DOCSLIB.ORG

Developmental Coordination of Gene Expression Between Synaptic Partners During Gabaergic Circuit Assembly in Cerebellar Cortex

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Developmental Coordination of Gene Expression Between Synaptic Partners During Gabaergic Circuit Assembly in Cerebellar Cortex ORIGINAL RESEARCH ARTICLE published: 26 June 2012 NEURAL CIRCUITS doi: 10.3389/fncir.2012.00037 Developmental coordination of gene expression between synaptic partners during GABAergic circuit assembly in cerebellar cortex Anirban Paul 1,Ying Cai 2,3, Gurinder S. Atwal 2 and Z. Josh Huang 1* 1 Cold Spring Harbor Laboratory, Neuroscience, Cold Spring Harbor, New York, NY, USA 2 Cold Spring Harbor Laboratory, Quantitative Biology, Cold Spring Harbor, NY, USA 3 Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY, USA Edited by: The assembly of neural circuits involves multiple sequential steps such as the specifica- Edward M. Callaway, California tion of cell-types, their migration to proper brain locations, morphological and physiological Institute of Technology, USA differentiation, and the formation and maturation of synaptic connections. This intricate Reviewed by: David J. Margolis, University of and often prolonged process is guided by elaborate genetic mechanisms that regulate Zurich, Switzerland each step. Evidence from numerous systems suggests that each cell-type, once speci- Yuanquan Song, University of fied, is endowed with a genetic program that unfolds in response to, and is regulated by, California San Francisco, USA extrinsic signals, including cell–cell and synaptic interactions. To a large extent, the exe- *Correspondence: cution of this intrinsic program is achieved by the expression of specific sets of genes Z. Josh Huang, Cold Spring Harbor Laboratory, One Bungtown Road, that support distinct developmental processes. Therefore, a comprehensive analysis of Cold Spring Harbor, New York, NY the developmental progression of gene expression in synaptic partners of neurons may 11724, USA. provide a basis for exploring the genetic mechanisms regulating circuit assembly. Here e-mail: [email protected] we examined the developmental gene expression profiles of well-defined cell-types in a stereotyped microcircuit of the cerebellar cortex. We found that the transcriptomes of Purkinje cell and stellate/basket cells are highly dynamic throughout postnatal develop- ment. We revealed “phasic expression” of transcription factors, ion channels, receptors, cell adhesion molecules, gap junction proteins, and identified distinct molecular pathways that might contribute to sequential steps of cerebellar inhibitory circuit formation. We fur- ther revealed a correlation between genomic clustering and developmental co-expression of hundreds of transcripts, suggesting the involvement of chromatin level gene regulation during circuit formation. Keywords: cerebellum, circuit assembly, development, GABAergic, gene expression, microcircuit, Purkinje cell, stellate basket cell INTRODUCTION All cerebellar neurons derive from progenitors that prolifer- The cerebellum, a primary center for motor coordination, is an ate in two germinal neuroepithelia: the ventricular zone (VZ) excellent system to study neural circuit assembly in the CNS due generates GABAergic neurons, whereas the rhombic lip is the to its highly stereotyped cytoarchitecture. For example, the cere- origin of glutamatergic neurons. Among VZ-derivatives, GABAer- bellar cortex is organized as a near lattice-like circuit architecture gic projection neurons and interneurons are generated according with a protracted period of circuit formation. At the focal position to different strategies. PC are produced at the onset of cerebel- in cerebellar cortex and as its sole output are the Purkinje neurons, lar neurogenesis by discrete progenitor pools located in distinct which receive multiple sets of excitatory and inhibitory inputs. The VZ microdomains; they are specified within the VZ and acquire glutamatergic parallel fibers synapse onto the slender spines of the mature phenotypes largely according to cell-autonomous pro- distal dendrite, while the climbing fibers prefer the stubby spines grams. On the other hand, the different classes of inhibitory of the more proximal dendrite. In addition, the GABAergic bas- interneurons, including basket (BkC) and stellate (StC) cells, ket interneurons target Purkinje cell (PC) soma and axon initial derive from a single population of precursors that delaminate segments (AIS), whereas the stellate interneurons innervate the into the prospective white matter (PWM), where they continue dendritic shafts. The integration of