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Uncovering the Determinants of Compositionally Distinct, Coexisting Biomolecular Condensates- [electronic resource]
Uncovering the Determinants of Compositionally Distinct, Coexisting Biomolecular Condensates- [electronic resource]
상세정보
- 자료유형
- 학위논문파일 국외
- 최종처리일시
- 20240214101923
- ISBN
- 9798380844109
- DDC
- 574
- 저자명
- Lin, Andrew Z.
- 서명/저자
- Uncovering the Determinants of Compositionally Distinct, Coexisting Biomolecular Condensates - [electronic resource]
- 발행사항
- [S.l.]: : Washington University in St. Louis., 2023
- 발행사항
- Ann Arbor : : ProQuest Dissertations & Theses,, 2023
- 형태사항
- 1 online resource(200 p.)
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-05, Section: B.
- 주기사항
- Advisor: Pappu, Rohit V.
- 학위논문주기
- Thesis (Ph.D.)--Washington University in St. Louis, 2023.
- 사용제한주기
- This item must not be sold to any third party vendors.
- 초록/해제
- 요약Biomolecular condensates form via the phase transitions of proteins and nucleic acids. What is unclear is how unique condensates achieve compositional specificity. Of particular interest is the ability of distinct condensates to have shared and distinct components. This is readily apparent in the multinucleate filamentous fungi Ashbya gossypii, where condensates share a common cytoplasm. In Ashbya, the RNA-binding protein Whi3 forms condensates with CLN3, BNI1, and SPA2. These RNAs are responsible for initiating the cell cycle, forming actin cables, and directing polarized cell growth respectively. However in vivo observations have shown that certain Whi3-RNA condensates are capable of mixing while others remain distinct, despite Whi3 being a common component of each condensate; Whi3-BNI1 condensates colocalize with Whi3- SPA2 condensates, whereas Whi3-CLN3 condensates do not colocalize with Whi3-BNI1 or Whi3- SPA2 condensates.Here I present findings that demonstrate how dynamical control establishes demixed and mixed condensates in vitro as well as in live Ashbya cells. We demonstrate in vitro how the order of operations with respect to RNA addition can engender mixed or demixed condensates, regardless of RNA identity. Analyses of Whi3-RNA phase boundaries reveal Whi3-RNA heterotypic interactions driving condensate formation, with temporal control of RNAs and RNA-RNA homotypic interactions contributing to the formation of demixed condensates in ternary mixtures. In a facsimile of our in vitro temporal control schema, we also demonstrate in vivo how perturbing wildtype expression patterns of RNAs that normally do not colocalize is sufficient for generating mixed condensates. We also observe measurable defects in fungal morphology, suggesting that dynamical control is necessary for proper cell growth.Over the course of this dissertation, I have developed a robust pipeline for the routine expression and fluorescent tagging of CLN3, BNI1, and SPA2 which cover a wide range of lengths and sequence complexity. The methods we have developed are applicable to other biologically-relevant RNAs and will be a useful addition to any RNA biochemistry toolbox. Finally, we demonstrate how the composition and patterning of the Whi3 glutamine-rich region is capable of modulating homotypic and heterotypic phase separation with BNI1 RNA. Collectively, our findings demonstrate how dynamical control is a likely mechanism for the demixing of condensates in vitro and in vivo, and how the composition and patterning of polar residues can affect homotypic and heterotypic phase behavior.
- 일반주제명
- Biochemistry.
- 일반주제명
- Biophysics.
- 일반주제명
- Molecular biology.
- 일반주제명
- Genetics.
- 키워드
- Ashbya gossypii
- 키워드
- Proteins
- 키워드
- Nucleic acids
- 기타저자
- Washington University in St. Louis Biology & Biomedical Sciences (Plant & Microbial Biosciences)
- 기본자료저록
- Dissertations Abstracts International. 85-05B.
