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The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2018 Nanoscale Organization of the Small GTPase Rac1 Kelsey Maxwell Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biochemistry Commons, Biophysics Commons, Medicine and Health Sciences Commons, and the Molecular Biology Commons Recommended Citation Maxwell, Kelsey, "Nanoscale Organization of the Small GTPase Rac1" (2018). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 851. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/851 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. NANOSCALE ORGANIZATION OF THE SMALL GTPASE RAC1 Approval Page by Kelsey Maxwell, B.S. APPROVED: _______________________________ John F. Hancock, M.A., M.B., B.Chir., Ph.D. Advisory ProFessor _______________________________ JeFFrey A. Frost, Ph.D. _______________________________ Alemayehu A. Gorfe, Ph.D. _______________________________ Kartik Venkatachalam, Ph.D. _______________________________ Darren F. Boehning, Ph.D. APPROVED: _______________________________ Dean, The University oF Texas MD AnDerson Cancer Center UTHealth GraDuate School oF BiomeDical Sciences NANOSCALE ORGANIZATION OF THE SMALL GTPASE RAC1 A DISSERTATION PresenteD to the Faculty oF The University oF Texas MD AnDerson Cancer Center UTHealth GraDuate School oF BiomeDical Sciences in Partial FulFillment oF the Requirements For the Degree oF DOCTOR OF PHILOSOPHY by Kelsey Nicole Maxwell, B.S. Houston, TX May 2018 Title Page Dedication To Matty, Mom, DaD, AmanDa anD William iii Acknowledgements I woulD like to thank my aDvisor, Dr. John Hancock, For his continueD guiDance anD support through my training. There are many challenges on the roaD toward a PhD— FaileD experiments, DeFining positive anD negative controls, and coming up with new ideas—to name a Few. I am a better scientist because oF his leaDership through these challenges anD more. I woulD also like to speciFically acknowleDge two others in our lab who have proviDeD many hours oF guiDance. First, Dr. Yong Zhou, who has been instrumental to the Development oF this project. Dr. Zhou has given many hours to brainstorming sessions, answering enDless questions, anD eDiting over the last six years. SeconD, Dr. Jin Cho, For not only helping me with technical aspects For possibly every experiment I ever laiD hanDs on but, more importantly, For simply being an encouraging FrienD. I woulD like to speciFically thank the members oF my aDvisory committee: Dr. Alemayehu Gorfe, Dr. JeFF Frost, Dr. Kartik Venkatachalam, Dr. John Putkey, anD Dr. Darren Boehning: thank you For caring about me, this project, anD For all oF your creative iDeas that maDe this work what it is toDay. Thank you also to the Faculty anD staFF oF the Department oF Integrative Biology anD Pharmacology For their open Doors, training, anD support. To Xiaoping Ma, Wei Chen, anD Hong Liang, thank you For helping me with so many experiments, always encouraging me, anD For all the canDies. The lab woulD Fall apart without you. To the University oF Texas MD AnDerson UTHealth GraDuate School oF BiomeDical Sciences Faculty, staFF, anD Deans, Dr. Michael Blackburn anD Dr. Michelle Barton: thank you For all that you Do For our school anD stuDents. I woulD also like to thank iv the Rosalie B. Hite GraDuate Fellowship in Cancer Research anD the Hite Fellowship Committee For FunDing me these past three years, anD Mrs. Roquemore For her generous contributions to GSBS through the FaDine Jackson Roquemore Scholarship in Cancer Research that has also proviDeD me with generous Financial support. Without the FrienDship oF my Fellow stuDents anD colleagues my time in Houston woulD have been very lonely. To Dr. RanDi Fitzgibbon, Dr. Dhananjay Thakur, Brittany Jewell, Mimi Le, Tanya BalDwin, Ashabari Mukherjee, Janani Subramanian, Rachel Tay, Sam Berkey, Courtney Olsen, Stephanie Bsaibes, anD Stephanie Tabor: thank you For all oF the laughter, venting sessions, soliDarity, lunch Dates, Ruben puppy-sitting, and motivation. I am lucky to have such Driven anD inspiring FrienDs! Most importantly, I woulD like to thank my Family. To my niece, Evelyn Milam, who was born During the writing oF this Dissertation –you’re only six ten weeks olD but you’re a great motivator! To my siblings, AmanDa Milam and William Maxwell, thank you For being my best FrienDs anD the amazing people that you are. To my brother-in-law Jason Milam, who actually reaD this entire thesis anD proviDeD eDits: thank you For Doing that! To my aunts, uncles, granDDaDDy Ervin Englert, anD granDma Marie Maxwell: thank you For always encouraging me to work hard anD believing that I have the capacity –and stubbornness– to achieve my goals. To my granD-mama GlaDys Englert anD granDpa Bill Maxwell, who unFortunately never got to see me enter graDuate school but were always supportive oF my eDucation: we miss you. To my in-laws: For loving me anD helping me along the way. To my parents: For their unconDitional love anD countless sacriFices For our eDucation, happiness, anD well-being, your eFForts are so appreciateD. To my liFe partner, Matt Acosta: thank you For your