these excitatory and inhibitory to divide until the end of the first postnatal week (Altman and inputs shapes PC outputs, which are transmitted to deep cerebel- Bayer, 1997; Leto et al., 2012). lar nuclei and regulate motor coordination (D’Angelo et al., 2011). Following their generation in the PWM, basket, and stellate While significant progress have been made in understanding the cells migrate toward cerebellar cortex and reach Purkinje cell layer development of excitatory connectivity (i.e., parallel fibers and (PCL) by approximately P7 and P10, respectively. During the climbing fibers), the development of inhibitory circuitry remains subsequent weeks, PC, BkC, and StC undergo profound morpho- poorly understood. logical and physiological differentiation while establishing specific Frontiers in Neural Circuits www.frontiersin.org June 2012 | Volume 6 | Article 37 | 1 Paul et al. Cell-type-specific transcriptome during circuit assembly synaptic connectivity (Cameron et al., 2009). For example, the amplified by two rounds of linear isothermal amplification steps PCs elaborate their dendrites with characteristic branching pat- using the MessageAmpII aRNA amplification kit (Ambion, USA terns while the BskCs extend their axon branches onto PCs. Each Cat#AM1751) followed by biotin labeling. BskC innervates 7–10 PCs at their soma and AIS (Figure 1A). The maturation of BskC axon arbor and pinceau synapses con- GENE EXPRESSION tinue into the fourth postnatal week (Ango et al., 2004, 2008). Labeled aRNA were hybridized to Affymetrix mouse 30 expres- The development of StCs lags behind that of BskCs by a few days. sion array MOE430.2 according to manufacturers fluid han- StC axons extend along the Bergmann glia fibers with characteris- dling, hybridization, and scanning protocols. CEL files from each tic trajectories to innervate PC dendrites. Using a candidate gene hybridization was converted to normalized expression values using approach, we have previously demonstrated that members of the Bioconductor package1 in R2 using GCRMA method. Further L1 family immunoglobulin cell adhesion molecules (IgCAMs), differential analysis, Principal Component Analysis (PCA), cross- neurofascin, and CHL1, contribute to the subcellular organization correlation analysis, and downstream visualization was done in of BskC and StC innervation,respectively (Ango et al.,2004,2008). dCHIP software3. Microarray data submitted to GEO; accession However, the genetic mechanisms that control the concerted dif- GSE37055. ferentiation and synaptic connectivity during inhibitory circuit formation remain poorly understood. IN SITU HYBRIDIZATION To explore the intrinsic genetic program that direct the dif- To generate in situ probes Trizol extracted total mouse brain RNA ferentiation and connectivity between PC and BskC/StC(S/BC) was used to perform RT-PCR using gene specific primers (Super- cells, here we examined the developmental dynamics of their spe- script III, Invitrogen, USA). RT product was subjected to nested cific gene expression profiles. We found (i) phasic developmental PCR with T3 tagged forward and T7 tagged reverse primers (see expression of transcription factors (TFs), ion channels, recep- primer list in Table A2 in Appendix). In vitro transcription using tors, cell adhesion molecules (CAMs), gap junction proteins, and with T7 and T3 driven RNA polymerase and DIG-labeled rNTPs novel cell-type specific transcripts,(ii) distinct molecular pathways generate the probes that was run on Bioanalyzer to ensure sin- that may contribute to different developmental stages for these gle RNA product of expected size. T7 produced antisense probes cell-types; (iii) correlation between genomic clustering and devel- and T3 generated the control sense probes. In situ hybridization opmental co-expression of hundreds of transcripts, suggesting the was performed at 61˚C on 15 mm thick sagittal cryo-sectioned involvement of chromatin level gene regulation during circuit for- brains from C57B6 male animals. Detection was done using anti- mation. Our dataset also provides a valuable resource for further DIG antibody and VectaRed detection reagent (Vector Labs, USA studying the molecular and developmental genetic mechanisms Cat#SK-5100). underlying cerebellar circuit formation. PERMUTATION TEST MATERIALS AND METHODS Permutation test was performed on normalized expression values MANUAL SORTING of PC and S/BC cells to find genes that are differentially expressed Amplified and labeled mRNA were prepared from 50 to 75 PC or across different time points. For each probe, a t-statistic T obs was between 125 and 150 S/BC cells that were manually sorted from computed as each developmental stage. While PCs are 40 mm in diameter and lie along the PCL the StC and the BskC are both 8–10 mm in diam- .Xi/ − Xj Tobs D r 2 eter and are distributed in the molecular layer. Although the BskC S2 S i C j cell bodies lie relatively closer to the PC it is not always apparent ni nj since StC and BskC are not uniformly distinct in layer separation. Also there is no molecular marker that distinguishes Bsk from Where <xi> is the mean of expression values of replicates at the StC. This prevented us from
