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hTERT-RPE1

RRID:CVCL_4388

Organism

Homo sapiens

Comments

Transfected with: HGNC; 11730; TERT. Omics: Deep proteome analysis. Omics: Deep RNAseq analysis. Misspelling: hTERT1-RPE1; Occasionally. DT Created: 04-04-12; Last updated: 25-02-19; Version: 17

Proper Citation

ATCC Cat# CRL-4000, RRID:CVCL_4388

Category

Telomerase immortalized cell line DT Created: 04-04-12; Last updated: 25-02-19; Version: 17

Sex

DT Created: 04-04-12; Last updated: 25-02-19; Version: 17

Synonyms

hTERT RPE1, hTERT-RPE-1, hTERT RPE-1, hTERT-RPE, TERT-RPE1, RPE-1, RPE1, RPE1-hTERT, RPE1-hTert, RPE-hTERT DT Created: 04-04-12, Last updated: 25-02-19, Version: 17

Vendor

ATCC

Cat Num

CRL-4000

Cross References

BTO; BTO:0004790 4DN; 4DNSRHGVFSRJ ATCC; CRL-4000 BioSample; SAMN03472089 Cell_Model_Passport; SIDM00409 ChEMBL-Cells; CHEMBL3307755 ChEMBL-Targets; CHEMBL615013 GEO; GSM2371252 PRIDE; PXD004915 Wikidata; Q54896633 DT Created: 04-04-12; Last updated: 25-02-19; Version: 17

Hierarchy

DT Created: 04-04-12; Last updated: 25-02-19; Version: 17

ZMYND10 functions in a chaperone relay during axonemal dynein assembly.

  • Mali GR
  • Elife
  • 2018 Jun 19

Literature context:


Abstract:

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of 'client' proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that Primary Ciliary Dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.

Funding information:
  • Medical Research Council - MRC_UU_12018/26()
  • NCI NIH HHS - R44 CA165312(United States)

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates.

  • Sepulveda G
  • Elife
  • 2018 Apr 30

Literature context:


Abstract:

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Funding information:
  • NIA NIH HHS - R01 AG039756(United States)
  • University of California, Davis - New Faculty Startup Funds()

Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length.

  • Liu S
  • Elife
  • 2018 Mar 29

Literature context:


Abstract:

Animal cells within a tissue typically display a striking regularity in their size. To date, the molecular mechanisms that control this uniformity are still unknown. We have previously shown that size uniformity in animal cells is promoted, in part, by size-dependent regulation of G1 length. To identify the molecular mechanisms underlying this process, we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression. Small cells display higher p38 activity and spend more time in G1 than larger cells. Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells, resulting in faster proliferation, smaller cell size and increased size heterogeneity. We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly, to increase cell size uniformity.

Funding information:
  • Biotechnology and Biological Sciences Research Council - 095831(United Kingdom)
  • Canadian Institutes of Health Research - FRN-343437()
  • National Institute of General Medical Sciences - GM26875()

A Precise Cdk Activity Threshold Determines Passage through the Restriction Point.

  • Schwarz C
  • Mol. Cell
  • 2018 Jan 18

Literature context:


Abstract:

At the restriction point (R), mammalian cells irreversibly commit to divide. R has been viewed as a point in G1 that is passed when growth factor signaling initiates a positive feedback loop of Cdk activity. However, recent studies have cast doubt on this model by claiming R occurs prior to positive feedback activation in G1 or even before completion of the previous cell cycle. Here we reconcile these results and show that whereas many commonly used cell lines do not exhibit a G1 R, primary fibroblasts have a G1 R that is defined by a precise Cdk activity threshold and the activation of cell-cycle-dependent transcription. A simple threshold model, based solely on Cdk activity, predicted with more than 95% accuracy whether individual cells had passed R. That a single measurement accurately predicted cell fate shows that the state of complex regulatory networks can be assessed using a few critical protein activities.

Funding information:
  • NICHD NIH HHS - HD55391(United States)
  • NIGMS NIH HHS - R01 GM092925()
  • NIGMS NIH HHS - T32 GM007276()

A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells.

