{"id":{"repo_id":"iastate","oai_identifier":"oai:dr.lib.iastate.edu:20.500.12876/2vaZ46Qr"},"canonical_url":"https://search.dev.ndltd.org/etd/iastate/oai:dr.lib.iastate.edu:20.500.12876/2vaZ46Qr","repository":{"repo_id":"iastate","name":"Iowa State University","base_url":"https://dr.lib.iastate.edu/server/oai/request"},"display":{"title":"Molecular and functional characterization of tubulin isotypes in budding yeast (Saccharomyces cerevisiae)","abstract":"Microtubules (MT) are dynamic cytoskeleton filaments essential for a wide range of cellular processes, including cell division, intracellular transport, axon formation, and cilia/flagella function. MTs are intrinsically dynamic polymers comprised of tubulin protein, which is a heterodimer of - and -subunits. It is proposed that one adaptation that allows MTs to achieve a wide range of functions is the expression of multiple tubulin isotypes. Indeed, most eukaryotes express multiple variants, or isotypes, of -and -tubulin that copolymerize to form individual MTs. Yet, how each isotype contributes to MT-dependent functions is extremely limited in any organism. The need for this knowledge is highlighted by the expanding set of mutations in specific human tubulin isotypes known to cause a wide range of fertility and neurological disorders, collectively referred to as tubulinopathies. This question is more tractable in simple model systems because, relative to humans, they encode fewer  and  tubulin variants, use less tubulin post-translational modifications, and are compatible with a range of genome-editing techniques. Yeast is an advantageous model for tubulin isotype study because, unlike higher eukaryotes, it uses just one tubulin (TUB2) and twotubulin isotypes (TUB1 and TUB3), without PTMs, to build MTs for distinct tasks. In this dissertation, I developed otherwise isogenic cells, expressing only one of the tubulin isotypes, termed Tub1-only and Tub3-only cells. This is the first study to generate a tub1 (null) haploid cell of budding yeast stably expressing Tub3 at levels comparable to total tubulin in wildtype. I show that Tub1- and Tub3-only cells are viable and express equivalent tubulin comparable to total wildtype levels. This controlled comparison allowed us to isolate the function of each isotype and provide compelling evidence for their specialized contribution to diverse MT-mediated functions. Previous studies suggested that the difference between the isotypes is simply quantitative, resulting from higher mRNA levels of TUB1 than TUB3. In contrast, our genetic and molecular characterization demonstrates that both isotypes normally contribute equal amounts to total transcripts and tubulin protein levels in wildtype cells. A significant strength of the yeast system is genetic tractability. Using genome-wide screening, we uncover diverse, opposing, and unexpected categories of genetic interactions between isotypes and demonstrate distinct roles in MT-mediated processes, particularly spindle positioning and effective spindle morphogenesis. The budding yeast mitotic spindle comprises three different MT populations that must be properly coordinated to ensure that the spindle is fully functional. We exploited the single isotype cells to show that tubulin isotypes influence the properties and function of specific MT classes during spindle positioning, spindle assembly, and spindle elongation at distinct stages of the cell cycle. Using functional assays, we find that relative to control cells harboring both isotypes, the effectiveness of spindle positioning and spindle function becomes enhanced or compromised in specific isotype cells in ways indicative of their specialized contribution to these processes. Furthermore, we demonstrate that one key mechanism underlying specialized isotype function is the differential localization of MAPs and regulatory proteins to microtubules — which correlates with the role of the isotype in each process. Collectively, our results dispel a long-standing paradigm that the two-budding yeast -tubulin isotypes are functionally interchangeable. Instead, we provide the first evidence for their unique contribution to spindle positioning, spindle function, and chromosome segregation through their differential interactions with MAPs, motors, and regulatory proteins. Overall, these findings provide novel mechanistic insights for understanding how tubulin isotypes in higher eukaryotes allow highly conserved microtubules to execute various and diverse cellular tasks.","abstract_html":"Microtubules (MT) are dynamic cytoskeleton filaments essential for a wide range of cellular processes, including cell division, intracellular transport, axon formation, and cilia/flagella function. MTs are intrinsically dynamic polymers comprised of tubulin protein, which is a heterodimer of - and -subunits. It is proposed that one adaptation that allows MTs to achieve a wide range of functions is the expression