Reticulon variation and correlation to Nuclear Size Regulation

Slides:

Advertisements

Similar presentations

Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry

Advertisements

Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly By Rasala et al. Presented by Laurent Palmatier BGGN 222.

Figure 5 Sample mobile shift assay gel. The 3 rd lane from the left shows a significant “shift” in the protein location, representing the proteins which.

LABORATORY 6 PART B PURIFICATION OF M FP FROM AN OVERNIGHT CULTURE.

Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2008 Jessian Resto 1 ; Kristen Johansen 2 ; Jorgen.

Introduction Determination of Chickens as a Useful Model for Antibody Production to Green Fluorescent Protein and a Tomato Proteinase Inhibitor II Peptide.

Vaccinia Virus G1L Protein Expression and Purification HHMI Summer Undergraduate Research Program 2004 Laboratory of Dr. Dennis Hruby Oregon State University.

Principles of purification of macromolecules Genetics 222 Method and Logic in Experimental Genetics Reference Protein Purification: Principles and Practice.

A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum By: Dorothee van Breevoort Panos Athanasopoulos Nika Strokappe.

Modifications of the Catalytic Properties of Butane Monooxygenase in P. butanovora Sarah Jean Mentors: Dr. Daniel J. Arp Dr. Luis Sayavedra-Soto Kim Halsey.

Isolation and Characterization of Cellular Complexes Containing the Histone Deacetylase SIRT1 Vincent Lew Howard Hughes Medical Institute Fellowship Mentor:

Supervisor: Dr. David Wishart

Complexity of Signaling Networks Old Protein, New Tricks Biplab Bose.

Undergraduate Research in Cancer Biology Mahima Venkatesh Mentor-Charmaine Ramlogan-Steel, MD (Postdoctoral Fellow) Principal Investigator- George Atweh,

Regulation of Contractile Forces within Cells Avi Kandel, Ryan Frei, Ken Prehoda Department of Chemistry, Institute of Molecular Biology, University of.

AP Bio Lab 8: Transformation We will start on TUESDAY!

Generation of Transcription Factor Constructs for Mammalian Transfection Leah Schumerth, Michael Farrell, and Winnifred Bryant Ph. D. Department of Biology.

Manufacture of Human Interleukin 13 Protein Using a Prokaryotic Expression System Ryan Rupp, York College of Pennsylvania, Department of Biological Sciences.

Preparing an Overnight Culture of E. coli Possible Problems When there is a mixed culture of red and white (nonexpressing) cells, the white cells.

Expression, Purification and Isolation of the MinE protein By Arsalan Wasim and Nicholas Wong.

Chapter 20 Experimental Systems Dr. Capers.  In vivo ○ Involve whole animal  In vitro ○ Defined populations of immune cells are studied under controlled.

Quantification and DNA Sequencing of IL-13Rα1 and IL-13Rα2 On Various Cancer Cell Lines Erin Dolac, Department of.

Overview Amgen Biotech Labs In this set of labs, students will:

A Study of Starch Metabolizing Proteins Pam Brewer-Michael 1, Tracie Hennen-Bierwagen 2, Myers /James Laboratory 2 1 Marshalltown Community School District,

Optimizing Purification and Quantitative Detection of Lysozyme and Avidin Tatiana Soboleva, Ryan Colakovic, Dr. Theresa Salerno (Faculty Mentor)

The effect of salts and chaperone proteins on Alkaline Phosphatase stability Aka: the results of 271 continuous enzyme activity assays Mary Klein Dhruve.

Taylor Bendt Faculty advisor: Dr. Gary Merrill. DNA Damage p53 DNA repairApoptosisp21 Cell cycle arrest Genome maintenance  Important for cancer prevention.

MOLECULAR BIOLOGY IN ACTION In this project, students will use what they have learned in the previous courses to complete a larger multi-step molecular.

RASSF2DSARAH-GF (b) MST1-F (a) M F G 70 kD a 55 kD b 36 kD

Amino Acids, Peptides, and Proteins

Extraction and Determination of Bacterial Proteins.

hSSB1 Bacterial Transformation and Expression

Fac. of Agriculture, Assiut Univ.

Volume 35, Issue 4, Pages (August 2009)

Volume 27, Issue 1, Pages (October 2013)

Volume 66, Issue 4, Pages (October 2004)

Purusharth Rajyaguru, Meipei She, Roy Parker Molecular Cell

Volume 14, Issue 9, Pages (March 2016)

Cdk2 Kinase Is Required for Entry into Mitosis as a Positive Regulator of Cdc2–Cyclin B Kinase Activity Thomas M Guadagno, John W Newport Cell Volume.

Nicolas Charlet-B, Gopal Singh, Thomas A. Cooper Molecular Cell

Natalie Galles S. N. Bose Research Exchange Summer 2016

Week 1: Tutorial Outline

1st LAB MEETING 김상범.