patience anD your enDless well oF encouragement. I love you! v NANOSCALE ORGANIZATION OF THE SMALL GTPASE RAC1 Abstract Kelsey Maxwell, B.S. Advisory ProFessor: John F. Hancock, M.A., M.B., B.Chir., Ph.D. Rac1 is a small, guanine-nucleotiDe binDing protein that cycles between an inactive GDP-bounD anD active GTP-bounD state to regulate actin-meDiateD motility, migration, anD aDhesion. Plasma membrane (PM) localization is essential For its biological activity. Rac1 PM targeting is DirecteD by a C-terminal membrane anchor that encompasses a geranylgeranyl-cysteine-methyl-ester, palmitoyl, anD a polybasic Domain (PBD) oF contiguous lysine anD arginine resiDues. Using high-resolution imaging combineD with spatial mapping analysis, I found that Rac1 Forms nanoclusters on the PM. Cycling between the GTP- and GDP-bounD states, Rac1 Forms nanoclusters that are non-overlapping, consequently unDergoing guanine nucleotiDe-dependent spatial segregation. I Further FounD that Rac1 selectively associates with phosphatiDic aciD (PA) anD phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enricheD in these anionic lipiDs. I FounD these lipiDs to be structurally important as Depleting the PM oF PA or PIP3 impaireD both Rac1 PM targeting anD nanoclustering. LipiD binDing speciFicity oF Rac1 is encoDeD in the C-terminal PBD amino aciD sequence in combination with the aDjacent lipiD anchors. Point mutations within the PBD, incluDing highly conserveD arginine to lysine substitutions or mutations exchanging the geranylgeranyl For Farnesyl, proFounDly altered Rac1 lipiD binDing speciFicity without changing electrostatics oF the protein anD resulted in impaireD macropinocytosis anD DecreaseD cell spreaDing. In this thesis, I proposeD that Rac1 nanoclusters act as lipiD-baseD signaling platForms emulating the spatiotemporal organization oF Ras proteins anD Further vi showed that the Rac1 PBD-prenyl anchor has a biological Function that extenDs beyonD simple electrostatic engagement with the PM. vii Table of Contents Approval Page ................................................................................................................ iii Title Page ....................................................................................................................... iv DeDication ...................................................................................................................... iii AcknowleDgements ........................................................................................................ iv Abstract .......................................................................................................................... vi List oF Illustrations .......................................................................................................... xii List oF Tables ................................................................................................................ xiv Abbreviations ................................................................................................................. xv Chapter 1 ......................................................................................................................... 1 IntroDuction ...................................................................................................................... 1 1.1 Membrane Domains anD plasma membrane complexity ....................................... 2 1.2 Signaling complexes .............................................................................................. 7 1.2.1 Ras nanoclusters ...........................................................................................
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  • PAR6 (PARD6A) (NM 016948) Human Tagged ORF Clone Product Data

    PAR6 (PARD6A) (NM 016948) Human Tagged ORF Clone Product Data

    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC221092L3 PAR6 (PARD6A) (NM_016948) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: PAR6 (PARD6A) (NM_016948) Human Tagged ORF Clone Tag: Myc-DDK Symbol: PARD6A Synonyms: PAR-6A; PAR6; PAR6alpha; PAR6C; TAX40; TIP-40 Vector: pLenti-C-Myc-DDK-P2A-Puro (PS100092) E. coli Selection: Chloramphenicol (34 ug/mL) Cell Selection: Puromycin ORF Nucleotide The ORF insert of this clone is exactly the same as(RC221092). Sequence: Restriction Sites: SgfI-MluI Cloning Scheme: ACCN: NM_016948 ORF Size: 1038 bp This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 PAR6 (PARD6A) (NM_016948) Human Tagged ORF Clone – RC221092L3 OTI Disclaimer: The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. More info OTI Annotation: This clone was engineered to express the complete ORF with an expression tag. Expression varies depending on the nature of the gene. RefSeq: NM_016948.1 RefSeq Size: 1269 bp RefSeq ORF: 1041 bp Locus ID: 50855 UniProt ID: Q9NPB6 Protein Families: Druggable Genome, Transcription Factors Protein Pathways: Endocytosis, Tight junction MW: 37.2 kDa Gene Summary: This gene is a member of the PAR6 family and encodes a protein with a PSD95/Discs- large/ZO1 (PDZ) domain and a semi-Cdc42/Rac interactive binding (CRIB) domain.