin-collagen co-gels for generating microvesselsex vivousing endothelial cell-lined microfluidics and multipotent stromal cell (MSC)-induced capillary morphogenesis.
Regulation of cellular senescence by extracellular matrix during chronic fibrotic diseases.

Nurse Pract. 2020 Nov;45(11): 41-47

Recognizing and treating autosomal dominant polycystic kidney disease.

Chigozie G Uko.

Autosomal dominant polycystic kidney disease causes chronic kidney disease and end-stage renal disease. Mechanisms include cyst production, multiplication, and enlargement leading to increased kidney size, and ultimately kidney failure. Although there is no known cure, NPs are uniquely positioned to help patients manage their symptoms and delay onset of kidney failure and need for dialysis.

DOI: https://doi.org/10.1097/01.NPR.0000718496.52494.30
Biomolecules. 2020 Oct 17. pii: E1452. [Epub ahead of print]10(10):

Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects.

Li Zheng, Sylvie Chenavas, Fabien Kieken, Andrew Trease, Sarah Brownell, Asokan Anbanandam, Paul L Sorgen, Gaelle Spagnol.

  The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

Keywords:  NMR; ODDD; calmodulin; circular dichroism; connexin43; cytoplasmic loop domain; gap junctions

DOI:  https://doi.org/10.3390/biom10101452
Front Cell Dev Biol. 2020 ;8 578239

Non-ciliary Roles of IFT Proteins in Cell Division and Polycystic Kidney Diseases.

Benjamin Vitre, Audrey Guesdon, Benedicte Delaval.

  Cilia are small organelles present at the surface of most differentiated cells where they act as sensors for mechanical or biochemical stimuli. Cilia assembly and function require the Intraflagellar Transport (IFT) machinery, an intracellular transport system that functions in association with microtubules and motors. If IFT proteins have long been studied for their ciliary roles, recent evidences indicate that their functions are not restricted to the cilium. Indeed, IFT proteins are found outside the ciliary compartment where they are involved in a variety of cellular processes in association with non-ciliary motors. Recent works also provide evidence that non-ciliary roles of IFT proteins could be responsible for the development of ciliopathies related phenotypes including polycystic kidney diseases. In this review, we will discuss the interactions of IFT proteins with microtubules and motors as well as newly identified non-ciliary functions of IFT proteins, focusing on their roles in cell division. We will also discuss the potential contribution of non-ciliary IFT proteins functions to the etiology of kidney diseases.

Keywords:  cell division; ciliopathies; intraflagellar transport; microtubule – associated proteins; molecular motor

DOI:  https://doi.org/10.3389/fcell.2020.578239
JMIR Res Protoc. 2020 Oct 19. 9(10): e22024

The Kidney Score Platform for Patient and Clinician Awareness, Communication, and Management of Kidney Disease: Protocol for a Mixed Methods Study.

Delphine S Tuot, Susan T Crowley, Lois A Katz, Joseph Leung, Delly K Alcantara-Cadillo, Christopher Ruser, Elizabeth Talbot-Montgomery, Joseph A Vassalotti.