  • Kleiner RE
  • Cell Chem Biol
  • 2018 Jan 18

Literature context:


Abstract:

Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics.

Funding information:
  • NIGMS NIH HHS - R01 GM098579()

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

  • Clift D
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Funding information:
  • NIDCD NIH HHS - P30 DC04657(United States)

New tools for "hot-wiring" clathrin-mediated endocytosis with temporal and spatial precision.

  • Wood LA
  • J. Cell Biol.
  • 2017 Dec 4

Literature context:


Abstract:

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment ("hook") is inducibly attached to an "anchor" protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook-anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it "hot-wiring." We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the β2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement.

Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability.

  • Dumitru AMG
  • Elife
  • 2017 Nov 20

Literature context:


Abstract:

The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.

Funding information:
  • NIA NIH HHS - AG012609(United States)

NuMA recruits dynein activity to microtubule minus-ends at mitosis.

  • Hueschen CL
  • Elife
  • 2017 Nov 29

Literature context:


Abstract:

To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin; both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves a C-terminal region outside NuMA's canonical microtubule-binding domain and is independent of minus-end binders γ-TuRC, CAMSAP1, and KANSL1/3. Both NuMA's minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

Funding information:
  • NIAID NIH HHS - U01 AI82226(United States)

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

  • Chao HX
  • Cell Syst
  • 2017 Nov 22

Literature context:


Abstract:

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none.

Funding information:
  • NICHD NIH HHS - DP2 HD091800()
  • NIGMS NIH HHS - K99 GM102372()
  • NIGMS NIH HHS - R00 GM102372()
  • NIGMS NIH HHS - R01 GM083024()
  • NIGMS NIH HHS - R01 GM102413()
  • NIGMS NIH HHS - T32 GM067553()

Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

  • Bosse KR
  • Cancer Cell
  • 2017 Sep 11

Literature context:


Abstract:

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Funding information:
  • NCI NIH HHS - T32 CA009615()
  • NIGMS NIH HHS - T32 GM008638()

Global Inhibition with Specific Activation: How p53 and MYC Redistribute the Transcriptome in the DNA Double-Strand Break Response.

  • Porter JR
  • Mol. Cell
  • 2017 Sep 21

Literature context:


Abstract:

In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.

Human Centromeres Produce Chromosome-Specific and Array-Specific Alpha Satellite Transcripts that Are Complexed with CENP-A and CENP-C.

  • McNulty SM
  • Dev. Cell
  • 2017 Aug 7

Literature context:


Abstract:

Human centromeres are defined by alpha satellite DNA arrays that are distinct and chromosome specific. Most human chromosomes contain multiple alpha satellite arrays that are competent for centromere assembly. Here, we show that human centromeres are defined by chromosome-specific RNAs linked to underlying organization of distinct alpha satellite arrays. Active and inactive arrays on the same chromosome produce discrete sets of transcripts in cis. Non-coding RNAs produced from active arrays are complexed with CENP-A and CENP-C, while inactive-array transcripts associate with CENP-B and are generally less stable. Loss of CENP-A does not affect transcript abundance or stability. However, depletion of array-specific RNAs reduces CENP-A and CENP-C at the targeted centromere via faulty CENP-A loading, arresting cells before mitosis. This work shows that each human alpha satellite array produces a unique set of non-coding transcripts, and RNAs present at active centromeres are necessary for kinetochore assembly and cell-cycle progression.

Funding information:
  • NIGMS NIH HHS - R01 GM098500()

RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks.

  • Dungrawala H
  • Mol. Cell
  • 2017 Aug 3

Literature context:


Abstract:

RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.

Funding information:
  • NCI NIH HHS - P01 CA092584()
  • NIGMS NIH HHS - R01 GM116616()

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

  • Krishnan S
  • Mol. Cell
  • 2017 Aug 17

Literature context:


Abstract:

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes.

Funding information:
  • Howard Hughes Medical Institute - R01 GM064844()
  • NCI NIH HHS - P30 CA016087()
  • NCI NIH HHS - R01 CA199652()
  • NIGMS NIH HHS - S10 RR023704()

Evaluation of ATM heterozygous mutations underlying individual differences in radiosensitivity using genome editing in human cultured cells.