Volume 16, Issue 6, Pages (June 2009)

Lipids up-regulate uncoupling protein 2 expression in rat hepatocytes

Dimers Probe the Assembly Status of Multimeric Membrane Proteins

Yingqun Huang, Renata Gattoni, James Stévenin, Joan A. Steitz

Protein Targeting: ER Leads the Way to the Inner Nuclear Envelope

Volume 1, Issue 7, Pages (June 1998)

The Spinal Muscular Atrophy Disease Gene Product, SMN, and Its Associated Protein SIP1 Are in a Complex with Spliceosomal snRNP Proteins Qing Liu, Utz.

Volume 17, Issue 6, Pages (December 1996)

Nuclear Size Is Regulated by Importin α and Ntf2 in Xenopus

NAD+-Dependent Modulation of Chromatin Structure and Transcription by Nucleosome Binding Properties of PARP-1 Mi Young Kim, Steven Mauro, Nicolas Gévry,

Volume 23, Issue 8, Pages (May 2018)

Volume 113, Issue 2, Pages (April 2003)

Volume 31, Issue 3, Pages (November 2014)

Volume 17, Issue 7, Pages (November 2016)

Volume 3, Issue 2, Pages (August 2002)

Andrei Kuzmichev, Thomas Jenuwein, Paul Tempst, Danny Reinberg

Mitogen- and Stress-Activated Protein Kinase 2 and Cyclic AMP Response Element Binding Protein are Activated in Lesional Psoriatic Epidermis Anne T.

Ruth Halaban, Elaine Cheng Journal of Investigative Dermatology

Volume 139, Issue 4, Pages (November 2009)

Purified proteins used for in vitro assay.

Volume 3, Issue 2, Pages (August 2002)

RECQL4 is a MAP with a spindle function.

Vidhya Ramachandran, Khyati H. Shah, Paul K. Herman Molecular Cell

The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3 by Jason Hatakeyama, Jessica H. Wald, Hanine.

Characterization of the complex formed at the Fra-1 RCE1.

Volume 26, Issue 19, Pages (October 2016)

Dual Toxic-Peptide-Coding Staphylococcus aureus RNA under Antisense Regulation Targets Host Cells and Bacterial Rivals Unequally Marie-Laure Pinel-Marie,

Presentation transcript:

Reticulon variation and correlation to Nuclear Size Regulation Amanda Johnson INBRE Undergraduate Research Summer Fellow Levy Lab University of Wyoming

Background:

Model Organism: xENOPUS LAEVIS Formation of a nuclear envelope within egg extracts following the addition of Xenopus sperm. Google images

Reticulons: Set of proteins exclusively in the ER tubules Contribute to ER bending and shaping Shift the balance from flat membrane sheets towards tubular network ER sheet Reticulons ER tubules

Reticulon Isoforms

RESEARCH objective: To evaluate the effective properties of various reticulon proteins on nuclear size regulation. To prompt further investigation on this area of research How altering the concentrations of these proteins might effect nuclear size in: embryonic development cancer progression

Experimental outline: Transformation of chemically competent E. coli BL21 cells to introduce the following plasmids with their specific reticulon proteins and isoforms: pET-30b Rtn4b (Kan resistance) pKW 197 Rtn4a #6 pKW 197 Rtn4a #8 (Amp resistance) pKW 197 Rtn4a #10

Experimental Outline: Overnight expression cultures (using IPTG) grown for each of the bacterial cultures 16° C 25° C Eliminated background on Western blots 37° C Gel electrophoresis and Western blotting to determine the presence and purity of the proteins Rtn4a size is around 130-135 kD Rtn4b size is around 39-45 kD

eXPERIMENTAL ouTLINE: Protein purification using His-binding resin Dialysis to further purify and concentrate desired reticulon proteins Quantification of protein (Rtn4b and Rtn4a #8) concentrations Image J Xenopus egg extract preparation

Experimental outline: Nuclear assembly in vitro using Xenopus laevis egg extract Addition of Rtn4b or Rtn4a #8 at varying concentrations Nuclear Immunofluorescence 1° antibody binds nuclear pore complexes 2° anti-mouse antibody binds 1° antibody Nuclear quantification Metamorph NPC

Quantifying rtn concentration using image j BSA standards Rtn 4a #8 pellet Rtn4a #8 supe Rtn4b supe Gel used to quantify concentrations Post-dialysis Rtn4a #8: 130kD (w/out tag) Rtn4b: 46kD (w/out tag) 250 150 100 75 50 37 25 20 15 10

QuantifyinG rTN Concentration using imagej Rtn4b: 0.1 mg/ml Rtn 4a #8: 0.003 mg/ml Molarity: Rtn4b: ~2uM Rtn4a #8: ~20nM

Rtn OVEREXPRESSION dECREASES NUCLEAR SIZE Total area of nuclei (um2) CTRL 50- 4b 100- 4b 200- 4b 2- 4a Concentration of reticulon protein (nM)

CONCLUSIOn: The overexpression of reticulons (either Rtn4a or Rtn4b) in the cell has a direct effect on nuclear size. The increase in reticulon proteins causes a decrease in nuclear size because of the decreased ER sheet availability.

Acknowledgments: Dan Levy, PI Karen White, lab manager Predrag Jevtic, PhD student Lisa Edens, PhD student Lidija Vukovic, PhD student