Publications by authors named "Jay Gopalakrishnan"

25 Publications

  • Page 1 of 1

Neurotropic Effects of SARS-CoV-2 Modeled by the Human Brain Organoids.

Stem Cell Reports 2021 03 12;16(3):373-384. Epub 2021 Feb 12.

Institute of Human Genetics, Universitätsklinikum Heinrich-Heine-Universität Düsseldorf, Universität Street 1, 40225 Düsseldorf, Germany. Electronic address:

COVID-19, caused by SARS-CoV-2, is a socioeconomic burden, which exhibits respiratory illness along with unexpected neurological complications. Concerns have been raised about whether the observed neurological symptoms are due to direct effects on CNS or associated with the virus's systemic effect. Recent SARS-CoV-2 infection studies using human brain organoids revealed that SARS-CoV-2 targets human neurons. Human brain organoids are stem cell-derived reductionist experimental systems that have highlighted the neurotropic effects of SARS-CoV-2. Here, we summarize the neurotoxic effects of SARS-CoV-2 using brain organoids and comprehensively discuss how brain organoids could further improve our understanding when they are fine-tuned.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2021.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879157PMC
March 2021

Trends and challenges in modeling glioma using 3D human brain organoids.

Cell Death Differ 2021 Jan 1;28(1):15-23. Epub 2020 Dec 1.

Institute of Human Genetics, University Hospital Düsseldorf, Heinrich-Heine-Universität, Universitätsstr. 1, 40225, Düsseldorf, Germany.

The human brain organoids derived from pluripotent cells are a new class of three-dimensional tissue systems that recapitulates several neural epithelial aspects. Brain organoids have already helped efficient modeling of crucial elements of brain development and disorders. Brain organoids' suitability in modeling glioma has started to emerge, offering another usefulness of brain organoids in disease modeling. Although the current state-of-the organoids mostly reflect the immature state of the brain, with their vast cell diversity, human brain-like cytoarchitecture, feasibility in culturing, handling, imaging, and tractability can offer enormous potential in reflecting the glioma invasion, integration, and interaction with different neuronal cell types. Here, we summarize the current trend of employing brain organoids in glioma modeling and discuss the immediate challenges. Solving them might lay a foundation for using brain organoids as a pre-clinical 3D substrate to dissect the glioma invasion mechanisms in detail.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41418-020-00679-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707134PMC
January 2021

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Nat Commun 2020 11 16;11(1):5816. Epub 2020 Nov 16.

Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen, Denmark.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-19658-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670429PMC
November 2020

Structure aware Runge-Kutta time stepping for spacetime tents.

SN Partial Differ Equ Appl 2020 28;1(4):19. Epub 2020 Jul 28.

Technische Universität Wien, Wiedner Hauptstraße 8-10, 1040 Wien, Austria.

We introduce a new class of Runge-Kutta type methods suitable for time stepping to propagate hyperbolic solutions within tent-shaped spacetime regions. Unlike standard Runge-Kutta methods, the new methods yield expected convergence properties when standard high order spatial (discontinuous Galerkin) discretizations are used. After presenting a derivation of nonstandard order conditions for these methods, we show numerical examples of nonlinear hyperbolic systems to demonstrate the optimal convergence rates. We also report on the discrete stability properties of these methods applied to linear hyperbolic equations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s42985-020-00020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446293PMC
July 2020

SARS-CoV-2 targets neurons of 3D human brain organoids.

EMBO J 2020 10 23;39(20):e106230. Epub 2020 Sep 23.

Institute of Human Genetics, University Hospital Düsseldorf, Heinrich-Heine-Universität, Düsseldorf, Germany.

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2020106230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560208PMC
October 2020

Rapid and Efficient Invasion Assay of Glioblastoma in Human Brain Organoids.

Cell Rep 2020 06;31(10):107738

Institute of Human Genetics, University Hospital Düsseldorf, Heinrich-Heine-Universität, Universitätsstrasse 1, 40225 Düsseldorf, Germany. Electronic address:

Glioblastoma (GBM) possesses glioma stem cells (GSCs) that exhibit aggressive invasion behavior in the brain. Current preclinical GBM invasion assays using mouse brain xenografts are time consuming and less efficient. Here, we demonstrate an array of methods that allow rapid and efficient assaying of GSCs invasion in human brain organoids. The assays are versatile to characterize various aspects of GSCs, such as invasion, integration, and interaction with mature neurons of brain organoids. Tissue clearing and quantitative 3D imaging of GSCs in host organoids reveal that invasiveness is inversely correlated with the organoids' age. Importantly, the described invasion assays can distinguish the invasive behaviors of primary and recurrent GSCs. The assays are also amenable to test pharmacological agents. As an example, we show that GI254023X, an inhibitor of ADAM10, could prevent the integration of GSCs into the organoids.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.107738DOI Listing
June 2020

Human Brain Organoids to Decode Mechanisms of Microcephaly.