   BACKGROUND: Patient awareness, clinician detection, and management of chronic kidney disease remain suboptimal, despite clinical practice guidelines and diverse education programs.
OBJECTIVE: This protocol describes a study to develop and investigate the impact of the National Kidney Foundation Kidney Score Platform on chronic kidney disease awareness, communication, and management, by leveraging the Behavior Change Wheel, an implementation science framework that helps identify behavioral intervention targets and functions that address barriers to behavior change.
METHODS: We interviewed 20 patients with chronic kidney disease and 11 clinicians to identify patient and clinician behaviors suitable for intervention and barriers to behavior change (eg, limited awareness of chronic kidney disease clinical practice guidelines within primary care settings, limited data analytics to highlight chronic kidney disease care gaps, asymptomatic nature of chronic kidney disease in conjunction with patient reliance on primary care clinicians to determine risk and order kidney testing). Leveraging the Behavior Change Wheel, the Kidney Score Platform was developed with a patient-facing online Risk Calculator and a clinician-facing Clinical Practice Toolkit. The Risk Calculator utilizes risk predictive analytics to provide interactive health information tailored to an individual's chronic kidney disease risk and health status. The Clinical Practice Toolkit assists clinicians in discussing chronic kidney disease with individuals at risk for and with kidney disease and in managing their patient population with chronic kidney disease. The Kidney Score Platform will be tested in 2 Veterans Affairs primary health care settings using a pre-post study design. Outcomes will include changes in patient self-efficacy for chronic kidney disease management (primary outcome), quality of communication with clinicians about chronic kidney disease, and practitioners' knowledge of chronic kidney disease guidelines. Process outcomes will identify usability and adoption of different elements of the Kidney Score Platform using qualitative and quantitative methods.
RESULTS: As of September 2020, usability studies are underway with veterans and clinicians to refine the patient-facing components of the Kidney Score Platform before study initiation. Results and subsequent changes to the Kidney Score Platform will be published at a later date. The study is expected to be completed by December 2021.
CONCLUSIONS: Results of this study will be used to inform integration of the Kidney Score Platform within primary care settings so that it can serve as a central component of the National Kidney Foundation public awareness campaign to educate, engage, and empower individuals at risk for and living with chronic kidney disease.
INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/22024.

Keywords:  CKD; awareness; behavioral change wheel, RE-AIM; chronic kidney disease; implementation science

DOI:  https://doi.org/10.2196/22024
PLoS Comput Biol. 2020 Oct 22. 16(10): e1008337

CystAnalyser: A new software tool for the automatic detection and quantification of cysts in Polycystic Kidney and Liver Disease, and other cystic disorders.

Adrián Cordido, Eva Cernadas, Manuel Fernández-Delgado, Miguel A García-González.

The Polycystic Kidney Disease (PKD) is characterized by progressive renal cyst development and other extrarenal manifestation including Polycystic Liver Disease (PLD). Phenotypical characterization of animal models mimicking human diseases are commonly used, in order to, study new molecular mechanisms and identify new therapeutic approaches. The main biomarker of disease progression is total volume of kidney and liver in both human and mouse, which correlates with organ function. For this reason, the estimation of the number and area of the tissue occupied by cysts, is critical for the understanding of physiological mechanisms underlying the disease. In this regard, cystic index is a robust parameter commonly used to quantify the severity of the disease. To date, the vast majority of biomedical researchers use ImageJ as a software tool to estimate the cystic index by quantifying the cystic areas of histological images after thresholding. This tool has imitations of being inaccurate, largely due to incorrectly identifying non-cystic regions. We have developed a new software, named CystAnalyser (register by Universidade de Santiago de Compostela-USC, and Fundación Investigación Sanitaria de Santiago-FIDIS), that combines automatic image processing with a graphical user friendly interface that allows investigators to oversee and easily correct the image processing before quantification. CystAnalyser was able to generate a cystic profile including cystic index, number of cysts and cyst size. In order to test the CystAnalyser software, 795 cystic kidney, and liver histological images were analyzed. Using CystAnalyser there were no differences calculating cystic index automatically versus user input, except in specific circumstances where it was necessary for the user to distinguish between mildly cystic from non-cystic regions. The sensitivity and specificity of the number of cysts detected by the automatic quantification depends on the type of organ and cystic severity, with values 76.84-78.59% and 76.96-89.66% for the kidney and 87.29-93.80% and 63.42-86.07% for the liver. CystAnalyser, in addition, provides a new tool for estimating the number of cysts and a more specific measure of the cystic index than ImageJ. This study proposes CystAnalyser is a new robust and freely downloadable software tool for analyzing the severity of disease by quantifying histological images of cystic organs for routine biomedical research. CystAnalyser can be downloaded from https://citius.usc.es/transferencia/software/cystanalyser (for Windows and Linux) for research purposes upon acceptance.

DOI: https://doi.org/10.1371/journal.pcbi.1008337
CRISPR J. 2020 Oct;3(5): 322-324

My CRISPR Book.

Kevin Davies.