Load more

Recommended publications
  • Rule Mining on Microrna Expression Profiles For
    Faculty of Engineering and Information Technology University of Technology, Sydney Rule Mining on MicroRNA Expression Profiles for Human Disease Understanding A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Renhua Song July 2016 CERTIFICATE OF AUTHORSHIP/ORIGINALITY I certify that the work in this thesis has not previously been submitted for a degree nor has it been submitted as part of requirements for a degree except as fully acknowledged within the text. I also certify that the thesis has been written by me. Any help that I have received in my research work and the preparation of the thesis itself has been acknowledged. In addition, I certify that all information sources and literature used are indicated in the thesis. Signature of Candidate i Acknowledgments First, and foremost, I would like to express my gratitude to my chief supervisor, Assoc. Prof. Jinyan Li, and to my co-supervisors Assoc. Prof. Paul Kennedy and Assoc. Prof. Daniel Catchpoole. I am extremely grateful for all the advice and guidance so unselfishly given to me over the last three and half years by these three distinguished academics. This research would not have been possible without their high order supervision, support, assistance and leadership. The wonderful support and assistance provided by many people during this research is very much appreciated by me and my family. I am very grateful for the help that I received from all of these people but there are some special individuals that I must thank by name. A very sincere thank you is definitely owed to my loving husband Jing Liu, not only for his tremendous support during the last three and half years, but also his unfailing and often sorely tested patience.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Exploring Prostate Cancer Genome Reveals Simultaneous Losses of PTEN, FAS and PAPSS2 in Patients with PSA Recurrence After Radical Prostatectomy
    Int. J. Mol. Sci. 2015, 16, 3856-3869; doi:10.3390/ijms16023856 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Exploring Prostate Cancer Genome Reveals Simultaneous Losses of PTEN, FAS and PAPSS2 in Patients with PSA Recurrence after Radical Prostatectomy Chinyere Ibeawuchi 1, Hartmut Schmidt 2, Reinhard Voss 3, Ulf Titze 4, Mahmoud Abbas 5, Joerg Neumann 6, Elke Eltze 7, Agnes Marije Hoogland 8, Guido Jenster 9, Burkhard Brandt 10 and Axel Semjonow 1,* 1 Prostate Center, Department of Urology, University Hospital Muenster, Albert-Schweitzer-Campus 1, Gebaeude 1A, Muenster D-48149, Germany; E-Mail: [email protected] 2 Center for Laboratory Medicine, University Hospital Muenster, Albert-Schweitzer-Campus 1, Gebaeude 1A, Muenster D-48149, Germany; E-Mail: [email protected] 3 Interdisciplinary Center for Clinical Research, University of Muenster, Albert-Schweitzer-Campus 1, Gebaeude D3, Domagkstrasse 3, Muenster D-48149, Germany; E-Mail: [email protected] 4 Pathology, Lippe Hospital Detmold, Röntgenstrasse 18, Detmold D-32756, Germany; E-Mail: [email protected] 5 Institute of Pathology, Mathias-Spital-Rheine, Frankenburg Street 31, Rheine D-48431, Germany; E-Mail: [email protected] 6 Institute of Pathology, Klinikum Osnabrueck, Am Finkenhuegel 1, Osnabrueck D-49076, Germany; E-Mail: [email protected] 7 Institute of Pathology, Saarbrücken-Rastpfuhl, Rheinstrasse 2, Saarbrücken D-66113, Germany; E-Mail: [email protected] 8 Department
    [Show full text]
  • Transcriptomic Analysis of Native Versus Cultured Human and Mouse Dorsal Root Ganglia Focused on Pharmacological Targets Short
    bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Transcriptomic analysis of native versus cultured human and mouse dorsal root ganglia focused on pharmacological targets Short title: Comparative transcriptomics of acutely dissected versus cultured DRGs Andi Wangzhou1, Lisa A. McIlvried2, Candler Paige1, Paulino Barragan-Iglesias1, Carolyn A. Guzman1, Gregory Dussor1, Pradipta R. Ray1,#, Robert W. Gereau IV2, # and Theodore J. Price1, # 1The University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, 800 W Campbell Rd. Richardson, TX, 75080, USA 2Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine # corresponding authors [email protected], [email protected] and [email protected] Funding: NIH grants T32DA007261 (LM); NS065926 and NS102161 (TJP); NS106953 and NS042595 (RWG). The authors declare no conflicts of interest Author Contributions Conceived of the Project: PRR, RWG IV and TJP Performed Experiments: AW, LAM, CP, PB-I Supervised Experiments: GD, RWG IV, TJP Analyzed Data: AW, LAM, CP, CAG, PRR Supervised Bioinformatics Analysis: PRR Drew Figures: AW, PRR Wrote and Edited Manuscript: AW, LAM, CP, GD, PRR, RWG IV, TJP All authors approved the final version of the manuscript. 1 bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Modulating Hallmarks of Cholangiocarcinoma
    University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-14-2018 Modulating Hallmarks of Cholangiocarcinoma Cody Wehrkamp University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Molecular Biology Commons Recommended Citation Wehrkamp, Cody, "Modulating Hallmarks of Cholangiocarcinoma" (2018). Theses & Dissertations. 337. https://digitalcommons.unmc.edu/etd/337 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. MODULATING HALLMARKS OF CHOLANGIOCARCINOMA by Cody J. Wehrkamp A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Biochemistry and Molecular Biology Graduate Program Under the Supervision of Professor Justin L. Mott University of Nebraska Medical Center Omaha, Nebraska November 2018 Supervisory Committee: Kaustubh Datta, Ph.D. Melissa Teoh‐Fitzgerald, Ph.D. Richard G. MacDonald, Ph.D. Acknowledgements This endeavor has led to scientific as well as personal growth for me. I am indebted to many for their knowledge, influence, and support along the way. To my mentor, Dr. Justin L. Mott, you have been an incomparable teacher and invaluable guide. You upheld for me the concept that science is intrepid, even when the experience is trying. Through my training, and now here at the end, I can say that it has been an honor to be your protégé. When you have shaped your future graduates to be and do great, I will be privileged to say that I was your first one.
    [Show full text]
  • Methods in and Applications of the Sequencing of Short Non-Coding Rnas" (2013)
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Methods in and Applications of the Sequencing of Short Non- Coding RNAs Paul Ryvkin University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Ryvkin, Paul, "Methods in and Applications of the Sequencing of Short Non-Coding RNAs" (2013). Publicly Accessible Penn Dissertations. 922. https://repository.upenn.edu/edissertations/922 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/922 For more information, please contact [email protected]. Methods in and Applications of the Sequencing of Short Non-Coding RNAs Abstract Short non-coding RNAs are important for all domains of life. With the advent of modern molecular biology their applicability to medicine has become apparent in settings ranging from diagonistic biomarkers to therapeutics and fields angingr from oncology to neurology. In addition, a critical, recent technological development is high-throughput sequencing of nucleic acids. The convergence of modern biotechnology with developments in RNA biology presents opportunities in both basic research and medical settings. Here I present two novel methods for leveraging high-throughput sequencing in the study of short non- coding RNAs, as well as a study in which they are applied to Alzheimer's Disease (AD). The computational methods presented here include High-throughput Annotation of Modified Ribonucleotides (HAMR), which enables researchers to detect post-transcriptional covalent modifications ot RNAs in a high-throughput manner. In addition, I describe Classification of RNAs by Analysis of Length (CoRAL), a computational method that allows researchers to characterize the pathways responsible for short non-coding RNA biogenesis.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Molecular Effects of Isoflavone Supplementation Human Intervention Studies and Quantitative Models for Risk Assessment