Front Cell Neurosci 2020 8;14:115. Epub 2020 May 8.

Laboratory for Centrosome and Cytoskeleton Biology, Institute für Humangenetik, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Düsseldorf, Germany.

Brain organoids are stem cell-based self-assembling 3D structures that recapitulate early events of human brain development. Recent improvements with patient-specific 3D brain organoids have begun to elucidate unprecedented details of the defective mechanisms that cause neurodevelopmental disorders of congenital and acquired microcephaly. In particular, brain organoids derived from primary microcephaly patients have uncovered mechanisms that deregulate neural stem cell proliferation, maintenance, and differentiation. Not only did brain organoids reveal unknown aspects of neurogenesis but also have illuminated surprising roles of cellular structures of centrosomes and primary cilia in regulating neurogenesis during brain development. Here, we discuss how brain organoids have started contributing to decoding the complexities of microcephaly, which are unlikely to be identified in the existing non-human models. Finally, we discuss the yet unresolved questions and challenges that can be addressed with the use of brain organoids as models of neurodevelopmental disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2020.00115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225330PMC
May 2020

Repurposing the serotonin agonist Tegaserod as an anticancer agent in melanoma: molecular mechanisms and clinical implications.

J Exp Clin Cancer Res 2020 Feb 21;39(1):38. Epub 2020 Feb 21.

Department of Molecular Medicine II, Medical Faculty, Heinrich-Heine-University, Universitätsstraße 1, 40225, Düsseldorf, Germany.

Background: New therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors.

Methods: Drug screening, IC50 determinations as well as synergy assays were detected by the MTT assay. Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry. TUNEL staining was performed using immunocytochemistry. Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry. To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used. Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors. Evaluation of immune infiltrates was carried out by flow cytometry.

Results: Using a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines. Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS). TM's anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition. Specifically, TM blunted S6 phosphorylation in both BRAF and BRAF wildtype (WT) melanoma cell lines. TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model. In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation. Furthermore TM decreased the infiltration of immune suppressive regulatory CD4CD25 T cells and FOXP3 and ROR-γt positive CD4 T cells. Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAF mutation and could be additively or synergistically combined with Cobimetinib in both BRAF and BRAF WT melanoma cell lines in inducing anti-cancer effects.

Conclusion: Taken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAF and BRAF WT melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13046-020-1539-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035645PMC
February 2020

Dzip1 and Fam92 form a ciliary transition zone complex with cell type specific roles in .

Elife 2019 12 10;8. Epub 2019 Dec 10.

Institut NeuroMyoGène, CNRS UMR 5310, INSERM U1217, Université Claude Bernard Lyon 1, Lyon, France.

Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. Here, we show that Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. We identify cell type specific roles of this functional module in two different tissues. While it is required for TZ assembly in all ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.49307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904220PMC
December 2019

The Emergence of Stem Cell-Based Brain Organoids: Trends and Challenges.

Bioessays 2019 08 5;41(8):e1900011. Epub 2019 Jul 5.

Institut für Humangenetik, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Universitätsstr. 1, 40225, Düsseldorf, Germany.

Recent developments in 3D cultures exploiting the self-organization ability of pluripotent stem cells have enabled the generation of powerful in vitro systems termed brain organoids. These 3D tissues recapitulate many aspects of human brain development and disorders occurring in vivo. When combined with improved differentiation methods, these in vitro systems allow the generation of more complex "assembloids," which are able to reveal cell diversities, microcircuits, and cell-cell interactions within their 3D organization. Here, the ways in which human brain organoids have contributed to demystifying the complexities of brain development and modeling of developmental disorders is reviewed and discussed. Furthermore, challenging questions that are yet to be addressed by emerging brain organoid research are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bies.201900011DOI Listing
August 2019

Plk1/Polo Phosphorylates Sas-4 at the Onset of Mitosis for an Efficient Recruitment of Pericentriolar Material to Centrosomes.