of multiple tubulin isotypes. Indeed, most eukaryotes express multiple variants, or isotypes, of -and -tubulin that copolymerize to form individual MTs. Yet, how each isotype contributes to MT-dependent functions is extremely limited in any organism. The need for this knowledge is highlighted by the expanding set of mutations in specific human tubulin isotypes known to cause a wide range of fertility and neurological disorders, collectively referred to as tubulinopathies. This question is more tractable in simple model systems because, relative to humans, they encode fewer  and  tubulin variants, use less tubulin post-translational modifications, and are compatible with a range of genome-editing techniques. Yeast is an advantageous model for tubulin isotype study because, unlike higher eukaryotes, it uses just one tubulin (TUB2) and twotubulin isotypes (TUB1 and TUB3), without PTMs, to build MTs for distinct tasks. In this dissertation, I developed otherwise isogenic cells, expressing only one of the tubulin isotypes, termed Tub1-only and Tub3-only cells. This is the first study to generate a tub1 (null) haploid cell of budding yeast stably expressing Tub3 at levels comparable to total tubulin in wildtype. I show that Tub1- and Tub3-only cells are viable and express equivalent tubulin comparable to total wildtype levels. This controlled comparison allowed us to isolate the function of each isotype and provide compelling evidence for their specialized contribution to diverse MT-mediated functions. Previous studies suggested that the difference between the isotypes is simply quantitative, resulting from higher mRNA levels of TUB1 than TUB3. In contrast, our genetic and molecular characterization demonstrates that both isotypes normally contribute equal amounts to total transcripts and tubulin protein levels in wildtype cells. A significant strength of the yeast system is genetic tractability. Using genome-wide screening, we uncover diverse, opposing, and unexpected categories of genetic interactions between isotypes and demonstrate distinct roles in MT-mediated processes, particularly spindle positioning and effective spindle morphogenesis. The budding yeast mitotic spindle comprises three different MT populations that must be properly coordinated to ensure that the spindle is fully functional. We exploited the single isotype cells to show that tubulin isotypes influence the properties and function of specific MT classes during spindle positioning, spindle assembly, and spindle elongation at distinct stages of the cell cycle. Using functional assays, we find that relative to control cells harboring both isotypes, the effectiveness of spindle positioning and spindle function becomes enhanced or compromised in specific isotype cells in ways indicative of their specialized contribution to these processes. Furthermore, we demonstrate that one key mechanism underlying specialized isotype function is the differential localization of MAPs and regulatory proteins to microtubules — which correlates with the role of the isotype in each process. Collectively, our results dispel a long-standing paradigm that the two-budding yeast -tubulin isotypes are functionally interchangeable. Instead, we provide the first evidence for their unique contribution to spindle positioning, spindle function, and chromosome segregation through their differential interactions with MAPs, motors, and regulatory proteins. Overall, these findings provide novel mechanistic insights for understanding how tubulin isotypes in higher eukaryotes allow highly conserved microtubules to execute various and diverse cellular tasks.","abstract_has_math":false,"creators":["Nsamba, Emmanuel"],"institution":"Iowa State University","degree_name":"Doctor of Philosophy","degree_level":"dissertation","degree_discipline":"Genetics","degree_department":"Department of Genetics, Development, and Cell Biology (LAS)","school":null,"contributors":[],"advisors":["Gupta, Mohan L.","Essner, Jeffrey J.","Johansen, Kristen M","Chen, Baoyu (Stone)","Schneider, Ian"],"committee_chairs":[],"committee_members":[],"year":2021,"date_issued":"2021-08","date_published":"2021-08","updated_at":"2026-07-24T02:38:23Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.31274/td-20240329-517"],"render_values":[{"text":"https://doi.org/10.31274/td-20240329-517","href":"https://doi.org/10.31274/td-20240329-517","code":true}]}]},"links":{"outbound_url":"https://dr.lib.iastate.edu/handle/20.500.12876/2vaZ46Qr","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Gupta, Mohan L.","Essner, Jeffrey J.","Johansen, Kristen M","Chen, Baoyu (Stone)","Schneider, Ian"]},{"key":"dc:contributor.department","label":"Department","values":["Department of Genetics, Development, and Cell Biology (LAS)"]},{"key":"dc:creator","label":"Author","values":["Nsamba, Emmanuel"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2022-11-09T02:27:56Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2022-11-09T02:27:56Z"]},{"key":"dc:date.issued","label":"Date","values":["2021-08"]},{"key":"dc:type","label":"Dc