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Cytoskeletal filament systems: assembly, regulation, and interplay in mammalian cells
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Martin Gullberg)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell represents the basic unit of structure and function for all life. The interior of a eukaryotic cell is organized by an extensive array of protein filaments – collectively referred to as the cytoskeleton. These filaments serve diverse essential functions, e.g. to provide mechanical resilience, facilitate intracellular transport, and enable cell polarization, locomotion and division. Here I have explored the mechanisms that regulate synthesis and assembly of two cytoskeletal filament systems – microtubules and septins – and how these interact in human cells. The present thesis is based on three principal discoveries. Firstly, we have found that the microtubule-destabilizing protein Op18/Stathmin also regulates synthesis of tubulin heterodimers, which are the building blocks for microtubules. Secondly, we have unraveled the general rules that govern assembly of mammalian septins into native polymerization-competent heterooligomers. Finally, our combined results point to a non-reciprocal interplay whereby interphase microtubules support a disc-like arrangement of septin filaments, which delineate static plasma membrane regions. I here discuss the physiological significance and implications of these findings.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, Institutionen för molekylärbiologi , 2011. , 68 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1450
National Category
Cell and Molecular Biology
Research subject
cellforskning
Identifiers
URN: urn:nbn:se:umu:diva-48508ISBN: 978-91-7459-301-3 (print)OAI: oai:DiVA.org:umu-48508DiVA: diva2:450376
Public defence
2011-11-11, Major Groove, Institutionen för Molekylärbiologi, Försörjningsvägen, Byggnad 6L, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-10-21 Created: 2011-10-20 Last updated: 2011-10-21Bibliographically approved
List of papers
1. Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line
Open this publication in new window or tab >>Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line
2008 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 314, no 6, 1367-77 p.Article in journal (Refereed) Published
Abstract [en]

Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.

Place, publisher, year, edition, pages
New York: Academic Press, 2008
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-20782 (URN)10.1016/j.yexcr.2007.12.018 (DOI)18262179 (PubMedID)
Available from: 2009-03-25 Created: 2009-03-25 Last updated: 2017-12-13Bibliographically approved
2. Global regulation of the interphase microtubule system by abundantly expressed Op18/stathmin.
Open this publication in new window or tab >>Global regulation of the interphase microtubule system by abundantly expressed Op18/stathmin.
2008 (English)In: Molecular biology of the cell, ISSN 1939-4586, Vol. 19, no 7, 2897-906 p.Article in journal (Refereed) Published
Abstract [en]

Op18/stathmin (Op18), a conserved microtubule-depolymerizing and tubulin heterodimer-binding protein, is a major interphase regulator of tubulin monomer-polymer partitioning in diverse cell types in which Op18 is abundant. Here, we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, whereas this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer-polymer partitioning, enables Op18 to exert global regulation of the microtubule system.

Identifiers
urn:nbn:se:umu:diva-20781 (URN)10.1091/mbc.E08-01-0058 (DOI)18434595 (PubMedID)
Available from: 2009-03-25 Created: 2009-03-25 Last updated: 2011-10-21
3. Deciphering the rules governing assembly order of mammalian septin complexes
Open this publication in new window or tab >>Deciphering the rules governing assembly order of mammalian septin complexes
2011 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 17, 3152-3164 p.Article in journal (Refereed) Published
Abstract [en]

Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals inter-dependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six-to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.

Place, publisher, year, edition, pages
Bethesda: The Soc.,, 2011
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-47388 (URN)10.1091/mbc.E11-03-0253 (DOI)
Available from: 2011-09-26 Created: 2011-09-20 Last updated: 2017-12-08
4. Microtubules support a disc-like septin arrangement at the plasma membrane of mammalian cells
Open this publication in new window or tab >>Microtubules support a disc-like septin arrangement at the plasma membrane of mammalian cells
2011 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 23, 4588-4601 p.Article in journal (Refereed) Published
Abstract [en]

Septin family proteins oligomerize through GTP-binding domains into core heteromers, which in turn polymerize at the cleavage furrow of dividing fungal and animal cells. Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. Here we developed protocols for visualization of authentic higher-order assemblies using tagged septins to effectively replace the endogenous gene-product within septin core heteromers in human cells. Our analysis revealed that septins assemble into microtubule-supported disc-like structures at the plasma membrane. In the absence of cell substrate-adhesion, this is the predominant higher-order arrangement in interphase cells and each one of the 7 to 8 septin family members expressed by the two analyzed cell types appears equally represented. However, studies of myeloid and lymphoid cell model systems revealed cell type specific alterations of higher-order septin arrangements in response to substrate-adhesion. Live-cell observations suggested that all higher-order septin assemblies are mutually exclusive with plasma membrane regions undergoing remodeling. The combined data point to a mechanism by which densely arranged cortical microtubules, which are typical for non-adhered spherical cells, support plasma membrane-bound disc-like septin assemblies.

Place, publisher, year, edition, pages
Bethesda: American Society for Cell Biology, 2011
Keyword
filament formation, budding yeast, saccharomyces-cerevisiae, organization, tubulin, actin, localization, cytoskeleton, interphase, dynamics
National Category
Cell and Molecular Biology
Research subject
cellforskning
Identifiers
urn:nbn:se:umu:diva-48430 (URN)10.1091/mbc.E11-09-0754 (DOI)000298140000011 ()
External cooperation:
Available from: 2011-10-20 Created: 2011-10-20 Last updated: 2017-12-08Bibliographically approved

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