Cell Rep 2018 12;25(13):3618-3630.e6

Institute of Human Genetics, Universitätsklinikum Heinrich-Heine-Universität Düsseldorf, Universität Str. 1, 40225 Düsseldorf, Germany; Center for Molecular Medicine Cologne, University of Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany; IUF-Leibniz-Institut für umweltmedizinische Forschung gGmbH, Auf'm Hennekamp 50, 40225 Düsseldorf, Germany. Electronic address:

Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.11.102DOI Listing
December 2018

Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells.

EMBO J 2019 01 10;38(2). Epub 2018 Dec 10.

Institute für Humangenetik, Universitätsklinikum Düsseldorf, Heinrich-Heine-Universität, Düsseldorf, Germany

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201899876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331730PMC
January 2019

Novel insights into SMALED2: BICD2 mutations increase microtubule stability and cause defects in axonal and NMJ development.

Hum Mol Genet 2018 05;27(10):1772-1784

Institute of Human Genetics, University of Cologne, 50931 Cologne, Germany.

Bicaudal D2 (BICD2) encodes a highly conserved motor adaptor protein that regulates the dynein-dynactin complex in different cellular processes. Heterozygous mutations in BICD2 cause autosomal dominant lower extremity-predominant spinal muscular atrophy-2 (SMALED2). Although, various BICD2 mutations have been shown to alter interactions with different binding partners or the integrity of the Golgi apparatus, the specific pathological effects of BICD2 mutations underlying SMALED2 remain elusive. Here, we show that the fibroblasts derived from individuals with SMALED2 exhibit stable microtubules. Importantly, this effect was observed regardless of where the BICD2 mutation is located, which unifies the most likely cellular mechanism affecting microtubules. Significantly, overexpression of SMALED2-causing BICD2 mutations in the disease-relevant cell type, motor neurons, also results in an increased microtubule stability which is accompanied by axonal aberrations such as collateral branching and overgrowth. To study the pathological consequences of BICD2 mutations in vivo, and to address the controversial debate whether two of these mutations are neuron or muscle specific, we generated the first Drosophila model of SMALED2. Strikingly, neuron-specific expression of BICD2 mutants resulted in reduced neuromuscular junction size in larvae and impaired locomotion of adult flies. In contrast, expressing BICD2 mutations in muscles had no obvious effect on motor function, supporting a primarily neurological etiology of the disease. Thus, our findings contribute to the better understanding of SMALED2 pathology by providing evidence for a common pathomechanism of BICD2 mutations that increase microtubule stability in motor neurons leading to increased axonal branching and to impaired neuromuscular junction development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddy086DOI Listing
May 2018

Losers of Primary Cilia Gain the Benefit of Survival.

Cancer Discov 2017 12;7(12):1374-1375

Center for Molecular Medicine Cologne and Institute of Biochemistry I of the University of Cologne, Cologne, Germany.

In this issue, Zhao and colleagues demonstrate that loss of primary cilia in medulloblastoma cells confers resistance to the Smoothened (SMO) inhibitor sonidegib. When treated with sonidegib, medulloblastoma cells lost their cilia and gained resistance. Surprisingly, loss of cilia is associated with recurrent mutations in ciliogenesis genes that are eventually able to drive drug resistance. These findings uncover a previously unknown mechanism of cancer cells in gaining a "persister-like" state against anticancer agents at the expense of losing primary cilia. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-17-1085DOI Listing
December 2017

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders.

J Vis Exp 2017 04 14(122). Epub 2017 Apr 14.

Center for Molecular Medicine Cologne, University of Cologne; Institute for Biochemistry I, Medical School of University of Cologne;

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/55372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564693PMC
April 2017

Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids.

Cell Stem Cell 2017 03 26;20(3):397-406.e5. Epub 2017 Jan 26.

Center for Molecular Medicine and Institute of Biochemistry I of the University of Cologne, Cologne 50931, Germany. Electronic address:

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2016.12.005DOI Listing
March 2017

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.

Nat Commun 2016 06 16;7:11874. Epub 2016 Jun 16.

Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China.

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912634PMC
June 2016

Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

Cell Cycle 2016 Aug 1;15(16):2216-2225. Epub 2016 Jun 1.

a Department of Epigenetics and Molecular Carcinogenesis , University of Texas MD Anderson Cancer Center , Houston , TX , USA.

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15384101.2016.1192728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993543PMC
August 2016

Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.

J Biomol Screen 2016 Jul 4;21(6):596-607. Epub 2016 Apr 4.