DOI: https://doi.org/10.1089/crispr.2020.29108.kda
Diabetes. 2020 Nov;69(11): 2229-2237

Acute Kidney Injury: A Bona Fide Complication of Diabetes.

Andrew Advani.

The landscape of kidney disease in diabetes has shifted. The classical dogma of "diabetic nephropathy" progressing through stages of albuminuria, leading to decline in glomerular filtration rate and end-stage kidney disease (ESKD), has been replaced by a more nuanced understanding of the complex and heterogeneous nature of kidney disease in diabetes. Paralleling this evolution, standardized definitions have resulted in a growing appreciation that acute kidney injury (AKI) is increasing in its incidence rapidly and that people with diabetes are much more likely to develop AKI than people without diabetes. Here, I propose that AKI should be considered a complication of diabetes alongside other complications that similarly do not fit neatly into the historical microvascular/macrovascular paradigm. In this article, we take a look at the evidence indicating that diabetes is a major risk factor for AKI and we review the causes of this increased risk. We consider the long-term implications of AKI in diabetes and its potential contribution to the future development of chronic kidney disease, ESKD, and mortality. Finally, we look toward the future at strategies to better identify people at risk for AKI and to develop new approaches to improve AKI outcomes. Recognizing AKI as a bona fide complication of diabetes should open up new avenues for investigation that may ultimately improve the outlook for people living with diabetes and at risk for kidney disease.

DOI: https://doi.org/10.2337/db20-0604
Commun Biol. 2020 Oct 23. 3(1): 601

A cell cycle-dependent CRISPR-Cas9 activation system based on an anti-CRISPR protein shows improved genome editing accuracy.

Daisuke Matsumoto, Hirokazu Tamamura, Wataru Nomura.

The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations. Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications. Although several methods have been reported to result in less off-target effects associated with the CRISPR technology, these often exhibit lower editing efficiency. Therefore, efficient, accurate, and innocuous CRISPR technology is still required. Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages. Here, the anti-CRISPR protein, AcrIIA4, was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2, the phases of the cell cycle when homology-directed repair (HDR) is dominant. Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects. Thus, the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing.

DOI: https://doi.org/10.1038/s42003-020-01340-2
Cell Rep. 2020 Oct 20. pii: S2211-1247(20)31273-0. [Epub ahead of print]33(3): 108284

A Regulation Loop between YAP and NR4A1 Balances Cell Proliferation and Apoptosis.

Lingli He, Liang Yuan, Wentao Yu, Yang Sun, Dan Jiang, Xiaodong Wang, Xue Feng, Zuoyun Wang, Jinjin Xu, Ruizeng Yang, Wenxiang Zhang, Hua Feng, Hang-Zi Chen, Yi Arial Zeng, Lijian Hui, Qiao Wu, Yonglong Zhang, Lei Zhang.

  The Hippo signaling pathway maintains organ size and tissue homeostasis via orchestration of cell proliferation and apoptosis. How this pathway triggers cell apoptosis remains largely unexplored. Here, we identify NR4A1 as a target of the Hippo pathway that mediates the pro-apoptotic and anti-tumor effects of the Hippo pathway whereby YAP regulates the transcription, phosphorylation, and mitochondrial localization of NR4A1. NR4A1, in turn, functions as a feedback inhibitor of YAP to promote its degradation, thereby inhibiting the function of YAP during liver regeneration and tumorigenesis. Our studies elucidate a regulatory loop between NR4A1 and YAP to coordinate Hippo signaling activity during liver regeneration and tumorigenesis and highlight NR4A1 as a marker of Hippo signaling, as well as a therapeutic target for hepatocellular carcinoma.

Keywords:  Hippo signaling pathway; NR4A1; apoptosis; hepatocellular carcinoma (HCC); liver regeneration

DOI:  https://doi.org/10.1016/j.celrep.2020.108284
BMC Biotechnol. 2020 Oct 23. 20(1): 57

Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination.

Sanum Bashir, Tu Dang, Jana Rossius, Johanna Wolf, Ralf Kühn.