Type","values":["dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Genetics","Cellular biology","Molecular biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Iowa State University"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.31274/td-20240329-517"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://dr.lib.iastate.edu/handle/20.500.12876/2vaZ46Qr"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Microtubules (MT) are dynamic cytoskeleton filaments essential for a wide range of cellular processes, including cell division, intracellular transport, axon formation, and cilia/flagella function. MTs are intrinsically dynamic polymers comprised of tubulin protein, which is a heterodimer of - and -subunits. It is proposed that one adaptation that allows MTs to achieve a wide range of functions is the expression of multiple tubulin isotypes. Indeed, most eukaryotes express multiple variants, or isotypes, of -and -tubulin that copolymerize to form individual MTs. Yet, how each isotype contributes to MT-dependent functions is extremely limited in any organism. The need for this knowledge is highlighted by the expanding set of mutations in specific human tubulin isotypes known to cause a wide range of fertility and neurological disorders, collectively referred to as tubulinopathies. This question is more tractable in simple model systems because, relative to humans, they encode fewer  and  tubulin variants, use less tubulin post-translational modifications, and are compatible with a range of genome-editing techniques. Yeast is an advantageous model for tubulin isotype study because, unlike higher eukaryotes, it uses just one tubulin (TUB2) and twotubulin isotypes (TUB1 and TUB3), without PTMs, to build MTs for distinct tasks. In this dissertation, I developed otherwise isogenic cells, expressing only one of the tubulin isotypes, termed Tub1-only and Tub3-only cells. This is the first study to generate a tub1 (null) haploid cell of budding yeast stably expressing Tub3 at levels comparable to total tubulin in wildtype. I show that Tub1- and Tub3-only cells are viable and express equivalent tubulin comparable to total wildtype levels. This controlled comparison allowed us to isolate the function of each isotype and provide compelling evidence for their specialized contribution to diverse MT-mediated functions. Previous studies suggested that the difference between the isotypes is simply quantitative, resulting from higher mRNA levels of TUB1 than TUB3. In contrast, our genetic and molecular characterization demonstrates that both isotypes normally contribute equal amounts to total transcripts and tubulin protein levels in wildtype cells. A significant strength of the yeast system is genetic tractability. Using genome-wide screening, we uncover diverse, opposing, and unexpected categories of genetic interactions between isotypes and demonstrate distinct roles in MT-mediated processes, particularly spindle positioning and effective spindle morphogenesis. The budding yeast mitotic spindle comprises three different MT populations that must be properly coordinated to ensure that the spindle is fully functional. We exploited the single isotype cells to show that tubulin isotypes influence the properties and function of specific MT classes during spindle positioning, spindle assembly, and spindle elongation at distinct stages of the cell cycle. Using functional assays, we find that relative to control cells harboring both isotypes, the effectiveness of spindle positioning and spindle function becomes enhanced or compromised in specific isotype cells in ways indicative of their specialized contribution to these processes. Furthermore, we demonstrate that one key mechanism underlying specialized isotype function is the differential localization of MAPs and regulatory proteins to microtubules — which correlates with the role of the isotype in each process. Collectively, our results dispel a long-standing paradigm that the two-budding yeast -tubulin isotypes are functionally interchangeable. Instead, we provide the first evidence for their unique contribution to spindle positioning, spindle function, and chromosome segregation through their differential interactions with MAPs, motors, and regulatory proteins. Overall, these findings provide novel mechanistic insights for understanding how tubulin isotypes in higher eukaryotes allow highly conserved microtubules to execute various and diverse cellular tasks."