  • Royba E
  • Sci Rep
  • 2017 Jul 20

Literature context:


Abstract:

Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), which are an initial step towards chromosomal aberrations and cell death. It has been suggested that there are individual differences in radiosensitivity within human populations, and that the variations in DNA repair genes might determine this heterogeneity. However, it is difficult to quantify the effect of genetic variants on the individual differences in radiosensitivity, since confounding factors such as smoking and the diverse genetic backgrounds within human populations affect radiosensitivity. To precisely quantify the effect of a genetic variation on radiosensitivity, we here used the CRISPR-ObLiGaRe (Obligate Ligation-Gated Recombination) method combined with the CRISPR/Cas9 system and a nonhomologous end joining (NHEJ)-mediated knock-in technique in human cultured cells with a uniform genetic background. We generated ATM heterozygous knock-out (ATM +/-) cell clones as a carrier model of a radiation-hypersensitive autosomal-recessive disorder, ataxia-telangiectasia (A-T). Cytokinesis-blocked micronucleus assay and chromosome aberration assay showed that the radiosensitivity of ATM +/- cell clones was significantly higher than that of ATM +/+ cells, suggesting that ATM gene variants are indeed involved in determining individual radiosensitivity. Importantly, the differences in radiosensitivity among the same genotype clones were small, unlike the individual differences in fibroblasts derived from A-T-affected family members.

Funding information:
  • NIGMS NIH HHS - T32 GM008471(United States)

The CEP19-RABL2 GTPase Complex Binds IFT-B to Initiate Intraflagellar Transport at the Ciliary Base.

  • Kanie T
  • Dev. Cell
  • 2017 Jul 10

Literature context:


Abstract:

Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes, and thus initiates ciliary entry of IFT.

A Class of Environmental and Endogenous Toxins Induces BRCA2 Haploinsufficiency and Genome Instability.

  • Tan SLW
  • Cell
  • 2017 Jun 1

Literature context:


Abstract:

Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

  • Maciejowski J
  • Dev. Cell
  • 2017 Apr 24

Literature context:


Abstract:

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Funding information:
  • NCI NIH HHS - K22 CA207458()
  • NCI NIH HHS - P30 CA008748()
  • NIGMS NIH HHS - R01 GM083988()
  • NIGMS NIH HHS - R01 GM094972()

Decoupling global biases and local interactions between cell biological variables.

  • Zaritsky A
  • Elife
  • 2017 Mar 13

Literature context:


Abstract:

Analysis of coupled variables is a core concept of cell biological inference, with co-localization of two molecules as a proxy for protein interaction being a ubiquitous example. However, external effectors may influence the observed co-localization independently from the local interaction of two proteins. Such global bias, although biologically meaningful, is often neglected when interpreting co-localization. Here, we describe DeBias, a computational method to quantify and decouple global bias from local interactions between variables by modeling the observed co-localization as the cumulative contribution of a global and a local component. We showcase four applications of DeBias in different areas of cell biology, and demonstrate that the global bias encapsulates fundamental mechanistic insight into cellular behavior. The DeBias software package is freely accessible online via a web-server at https://debias.biohpc.swmed.edu.

Funding information:
  • NIGMS NIH HHS - P01 GM096971()
  • NIGMS NIH HHS - P01 GM103723()
  • NIGMS NIH HHS - R01 GM073165()

Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion.

  • Phillips AF
  • Mol. Cell
  • 2017 Feb 2

Literature context:


Abstract:

Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.

Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

  • Phua SC
  • Cell
  • 2017 Jan 12

Literature context:


Abstract:

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Funding information:
  • NIAMS NIH HHS - R01 AR054396()
  • NIDDK NIH HHS - R01 DK102910()
  • NIGMS NIH HHS - DP2 GM105448()
  • NIGMS NIH HHS - R01 GM095941()
  • NIGMS NIH HHS - R01 GM112988()

NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies.

  • Moutel S
  • Elife
  • 2016 Jul 19

Literature context:


Abstract:

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.