- 기본자료저록
- Dissertation Abstract International
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520240214101923
■006m o d
■007cr#unu||||||||
■020 ▼a9798380844109
■035 ▼a(MiAaPQ)AAI30691441
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a574
■1001 ▼aLin, Andrew Z.▼0(orcid)0000-0002-7877-6285
■24510▼aUncovering the Determinants of Compositionally Distinct, Coexisting Biomolecular Condensates▼h[electronic resource]
■260 ▼a[S.l.]:▼bWashington University in St. Louis. ▼c2023
■260 1▼aAnn Arbor :▼bProQuest Dissertations & Theses, ▼c2023
■300 ▼a1 online resource(200 p.)
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-05, Section: B.
■500 ▼aAdvisor: Pappu, Rohit V.
■5021 ▼aThesis (Ph.D.)--Washington University in St. Louis, 2023.
■506 ▼aThis item must not be sold to any third party vendors.
■520 ▼aBiomolecular condensates form via the phase transitions of proteins and nucleic acids. What is unclear is how unique condensates achieve compositional specificity. Of particular interest is the ability of distinct condensates to have shared and distinct components. This is readily apparent in the multinucleate filamentous fungi Ashbya gossypii, where condensates share a common cytoplasm. In Ashbya, the RNA-binding protein Whi3 forms condensates with CLN3, BNI1, and SPA2. These RNAs are responsible for initiating the cell cycle, forming actin cables, and directing polarized cell growth respectively. However in vivo observations have shown that certain Whi3-RNA condensates are capable of mixing while others remain distinct, despite Whi3 being a common component of each condensate; Whi3-BNI1 condensates colocalize with Whi3- SPA2 condensates, whereas Whi3-CLN3 condensates do not colocalize with Whi3-BNI1 or Whi3- SPA2 condensates.Here I present findings that demonstrate how dynamical control establishes demixed and mixed condensates in vitro as well as in live Ashbya cells. We demonstrate in vitro how the order of operations with respect to RNA addition can engender mixed or demixed condensates, regardless of RNA identity. Analyses of Whi3-RNA phase boundaries reveal Whi3-RNA heterotypic interactions driving condensate formation, with temporal control of RNAs and RNA-RNA homotypic interactions contributing to the formation of demixed condensates in ternary mixtures. In a facsimile of our in vitro temporal control schema, we also demonstrate in vivo how perturbing wildtype expression patterns of RNAs that normally do not colocalize is sufficient for generating mixed condensates. We also observe measurable defects in fungal morphology, suggesting that dynamical control is necessary for proper cell growth.Over the course of this dissertation, I have developed a robust pipeline for the routine expression and fluorescent tagging of CLN3, BNI1, and SPA2 which cover a wide range of lengths and sequence complexity. The methods we have developed are applicable to other biologically-relevant RNAs and will be a useful addition to any RNA biochemistry toolbox. Finally, we demonstrate how the composition and patterning of the Whi3 glutamine-rich region is capable of modulating homotypic and heterotypic phase separation with BNI1 RNA. Collectively, our findings demonstrate how dynamical control is a likely mechanism for the demixing of condensates in vitro and in vivo, and how the composition and patterning of polar residues can affect homotypic and heterotypic phase behavior.
■590 ▼aSchool code: 0252.
■650 4▼aBiochemistry.
■650 4▼aBiophysics.
■650 4▼aMolecular biology.
■650 4▼aGenetics.
■653 ▼aBiomolecular condensates
■653 ▼aAshbya gossypii
■653 ▼aProteins
■653 ▼aNucleic acids
■653 ▼aFungal morphology
■690 ▼a0487
■690 ▼a0786
■690 ▼a0369
■690 ▼a0307
■71020▼aWashington University in St. Louis▼bBiology & Biomedical Sciences (Plant & Microbial Biosciences).
■7730 ▼tDissertations Abstracts International▼g85-05B.
■773 ▼tDissertation Abstract International
■790 ▼a0252
■791 ▼aPh.D.
■792 ▼a2023
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T16935359▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
■980 ▼a202402▼f2024