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["PDF"]},{"key":"dc:title","label":"Title","values":["Molecular and functional characterization of tubulin isotypes in budding yeast (Saccharomyces cerevisiae)"]}]}],"canonical_facts":{"dc:contributor.advisor":["Gupta, Mohan L.","Essner, Jeffrey J.","Johansen, Kristen M","Chen, Baoyu (Stone)","Schneider, Ian"],"dc:contributor.department":["Department of Genetics, Development, and Cell Biology (LAS)"],"dc:creator":["Nsamba, Emmanuel"],"dc:date.accessioned":["2022-11-09T02:27:56Z"],"dc:date.available":["2022-11-09T02:27:56Z"],"dc:date.issued":["2021-08"],"dc:description.abstract":["Microtubules (MT) are dynamic cytoskeleton filaments essential for a wide range of cellular processes, including cell division, intracellular transport, axon formation, and cilia/flagella function. MTs are intrinsically dynamic polymers comprised of tubulin protein, which is a heterodimer of - and -subunits. It is proposed that one adaptation that allows MTs to achieve a wide range of functions is the expression of multiple tubulin isotypes. Indeed, most eukaryotes express multiple variants, or isotypes, of -and -tubulin that copolymerize to form individual MTs. Yet, how each isotype contributes to MT-dependent functions is extremely limited in any organism. The need for this knowledge is highlighted by the expanding set of mutations in specific human tubulin isotypes known to cause a wide range of fertility and neurological disorders, collectively referred to as tubulinopathies. This question is more tractable in simple model systems because, relative to humans, they encode fewer  and  tubulin variants, use less tubulin post-translational modifications, and are compatible with a range of genome-editing techniques. Yeast is an advantageous model for tubulin isotype study because, unlike higher eukaryotes, it uses just one tubulin (TUB2) and twotubulin isotypes (TUB1 and TUB3), without PTMs, to build MTs for distinct tasks. In this dissertation, I developed otherwise isogenic cells, expressing only one of the tubulin isotypes, termed Tub1-only and Tub3-only cells. This is the first study to generate a tub1 (null) haploid cell of budding yeast stably expressing Tub3 at levels comparable to total tubulin in wildtype. I show that Tub1- and Tub3-only cells are viable and express equivalent tubulin comparable to total wildtype levels. This controlled comparison allowed us to isolate the function of each isotype and provide compelling evidence for their specialized contribution to diverse MT-mediated functions. Previous studies suggested that the difference between the isotypes is simply quantitative, resulting from higher mRNA levels of TUB1 than TUB3. In contrast, our genetic and molecular characterization demonstrates that both isotypes normally contribute equal amounts to total transcripts and tubulin protein levels in wildtype cells. A significant strength of the yeast system is genetic tractability. Using genome-wide screening, we uncover diverse, opposing, and unexpected categories of genetic interactions between isotypes and demonstrate distinct roles in MT-mediated processes, particularly spindle positioning and effective spindle morphogenesis. The budding yeast mitotic spindle comprises three different MT populations that must be properly coordinated to ensure that the spindle is fully functional. We exploited the single isotype cells to show that tubulin isotypes influence the properties and function of specific MT classes during spindle positioning, spindle assembly, and spindle elongation at distinct stages of the cell cycle. Using functional assays, we find that relative to control cells harboring both isotypes, the effectiveness of spindle positioning and spindle function becomes enhanced or compromised in specific isotype cells in ways indicative of their specialized contribution to these processes. Furthermore, we demonstrate that one key mechanism underlying specialized isotype function is the differential localization of MAPs and regulatory proteins to microtubules — which correlates with the role of the isotype in each process. Collectively, our results dispel a long-standing paradigm that the two-budding yeast -tubulin isotypes are functionally interchangeable. Instead, we provide the first evidence for their unique contribution to spindle positioning, spindle function, and chromosome segregation through their differential interactions with MAPs, motors, and regulatory proteins. Overall, these findings provide novel mechanistic insights for understanding how tubulin isotypes in higher eukaryotes allow highly conserved microtubules to execute various and diverse cellular tasks."],"dc:format.mimetype":["PDF"],"dc:identifier.doi":["https://doi.org/10.31274/td-20240329-517"],"dc:identifier.uri":["https://dr.lib.iastate.edu/handle/20.500.12876/2vaZ46Qr"],"dc:language.iso":["en"],"dc:title":["Molecular and functional characterization of tubulin isotypes in budding yeast (Saccharomyces cerevisiae)"],"dc:type":["dissertation"],"thesis:degree_discipline":["Genetics","Cellular biology","Molecular biology"],"thesis:degree_level":["dissertation"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Iowa State University"]},"updated_at":"2026-07-24T02:38:23Z"}