2020 |
Bodrug, Tatyana; Wilson-Kubalek, Elizabeth M; Nithianantham, Stanley; Thompson, Alex F; Alfieri, April; Gaska, Ignas; Major, Jennifer; Debs, Garrett; Inagaki, Sayaka; Gutierrez, Pedro; Gheber, Larisa; McKenney, Richard J; Sindelar, Charles Vaughn; Milligan, Ronald; Stumpff, Jason; Rosenfeld, Steven S; Forth, Scott T; Al-Bassam, Jawdat The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding Journal Article eLife, 9 (e51131), pp. 1-36, 2020. @article{Al-Bassam20202, title = {The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding}, author = {Tatyana Bodrug and Elizabeth M Wilson-Kubalek and Stanley Nithianantham and Alex F Thompson and April Alfieri and Ignas Gaska and Jennifer Major and Garrett Debs and Sayaka Inagaki and Pedro Gutierrez and Larisa Gheber and Richard J McKenney and Charles Vaughn Sindelar and Ronald Milligan and Jason Stumpff and Steven S Rosenfeld and Scott T Forth and Jawdat Al-Bassam}, url = {https://elifesciences.org/download/aHR0cHM6Ly9jZG4uZWxpZmVzY2llbmNlcy5vcmcvYXJ0aWNsZXMvNTExMzEvZWxpZmUtNTExMzEtdjIucGRm/elife-51131-v2.pdf?_hash=w64w%2FC%2BfRDo9OHJ0V66v5yr9%2FFaPfhjUhET%2BB0b3ZkM%3D}, doi = {10.7554/eLife.51131}, year = {2020}, date = {2020-10-20}, journal = {eLife}, volume = {9}, number = {e51131}, pages = {1-36}, abstract = {Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends. |
2019 |
Cook, Brian D; Chang, Fred C; Flor-parra, Ignacio; Jawdat, Al-Bassam Microtubule Polymerase and Processive Plus-end Tracking functions originate from distinct features within TOG domain arrays Journal Article Mol Biol Cell, 2019. @article{Cook2019, title = {Microtubule Polymerase and Processive Plus-end Tracking functions originate from distinct features within TOG domain arrays}, author = {Brian D Cook and Fred C Chang and Ignacio Flor-parra and Jawdat, Al-Bassam}, url = {https://www.molbiolcell.org/doi/10.1091/mbc.E19-02-0093}, doi = {10.1091/mbc.E19-02-0093}, year = {2019}, date = {2019-04-10}, journal = {Mol Biol Cell}, abstract = {XMAP215/Stu2/Alp14 accelerates tubulin polymerization while processively tracking microtubule plus-ends via Tumor Overexpressed Gene (TOG) domain arrays. It remains poorly understood how these functions arise from tubulin recruitment, mediated by the distinct TOG1 and TOG2 domains, or the assembly of these arrays into large square complexes. Here, we describe a relationship between microtubule plus-end tracking and polymerase functions revealing their distinct origin within TOG arrays. We study Alp14 mutants designed based on structural models, with defects in either tubulin recruitment or self-organization. Using in vivo live imaging in fission yeast and in vitro microtubule dynamics assays, we show that tubulins recruited by TOG1 and TOG2 serve concerted, yet distinct, roles in microtubule plus-end tracking and polymerase functions. TOG1 is critical for processive plus-end tracking while TOG2 is critical for accelerating tubulin polymerization. Inactivating interfaces that stabilize square complexes lead to defects in both processive microtubule plus-end tracking and polymerase. Our studies suggest that a dynamic cycle between square and unfurled TOG array states gives rise to processive polymerase activity at microtubule plus-ends.}, keywords = {}, pubstate = {published}, tppubtype = {article} } XMAP215/Stu2/Alp14 accelerates tubulin polymerization while processively tracking microtubule plus-ends via Tumor Overexpressed Gene (TOG) domain arrays. It remains poorly understood how these functions arise from tubulin recruitment, mediated by the distinct TOG1 and TOG2 domains, or the assembly of these arrays into large square complexes. Here, we describe a relationship between microtubule plus-end tracking and polymerase functions revealing their distinct origin within TOG arrays. We study Alp14 mutants designed based on structural models, with defects in either tubulin recruitment or self-organization. Using in vivo live imaging in fission yeast and in vitro microtubule dynamics assays, we show that tubulins recruited by TOG1 and TOG2 serve concerted, yet distinct, roles in microtubule plus-end tracking and polymerase functions. TOG1 is critical for processive plus-end tracking while TOG2 is critical for accelerating tubulin polymerization. Inactivating interfaces that stabilize square complexes lead to defects in both processive microtubule plus-end tracking and polymerase. Our studies suggest that a dynamic cycle between square and unfurled TOG array states gives rise to processive polymerase activity at microtubule plus-ends. |
2018 |