    Molecular effects of isoflavone supplementation Human intervention studies and quantitative models for risk assessment Vera van der Velpen Thesis committee Promotors Prof. Dr Pieter van ‘t Veer Professor of Nutritional Epidemiology Wageningen University Prof. Dr Evert G. Schouten Emeritus Professor of Epidemiology and Prevention Wageningen University Co-promotors Dr Anouk Geelen Assistant professor, Division of Human Nutrition Wageningen University Dr Lydia A. Afman Assistant professor, Division of Human Nutrition Wageningen University Other members Prof. Dr Jaap Keijer, Wageningen University Dr Hubert P.J.M. Noteborn, Netherlands Food en Consumer Product Safety Authority Prof. Dr Yvonne T. van der Schouw, UMC Utrecht Dr Wendy L. Hall, King’s College London This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Molecular effects of isoflavone supplementation Human intervention studies and quantitative models for risk assessment Vera van der Velpen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 20 June 2014 at 13.30 p.m. in the Aula. Vera van der Velpen Molecular effects of isoflavone supplementation: Human intervention studies and quantitative models for risk assessment 154 pages PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in Dutch and English ISBN: 978-94-6173-952-0 ABSTRact Background: Risk assessment can potentially be improved by closely linked experiments in the disciplines of epidemiology and toxicology.
    [Show full text]
  • The Centriole Protein CEP76 Negatively Regulates PLK1 Activity in the Cytoplasm for Proper Mitotic Progression
    © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs241281. doi:10.1242/jcs.241281 RESEARCH ARTICLE The centriole protein CEP76 negatively regulates PLK1 activity in the cytoplasm for proper mitotic progression Yutaka Takeda, Kaho Yamazaki, Kaho Hashimoto, Koki Watanabe, Takumi Chinen*,‡ and Daiju Kitagawa*,‡ ABSTRACT are important for accurate mitotic progression (Golsteyn, 1995; Polo-like kinase 1 (PLK1) dynamically changes its localization and Schmucker and Sumara, 2014). Several factors regulate PLK1 plays important roles in proper mitotic progression. In particular, strict localization during mitosis. PLK1 is recruited to centrosomes in a control of cytoplasmic PLK1 is needed to prevent mitotic defects. CDK1-dependent manner (Lee et al., 2014). The initial recruitment However, the regulation of cytoplasmic PLK1 is not fully understood. In of PLK1 to kinetochores is mediated by its interaction with PBIP1 this study, we show that CEP76, a centriolar protein, physically (also known as CENPU; Kang et al., 2006). During anaphase, PLK1 interacts with PLK1 and tightly controls the activation of cytoplasmic binds to PRC1 and changes its localization to the midbody (Hu et al., PLK1 during mitosis in human cells. We found that removal of 2012). In addition, strict control of PLK1 in the cytoplasm is needed centrosomes induced ectopic aggregation of PLK1, which is highly to prevent its aggregation and mitotic defects (Mukhopadhyay and phosphorylated, in the cytoplasm during mitosis. Importantly, a Dasso, 2010; Schmit et al., 2012; Zhao et al., 2016). However, the targeted RNAi screen revealed that depletion of CEP76 resulted in a detailed mechanisms through which the localization, expression and similar phenotype.
    [Show full text]
  • Transcriptomic Profiling of Ca Transport Systems During
    cells Article Transcriptomic Profiling of Ca2+ Transport Systems during the Formation of the Cerebral Cortex in Mice Alexandre Bouron Genetics and Chemogenomics Lab, Université Grenoble Alpes, CNRS, CEA, INSERM, Bâtiment C3, 17 rue des Martyrs, 38054 Grenoble, France; [email protected] Received: 29 June 2020; Accepted: 24 July 2020; Published: 29 July 2020 Abstract: Cytosolic calcium (Ca2+) transients control key neural processes, including neurogenesis, migration, the polarization and growth of neurons, and the establishment and maintenance of synaptic connections. They are thus involved in the development and formation of the neural system. In this study, a publicly available whole transcriptome sequencing (RNA-Seq) dataset was used to examine the expression of genes coding for putative plasma membrane and organellar Ca2+-transporting proteins (channels, pumps, exchangers, and transporters) during the formation of the cerebral cortex in mice. Four ages were considered: embryonic days 11 (E11), 13 (E13), and 17 (E17), and post-natal day 1 (PN1). This transcriptomic profiling was also combined with live-cell Ca2+ imaging recordings to assess the presence of functional Ca2+ transport systems in E13 neurons. The most important Ca2+ routes of the cortical wall at the onset of corticogenesis (E11–E13) were TACAN, GluK5, nAChR β2, Cav3.1, Orai3, transient receptor potential cation channel subfamily M member 7 (TRPM7) non-mitochondrial Na+/Ca2+ exchanger 2 (NCX2), and the connexins CX43/CX45/CX37. Hence, transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1), transmembrane protein 165 (TMEM165), and Ca2+ “leak” channels are prominent intracellular Ca2+ pathways. The Ca2+ pumps sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and plasma membrane Ca2+ ATPase 1 (PMCA1) control the resting basal Ca2+ levels.