   BACKGROUND: Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology.
RESULTS: For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci.
CONCLUSIONS: Our findings provide a novel efficient approach to promote precise gene editing in human cells.

Keywords:  BRCA1; CRISPR; Cas9. Genome editing; Gal4; HR; Precise gene editing; RNF169; Rad18; tetR

DOI:  https://doi.org/10.1186/s12896-020-00650-x
Dev Cell. 2020 Oct 16. pii: S1534-5807(20)30762-0. [Epub ahead of print]

Live Visualization of ERK Activity in the Mouse Blastocyst Reveals Lineage-Specific Signaling Dynamics.

Claire S Simon, Shahadat Rahman, Dhruv Raina, Christian Schröter, Anna-Katerina Hadjantonakis.

  FGF/ERK signaling is crucial for the patterning and proliferation of cell lineages that comprise the mouse blastocyst. However, ERK signaling dynamics have never been directly visualized in live embryos. To address whether differential signaling is associated with particular cell fates and states, we generated a targeted mouse line expressing an ERK-kinase translocation reporter (KTR) that enables live quantification of ERK activity at single-cell resolution. 3D time-lapse imaging of this biosensor in embryos revealed spatially graded ERK activity in the trophectoderm prior to overt polar versus mural differentiation. Within the inner cell mass (ICM), all cells relayed FGF/ERK signals with varying durations and magnitude. Primitive endoderm cells displayed higher overall levels of ERK activity, while pluripotent epiblast cells exhibited lower basal activity with sporadic pulses. These results constitute a direct visualization of signaling events during mammalian pre-implantation development and reveal the existence of spatial and temporal lineage-specific dynamics.

Keywords:  ERK; FGF; biosensor; blastocyst; dynamics; inner cell mass; signaling; trophectoderm

DOI:  https://doi.org/10.1016/j.devcel.2020.09.030
Front Med (Lausanne). 2020 ;7 499

Near-Single-Cell Proteomics Profiling of the Proximal Tubular and Glomerulus of the Normal Human Kidney.

Kidney Precision Medicine Project (KPMP) Consortium

  Molecular assessments at the single cell level can accelerate biological research by providing detailed assessments of cellular organization and tissue heterogeneity in both disease and health. The human kidney has complex multi-cellular states with varying functionality, much of which can now be completely harnessed with recent technological advances in tissue proteomics at a near single-cell level. We discuss the foundational steps in the first application of this mass spectrometry (MS) based proteomics method for analysis of sub-sections of the normal human kidney, as part of the Kidney Precision Medicine Project (KPMP). Using ~30-40 laser captured micro-dissected kidney cells, we identified more than 2,500 human proteins, with specificity to the proximal tubular (PT; n = 25 proteins) and glomerular (Glom; n = 67 proteins) regions of the kidney and their unique metabolic functions. This pilot study provides the roadmap for application of our near-single-cell proteomics workflow for analysis of other renal micro-compartments, on a larger scale, to unravel perturbations of renal sub-cellular function in the normal kidney as well as different etiologies of acute and chronic kidney disease.

Keywords:  glomerulus; kidney; mass spectrometry; proteomics; single cell analysis

DOI:  https://doi.org/10.3389/fmed.2020.00499
Nephrology (Carlton). 2020 Oct 19.

Advances in Understanding the Role of Adiponectin in Renal Fibrosis.

Dan Zhao, Xiaoyu Zhu, Lili Jiang, Xiu Huang, Yangyang Zhang, Xuejiao Wei, Xiaoxia Zhao.