Al-Bassam, Jawdat; Nithianantham, Stanley Malleable folding of coiled-coils regulates kinesin-3 dimerization Journal Article Proc Natl Acad Sci U S A, 115 (51), pp. 12845-12847, 2018. @article{Al-Bassam2108, title = {Malleable folding of coiled-coils regulates kinesin-3 dimerization}, author = {Jawdat Al-Bassam and Stanley Nithianantham}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/12845.full_.pdf, PDF}, doi = {10.1073/pnas.1818758115.}, year = {2018}, date = {2018-12-18}, journal = {Proc Natl Acad Sci U S A}, volume = {115}, number = {51}, pages = {12845-12847}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Nithianantham, Stanley; Cook, Brian D; Beans, Madeleine; Guo, Fei; Chang, Fred C; Al-Bassam, Jawdat Structural Basis of tubulin recruitment and assembly by microtubule polymerases with Tumor Overexpressed Gene (TOG) domain arrays Journal Article eLife, e38922 , pp. 1-33, 2018. @article{3, title = {Structural Basis of tubulin recruitment and assembly by microtubule polymerases with Tumor Overexpressed Gene (TOG) domain arrays}, author = {Stanley Nithianantham and Brian D Cook and Madeleine Beans and Fei Guo and Fred C Chang and Jawdat Al-Bassam}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/elife-38922-v2.pdf, PDF}, doi = {10.7554/eLife.38922}, year = {2018}, date = {2018-11-03}, urldate = {2018-11-13}, journal = {eLife}, volume = {e38922}, pages = {1-33}, institution = {University of California, Davis}, abstract = {XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of tumor overexpressed gene (TOG) domains, yet their mechanism remains unknown. Here, we describe the biochemical and structural basis for TOG arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 subunits, in which each domain exhibits a distinct exchange rate for tubulin. X-ray structures revealed square-shaped assemblies composed of pseudo-dimeric TOG1-TOG2 subunits assembled head-to-tail, positioning four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking and electron microscopy show Alp14-tubulin forms square assemblies in solution, and inactivating their interfaces destabilize this organization without influencing tubulin binding. An X-ray structure determined using approach to modulate tubulin polymerization revealed an unfurled assembly, in which TOG1-TOG2 uniquely bind to two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies that then unfurl, facilitating their concerted polymerization into protofilaments.}, keywords = {}, pubstate = {published}, tppubtype = {article} } XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of tumor overexpressed gene (TOG) domains, yet their mechanism remains unknown. Here, we describe the biochemical and structural basis for TOG arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 subunits, in which each domain exhibits a distinct exchange rate for tubulin. X-ray structures revealed square-shaped assemblies composed of pseudo-dimeric TOG1-TOG2 subunits assembled head-to-tail, positioning four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking and electron microscopy show Alp14-tubulin forms square assemblies in solution, and inactivating their interfaces destabilize this organization without influencing tubulin binding. An X-ray structure determined using approach to modulate tubulin polymerization revealed an unfurled assembly, in which TOG1-TOG2 uniquely bind to two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies that then unfurl, facilitating their concerted polymerization into protofilaments. |
Nithianantham, Stanley; McNally, Francis J; Al-Bassam, Jawdat Structural Basis for Disassembly of Katanin Heterododecamers Journal Article J Biol Chem, 293 , pp. 10590-10605, 2018. @article{S2018, title = {Structural Basis for Disassembly of Katanin Heterododecamers}, author = {Stanley Nithianantham and Francis J McNally and Jawdat Al-Bassam}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/2018-Nithianantham.pdf, PDF}, doi = {10.1074/jbc.RA117.001215}, year = {2018}, date = {2018-05-11}, journal = {J Biol Chem}, volume = {293}, pages = {10590-10605}, abstract = {The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism. |