    [Show full text]
  • Is Cep70, a Centrosomal Protein with New Roles in Breast Cancer Dissemination and Metastasis, a Facilitator of Epithelial-Mesenchymal Transition?
    Is Cep70, a centrosomal protein with new roles in breast cancer dissemination and metastasis, a facilitator of epithelial-mesenchymal transition? Pedro A. Lazo 1,2 1 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Salamanca, Spain 2 Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, Spain Running title: Cep70 and EMT Disclosures: None declared. Contact address: [email protected] 1 Introduction Microtubules are driving mechanisms of chromosomes, intracellular organelle movement, and cell shape and motility. Microtubules are organized on centrosomes, which are assembled in microtubule-organizing centers (MTOC). In mitosis there is no nuclear envelope and centromeres associated to microtubules are mainly involved in chromosome redistribution into daughter cells. In differentiated cells the microtubule- organizing centers are dispersed in the cytoplasm (non centrosomal (ncMTOC)) and interact with the minus end of microtubules through γ-tubulin. 1 However, it is not known if the microtubule contribution to tumor biology is only by facilitating tumor aneuploidy. During tumor dissemination, a process not linked to cell division, important changes take place in cell shape and motility. Microtubules are very dynamic because of their inherent structural instability, and this plasticity facilitates their reorganization during the epithelial-mesenchymal
    [Show full text]
  • Early Growth Response 1 Regulates Hematopoietic Support and Proliferation in Human Primary Bone Marrow Stromal Cells
    Hematopoiesis SUPPLEMENTARY APPENDIX Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells Hongzhe Li, 1,2 Hooi-Ching Lim, 1,2 Dimitra Zacharaki, 1,2 Xiaojie Xian, 2,3 Keane J.G. Kenswil, 4 Sandro Bräunig, 1,2 Marc H.G.P. Raaijmakers, 4 Niels-Bjarne Woods, 2,3 Jenny Hansson, 1,2 and Stefan Scheding 1,2,5 1Division of Molecular Hematology, Department of Laboratory Medicine, Lund University, Lund, Sweden; 2Lund Stem Cell Center, Depart - ment of Laboratory Medicine, Lund University, Lund, Sweden; 3Division of Molecular Medicine and Gene Therapy, Department of Labora - tory Medicine, Lund University, Lund, Sweden; 4Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands and 5Department of Hematology, Skåne University Hospital Lund, Skåne, Sweden ©2020 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2019.216648 Received: January 14, 2019. Accepted: July 19, 2019. Pre-published: August 1, 2019. Correspondence: STEFAN SCHEDING - [email protected] Li et al.: Supplemental data 1. Supplemental Materials and Methods BM-MNC isolation Bone marrow mononuclear cells (BM-MNC) from BM aspiration samples were isolated by density gradient centrifugation (LSM 1077 Lymphocyte, PAA, Pasching, Austria) either with or without prior incubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada) for lineage depletion (CD3, CD14, CD19, CD38, CD66b, glycophorin A). BM-MNCs from fetal long bones and adult hip bones were isolated as reported previously 1 by gently crushing bones (femora, tibiae, fibulae, humeri, radii and ulna) in PBS+0.5% FCS subsequent passing of the cell suspension through a 40-µm filter.
    [Show full text]