  Renal fibrosis is characterized by the proliferation of renal intrinsic cells, activation of renal interstitial fibroblasts, and deposition of extracellular matrix, processes that lead to the progressive loss of renal function. Renal fibrosis is characterized by the proliferation of renal intrinsic cells, activation of renal interstitial fibroblasts, and septal fibrosis is recognized as a marker for the progression of chronic kidney disease (CKD), a condition that is associated with high morbidity and mortality and is a significant public health burden. Despite extensive studies, there are no effective treatments for renal fibrosis. Adiponectin (APN) is a protein mainly produced by adipocytes that has anti-inflammatory and anti-atherosclerotic effects, improves insulin resistance, and provides other salutary effects. Recent studies found that APN can inhibit extracellular matrix deposition by inhibiting inflammation and oxidative stress, and by regulating the TGF-β, AMPK, MCP-1, and other signaling pathways. Many recent studies have examined the roles of these pathways in the pathogenesis of renal fibrosis. In thisarticle, we review the pathogenic mechanism of APN in renal fibrosis and provide a theoretical basis for delaying and blocking renal fibrosis by alteration of APN activity.

Keywords:  AMPK; TGF-β; adiponectin; chronic kidney disease; renal fibrosis

DOI:  https://doi.org/10.1111/nep.13808
Nucleic Acids Res. 2020 Oct 17. pii: gkaa897. [Epub ahead of print]

A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.

Rebeka Eki, Jane She, Mahmut Parlak, Mouadh Benamar, Kang-Ping Du, Pankaj Kumar, Tarek Abbas.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

DOI: https://doi.org/10.1093/nar/gkaa897
Biophys J. 2020 Sep 23. pii: S0006-3495(20)30723-2. [Epub ahead of print]

ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-β1.

Daniel P Maruri, Miguel Miron-Mendoza, Pouriska B Kivanany, Joshua M Hack, David W Schmidtke, W Matthew Petroll, Victor D Varner.

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-β1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.

DOI: https://doi.org/10.1016/j.bpj.2020.08.040
Biophys J. 2020 Oct 06. pii: S0006-3495(20)30767-0. [Epub ahead of print]

Polymer Stiffness Regulates Multivalent Binding and Liquid-Liquid Phase Separation.

Emiko Zumbro, Alfredo Alexander-Katz.

Multivalent binding is essential to many biological processes because it builds high-affinity bonds by using several weak binding interactions simultaneously. Multivalent polymers have shown promise as inhibitors of toxins and other pathogens, and they are important components in the formation of biocondensates. Explaining how structural features of these polymers change their binding and subsequent control of phase separation is critical to designing better pathogen inhibitors and also to understanding diseases associated with membraneless organelles. In this work, we will examine the binding of a multivalent polymer to a small target. This scenario could represent a polymeric inhibitor binding to a toxic protein or RNA binding to an RNA-binding protein in the case of liquid-liquid phase separation. We use simulation and theory to show that flexible random-coil polymers bind more strongly than stiff rod-like polymers and that flexible polymers nucleate condensed phases at lower binding energies than their rigid analogs. We hope these results will provide insight into the rational design of polymeric inhibitors and improve our understanding of phase separation in cells and membraneless organelles.

DOI: https://doi.org/10.1016/j.bpj.2020.09.035
DNA Repair (Amst). 2020 Sep;pii: S1568-7864(20)30163-4. [Epub ahead of print]93 102915

Roles for 53BP1 in the repair of radiation-induced DNA double strand breaks.

Atsushi Shibata, Penny A Jeggo.

  In mammalian cells, the mediator protein, 53BP1, exerts distinct impacts on the repair of DNA double strand breaks (DSBs) depending on the setting, for example whether the DSBs arise at telomeres or during replication or class switch recombination. Here, we focus on two roles of 53BP1 in response to ionising radiation (IR)-induced DSBs (IR-DSBs). Canonical DNA non-homologous end-joining (c-NHEJ) is the major DSB repair pathway with homologous recombination (HR) contributing to DSB repair in S/G2 phase. ATM signalling promotes histone modifications and protein assembly in the DSB vicinity, which can be visualised as irradiation induced foci (IRIF). 53BP1 assembles at DSBs in a complex manner involving the formation of nano-domains. In G1 and G2 phase, X- or gamma-ray induced DSBs are repaired with biphasic kinetics. 70-80 % of DSBs are repaired with fast kinetics in both cell cycle phases by c-NHEJ; the remaining DSBs are repaired with slower kinetics in G2 phase via HR and in G1 by a specialised form of c-NHEJ termed Artemis and resection-dependent c-NHEJ, due to a specific requirement for the nuclease, Artemis and resection factors. 53BP1 is essential for the repair of DSBs rejoined with slow kinetics in G1 and G2 phase. This 53BP1 function requires its tandem BRCT domain and interaction with NBS1. As a distinct function, 53BP1 suppresses resection during both HR and Artemis and resection-dependent c-NHEJ. This latter role requires RIF1 and is counteracted by BRCA1. 53BP1 appears to be dispensable for the rejoining of the fast c-NHEJ repair process.

