bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2020‒12‒27
seven papers selected by
Céline Gagnieux
École Polytechnique Fédérale de Lausanne (EPFL)

  1. Neoplasia. 2021 Jan;pii: S1476-5586(20)30183-4. [Epub ahead of print]23(1): 169-180
      Primary cilium is a microtubule-based organelle that projects from the surfaces of most mammalian cell types and protrudes into the extracellular milieu as an antenna-like sensor to senses extracellular physical and biochemical signals, and then transmits signals into cytoplasm or nucleus to regulate numerous physical and developmental processes. Therefore, loss of primary cilia is associated to multiple cancer progression, including skin, breast, pancreas, ovarian, prostate, and kidney cancers. Our previous studies demonstrate that high prevalent loss of DAB2 Interacting Protein (DAB2IP) is associated with renal cell carcinoma, and we found a kinesin-like protein, kinesin family member 3A (KIF3a), was significantly increased in DAB2IP-interacting protein fraction. KIF3 is one of the most abundant kinesin-2 family proteins expressed in cells, and it is necessary for ciliogenesis. In this study, we observed that loss of DAB2IP in normal kidney epithelial cell significantly impair primary cilia formation. We unveiled a new mechanism of primary cilia stability via DAB2IP and KIF3a physical interaction at DAB2IP-PH domain. Furthermore, we found that KIF3a also act as a tumor suppressor in renal cell carcinoma, affect tumor development and patient survival.
    Keywords:  DAB2IP; KIF3a; Primary cilia; Renal cell carcinoma; Tumor suppressor gene
  2. Int J Nephrol. 2020 ;2020 4108418
      The final dilution of urine is regulated via aquaporin-2 water channels in the distal part of the nephron. It is unclear whether urine dilution ability in autosomal dominant polycystic kidney disease patients (ADPKD patients) differs from other patients with similar degree of impaired renal function (non-ADPKD patients). The purpose of this case control study was to measure urine dilution ability in ADPKD patients compared to non-ADPKD patients and healthy controls. Methods. Eighteen ADPKD, 16 non-ADPKD patients (both with chronic kidney disease, stage I-IV), and 18 healthy controls received an oral water load of 20 ml/kg body weight. Urine was collected in 7 consecutive periods. We measured free water clearance (CH2O), urine osmolality, urine output, fractional excretion of sodium, urine aquaporin2 (u-AQP2), and urine epithelial sodium channel (u-ENaC). Blood samples were drawn four times (at baseline, 2 h, 4 h, and 6 hours after the water load) for analyses of plasma osmolality, vasopressin, renin, angiotensin II, and aldosterone. Brachial and central blood pressure was measured regularly during the test. Results. The three groups were age and gender matched, and the patient groups had similar renal function. One hour after water load, the ADPKD patients had an increased CH2O compared to non-ADPKD patients (2.97 ± 2.42 ml/min in ADPKD patients vs. 1.31 ± 1.50 ml/min in non-ADPKD patients, p0.029). The reduction in u-AQP2 and u-ENaC occurred earlier in ADPKD than in non-ADPKD patients. Plasma concentrations of vasopressin, renin, angiotensin II, and aldosterone and blood pressure measurements did not show any differences that could explain the deviation in urine dilution capacity between the patient groups. Conclusions. ADPKD patients had a higher CH2O than non-ADPKD patients after an oral water load, and u-AQP2 and u-ENaC were more rapidly reduced than in non-ADPKD patients. Thus, urine-diluting capacity may be better preserved in ADPKD patients than in non-ADPKD patients.
  3. J Neurosci Res. 2020 Dec 17.
      Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein-coupled receptor 37-like 1 (Gpr37l1) is expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri-ciliary membranes. Cerebellar primary astrocyte cultures from wild-type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild-type cells, Gpr37l1-/- astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild-type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1-Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling.
    Keywords:  G protein‐coupled receptor; RRID:AB_10709580; RRID:AB_10844948; RRID:AB_11000053; RRID:AB_11204167; RRID:AB_11205039; RRID:AB_141373; RRID:AB_141607; RRID:AB_141788; RRID:AB_162543; RRID:AB_1839970; RRID:AB_1904103; RRID:AB_2059853; RRID:AB_2060867; RRID:AB_2072166; RRID:AB_2109645; RRID:AB_2174039; RRID:AB_2174045; RRID:AB_2239686; RRID:AB_2245173; RRID:AB_2300649; RRID:AB_2534102; RRID:AB_2535792; RRID:AB_2536180; RRID:AB_2857918; RRID:AB_330744; RRID:AB_331646; RRID:AB_396365; RRID:AB_631728; RRID:AB_632416; RRID:AB_772207; RRID:AB_772210; RRID:AB_839154; RRID:AB_839504; RRID:MGI:5512669; RRID:SCR_002789; RRID:SCR_003070; RRID:SCR_003238; RRID:SCR_007370; RRID:SCR_010279; RRID:SCR_013673; RRID:SCR_014199; RRID:SCR_014210; cholesterol; mouse mutant; primary cilium
  4. CRISPR J. 2020 Dec;3(6): 550-561
      CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. The number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.
  5. CRISPR J. 2020 Dec;3(6): 440-453
      The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.
  6. J Vis Exp. 2020 Dec 05.
      Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).
  7. Am J Pathol. 2020 Dec 17. pii: S0002-9440(20)30562-9. [Epub ahead of print]
      Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells (LSECs) and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC initiating factors apart from paracrine stimuli may promote activation. Here we have explored the dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs. We establish E-cadherin as an adhesion molecule linking hepatocytes and HSCs. In vivo following CCl4-liver injury, HSCs lose their spines and dissociate from adherens junctions in early stages of injury, followed by activation, associated with enhanced YAP/TAZ expression. After liver injury is abrogated, HSCs reconstruct their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesces HSCs, accompanied by suppression of TAZ expression. In addition, following the increase of TAZ expression, HSCs are activated by autocrine stimulation of TGF-β, which is one mechanism of spontaneous activation. We have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechano-transduction following loss of adherens junctions between HSCs and hepatocytes.
    Keywords:  E-cadherin; Hepatic stellate cells; YAP/TAZ; adherens junctions; initiation