Helmholtz Zentrum München für Gesundheit und Umwelt (GmbH), Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Neuherberg, Germany

In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057116639992DOI Listing
July 2016

New Colchicine-Derived Triazoles and Their Influence on Cytotoxicity and Microtubule Morphology.

ACS Med Chem Lett 2016 Feb 29;7(2):188-91. Epub 2015 Dec 29.

Department of Chemistry, University of Cologne , Greinstr. 4, 50939 Cologne, Germany.

A series of new colchicinoids with a variable triazole unit at C-7 was synthesized through Cu(I)-catalyzed 1,3-dipolar cycloaddition (click-chemistry) of a colchicine-derived azide with various alkynes and the cytotoxicity against THP-1 and Jurkat cancer cell lines was used for structural optimization. Three particularly active compounds (IC50 ≤ 5 nM) were additionally investigated with respect to their efficacy against relevant solid tumor cell lines (HeLa, A549, and SK MES 1). Besides distorting the microtubule morphology by tubulin depolymerization, one compound also exhibited a pronounced centrosome declustering effect in triple negative breast cancer cells (MDA-MB-231) and nonsmall cell lung cancer cells (H1975).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsmedchemlett.5b00418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753549PMC
February 2016

CPAP promotes timely cilium disassembly to maintain neural progenitor pool.

EMBO J 2016 Apr 29;35(8):803-19. Epub 2016 Feb 29.

Center for Molecular Medicine and Institute for Biochemistry I of the University of Cologne, Cologne, Germany

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201593679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972140PMC
April 2016

Mathematical model for bone mineralization.

Front Cell Dev Biol 2015 21;3:51. Epub 2015 Aug 21.

Department of Mathematics, High Point University High Point, NC, USA.

Defective bone mineralization has serious clinical manifestations, including deformities and fractures, but the regulation of this extracellular process is not fully understood. We have developed a mathematical model consisting of ordinary differential equations that describe collagen maturation, production and degradation of inhibitors, and mineral nucleation and growth. We examined the roles of individual processes in generating normal and abnormal mineralization patterns characterized using two outcome measures: mineralization lag time and degree of mineralization. Model parameters describing the formation of hydroxyapatite mineral on the nucleating centers most potently affected the degree of mineralization, while the parameters describing inhibitor homeostasis most effectively changed the mineralization lag time. Of interest, a parameter describing the rate of matrix maturation emerged as being capable of counter-intuitively increasing both the mineralization lag time and the degree of mineralization. We validated the accuracy of model predictions using known diseases of bone mineralization such as osteogenesis imperfecta and X-linked hypophosphatemia. The model successfully describes the highly nonlinear mineralization dynamics, which includes an initial lag phase when osteoid is present but no mineralization is evident, then fast primary mineralization, followed by secondary mineralization characterized by a continuous slow increase in bone mineral content. The developed model can potentially predict the function for a mutated protein based on the histology of pathologic bone samples from mineralization disorders of unknown etiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2015.00051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544393PMC
September 2015

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis.

Proc Natl Acad Sci U S A 2014 Jan 2;111(3):E354-63. Epub 2014 Jan 2.

Ministry of Education Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences, and Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, People's Republic of China.

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th β-strands (β9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1317535111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903230PMC
January 2014

Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens.

J Biomol Screen 2014 Jun 26;19(5):715-26. Epub 2013 Dec 26.

Helmholtz Zentrum München für Gesundheit und Umwelt (HMGU), Institute of Molecular Toxicology and Pharmacology, Assay Development and Screening Platform, Neuherberg, Germany

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)-compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni(2+)-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057113516861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4153540PMC
June 2014

Instability in a generalized Keller-Segel model.

J Biol Dyn 2012 ;6:974-91

Mathematics, University of Florida, PO Box 118105, Gainesville, FL 32611-8105, USA.

We present a generalized Keller-Segel model where an arbitrary number of chemical compounds react, some of which are produced by a species, and one of which is a chemoattractant for the species. To investigate the stability of homogeneous stationary states of this generalized model, we consider the eigenvalues of a linearized system. We are able to reduce this infinite dimensional eigenproblem to a parametrized finite dimensional eigenproblem. By matrix theoretic tools, we then provide easily verifiable sufficient conditions for destabilizing the homogeneous stationary states. In particular, one of the sufficient conditions is that the chemotactic feedback is sufficiently strong. Although this mechanism was already known to exist in the original Keller-Segel model, here we show that it is more generally applicable by significantly enlarging the class of models exhibiting this instability phenomenon which may lead to pattern formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/17513758.2012.714478DOI Listing
December 2012