Keywords:  53BP1; Chromatin; DNA double-strand break repair; Homologous recombination; Ionising radiation; Non-homologous end-joining

DOI:  https://doi.org/10.1016/j.dnarep.2020.102915
Nat Commun. 2020 10 20. 11(1): 5312

Siah2 integrates mitogenic and extracellular matrix signals linking neuronal progenitor ciliogenesis with germinal zone occupancy.

Taren Ong, Niraj Trivedi, Randall Wakefield, Sharon Frase, David J Solecki.

Evidence is lacking as to how developing neurons integrate mitogenic signals with microenvironment cues to control proliferation and differentiation. We determine that the Siah2 E3 ubiquitin ligase functions in a coincidence detection circuit linking responses to the Shh mitogen and the extracellular matrix to control cerebellar granule neurons (CGN) GZ occupancy. We show that Shh signaling maintains Siah2 expression in CGN progenitors (GNPs) in a Ras/Mapk-dependent manner. Siah2 supports ciliogenesis in a feed-forward fashion by restraining cilium disassembly. Efforts to identify sources of the Ras/Mapk signaling led us to discover that GNPs respond to laminin, but not vitronectin, in the GZ microenvironment via integrin β1 receptors, which engages the Ras/Mapk cascade with Shh, and that this niche interaction is essential for promoting GNP ciliogenesis. As GNPs leave the GZ, differentiation is driven by changing extracellular cues that diminish Siah2-activity leading to primary cilia shortening and attenuation of the mitogenic response.

DOI: https://doi.org/10.1038/s41467-020-19063-7
Front Physiol. 2020 ;11 548055

Polycystin-1 Downregulation Induced Vascular Smooth Muscle Cells Phenotypic Alteration and Extracellular Matrix Remodeling in Thoracic Aortic Dissection.

Jing Zhang, Fei Liu, Yu-Bin He, Wei Zhang, Wen-Rui Ma, Jie Xing, Li-Xin Wang.

   Objective: Polycystin-1 (PC-1) is a protein encoded by the gene of polycystic kidney disease-1 (PKD-1). This study was designed to investigate the regulatory mechanisms of PC-1 on phenotypes of aortic vascular smooth muscle cells (VSMCs) and functions of extracellular matrix (ECM) in thoracic aortic dissection (TAD).
Methods: Aortic tissues from patients with TAD and healthy controls were collected, primary aortic VSMCs were also isolated. Immunohistochemistry, immunofluorescence, and immunocytochemistry was used to visualize the target proteins. Western blot and RT-qPCR were used to examine the expression of mRNA and proteins. Lentivirus infection was used to downregulate or overexpress PC-1.
Results: Compared with the control group, expression of PC-1 and the contractile phenotypic markers of VSMCs were decreased in TAD group, whereas expression of the synthetic markers of VSMCs, matrix metalloproteinase (MMP)-2, collagen I and collagen III were increased. The phosphorylation of mTOR, S6K and S6 were also elevated in TAD group. PC-1 downregulation of aortic VSMCs inhibited the expression of the contractile markers, but elevated the expression of the synthetic markers, MMP-2, collagen I and collagen III compared with the control group. The phosphorylation of mTOR, S6K and S6 were also increased in PKD-1-knockdown VSMCs. PC-1 upregulation reversed all these expression characteristics in aortic VSMCs. Furthermore, rapamycin treatment to PKD-1-knockdown VSMCs inhibited the effects caused by PC-1 downregulation.
Conclusion: Our study revealed PC-1 downregulation induces aortic VSMCs phenotypic alteration and ECM remodeling via activation of mTOR/S6K/S6 signaling pathway. Downregulation of PC-1 might be a potential mechanism for the development and progression of TAD. Rapamycin might be a potential inhibitor to attenuate the development and progression of TAD.

Keywords:  aortic dissection; extracellular matrix; phenotype; polycystin-1; rapamycin; vascular smooth muscle cells

DOI:  https://doi.org/10.3389/fphys.2020.548055
Biomed Mater. 2020 Oct 21.

Assessment of fibrin-collagen co-gels for generating microvesselsex vivousing endothelial cell-lined microfluidics and multipotent stromal cell (MSC)-induced capillary morphogenesis.

Ross E B Fitzsimmons, Ronald G Ireland, Aileen Zhong, Agnes Soos, Craig A Simmons.

  One aspect of the challenge of engineering viable tissues ex vivo is the generation of perfusable microvessels of varying diameters. In this work, we take the approach of using hydrogel-based microfluidics seeded with endothelial cells (ECs) to form small artery/vein-like vessels, in conjunction with using the self-assembly behavior of ECs to form capillary-like vessels when co-cultured with multipotent stromal cells (MSCs). In exploring this approach, we focused on investigating collagen, fibrin, and various collagen-fibrin co-gel formulations for their potential suitability as serving as scaffold materials by surveying their angiogencity and mechanical properties. Fibrin and co-gels successfully facilitated multicellular EC sprouting, whereas collagen elicited a migration response of individual ECs, unless supplemented with the PKC (protein kinase C)-activator, phorbol 12-myristate 13-acetate. Collagen scaffolds were also found to severely contract when embedded with mesenchymal cells, but this contraction could be abrogated with the addition of fibrin. Increasing collagen content within co-gel formulations, however, imparted a higher compressive modulus and allowed for the reliable formation of intact hydrogel-based microchannels which could then be perfused. Given the bioactivity and mechanical benefits of fibrin and collagen, respectively, collagen-fibrin co-gels are a promising scaffold option for generating vascularized tissue constructs.

Keywords:  Angiogenesis; Collagen; Endothelial cell; Fibrin; Hydrogel; Mesenchymal stromal cell

DOI:  https://doi.org/10.1088/1748-605X/abc38f
Clin Sci (Lond). 2020 Oct 30. 134(20): 2681-2706

Regulation of cellular senescence by extracellular matrix during chronic fibrotic diseases.

Kaj E C Blokland, Simon D Pouwels, Michael Schuliga, Darryl A Knight, Janette K Burgess.

  The extracellular matrix (ECM) is a complex network of macromolecules surrounding cells providing structural support and stability to tissues. The understanding of the ECM and the diverse roles it plays in development, homoeostasis and injury have greatly advanced in the last three decades. The ECM is crucial for maintaining tissue homoeostasis but also many pathological conditions arise from aberrant matrix remodelling during ageing. Ageing is characterised as functional decline of tissue over time ultimately leading to tissue dysfunction, and is a risk factor in many diseases including cardiovascular disease, diabetes, cancer, dementia, glaucoma, chronic obstructive pulmonary disease (COPD) and fibrosis. ECM changes are recognised as a major driver of aberrant cell responses. Mesenchymal cells in aged tissue show signs of growth arrest and resistance to apoptosis, which are indicative of cellular senescence. It was recently postulated that cellular senescence contributes to the pathogenesis of chronic fibrotic diseases in the heart, kidney, liver and lung. Senescent cells negatively impact tissue regeneration while creating a pro-inflammatory environment as part of the senescence-associated secretory phenotype (SASP) favouring disease progression. In this review, we explore and summarise the current knowledge around how aberrant ECM potentially influences the senescent phenotype in chronic fibrotic diseases. Lastly, we will explore the possibility for interventions in the ECM-senescence regulatory pathways for therapeutic potential in chronic fibrotic diseases.

Keywords:  Antifibrotics; DAMPs; Senescence; Senolytics; extracellular matrix; fibrosis

DOI:  https://doi.org/10.1042/CS20190893