Singh, Sudhir Kumar; Pandey, Himanshu; Al-Bassam, Jawdat; Gheber, Larisa Bidirectional motility of kinesin-5 motor proteins: structural determinants, cumulative functions and physiological roles Journal Article Cell Mol Life Sc, 75 (10), pp. 1757-1771, 2018. @article{Singh2018, title = {Bidirectional motility of kinesin-5 motor proteins: structural determinants, cumulative functions and physiological roles}, author = {Sudhir Kumar Singh and Himanshu Pandey and Jawdat Al-Bassam and Larisa Gheber}, url = { https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/Singh2018_Article_BidirectionalMotilityOfKinesin.pdf, PDF}, doi = {10.1007/s00018-018-2754-7}, year = {2018}, date = {2018-05-01}, journal = {Cell Mol Life Sc}, volume = {75}, number = {10}, pages = {1757-1771}, abstract = {Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942-4954, 2011; Roostalu et al., Science 332:94-99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Mitotic kinesin-5 bipolar motor proteins perform essential functions in mitotic spindle dynamics by crosslinking and sliding antiparallel microtubules (MTs) apart within the mitotic spindle. Two recent studies have indicated that single molecules of Cin8, the Saccharomyces cerevisiae kinesin-5 homolog, are minus end-directed when moving on single MTs, yet switch directionality under certain experimental conditions (Gerson-Gurwitz et al., EMBO J 30:4942-4954, 2011; Roostalu et al., Science 332:94-99, 2011). This finding was unexpected since the Cin8 catalytic motor domain is located at the N-terminus of the protein, and such kinesins have been previously thought to be exclusively plus end-directed. In addition, the essential intracellular functions of kinesin-5 motors in separating spindle poles during mitosis can only be accomplished by plus end-directed motility during antiparallel sliding of the spindle MTs. Thus, the mechanism and possible physiological role of the minus end-directed motility of kinesin-5 motors remain unclear. Experimental and theoretical studies from several laboratories in recent years have identified additional kinesin-5 motors that are bidirectional, revealed structural determinants that regulate directionality, examined the possible mechanisms involved and have proposed physiological roles for the minus end-directed motility of kinesin-5 motors. Here, we summarize our current understanding of the remarkable ability of certain kinesin-5 motors to switch directionality when moving along MTs. |
2017 |
Ofer Shapira Alina Goldstein, Jawdat Al-Bassam Larisa Gheber A potential physiological role for bi-directional motility and motor clustering of mitotic kinesin-5 Cin8 in yeast mitosis Journal Article J Cell Sci, 130 , pp. 725-734, 2017. @article{Shapira2017, title = {A potential physiological role for bi-directional motility and motor clustering of mitotic kinesin-5 Cin8 in yeast mitosis}, author = {Ofer Shapira, Alina Goldstein, Jawdat Al-Bassam, Larisa Gheber}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/yeast_kinesin_5_paper.pdf, PDF}, doi = {10.1242/jcs.195040}, year = {2017}, date = {2017-02-15}, journal = {J Cell Sci}, volume = {130}, pages = {725-734}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Al-Bassam, Jawdat Revisiting the tubulin cofactors and Arl2 in the regulation of soluble aß-tubulin pools and their effect on microtubule dynamics Journal Article Mol Biol Cell , 28 (3), pp. 359-363, 2017. @article{Al-Bassam2017, title = {Revisiting the tubulin cofactors and Arl2 in the regulation of soluble aß-tubulin pools and their effect on microtubule dynamics}, author = {Jawdat Al-Bassam}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/mbc.E15-10-0694.pdf, PDF}, doi = {10.1091/mbc.E15-10-0694}, year = {2017}, date = {2017-02-01}, journal = {Mol Biol Cell }, volume = {28}, number = {3}, pages = {359-363}, abstract = {Soluble αβ-tubulin heterodimers are maintained at high concentration inside eukaryotic cells, forming pools that fundamentally drive microtubule dynamics. Five conserved tubulin cofactors and ADP ribosylation factor-like 2 regulate the biogenesis and degradation of αβ-tubulins to maintain concentrated soluble pools. Here I describe a revised model for the function of three tubulin cofactors and Arl2 as a multisubunit GTP-hydrolyzing catalytic chaperone that cycles to promote αβ-tubulin biogenesis and degradation. This model helps explain old and new data indicating these activities enhance microtubule dynamics in vivo via repair or removal of αβ-tubulins from the soluble pools.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Soluble αβ-tubulin heterodimers are maintained at high concentration inside eukaryotic cells, forming pools that fundamentally drive microtubule dynamics. Five conserved tubulin cofactors and ADP ribosylation factor-like 2 regulate the biogenesis and degradation of αβ-tubulins to maintain concentrated soluble pools. Here I describe a revised model for the function of three tubulin cofactors and Arl2 as a multisubunit GTP-hydrolyzing catalytic chaperone that cycles to promote αβ-tubulin biogenesis and degradation. This model helps explain old and new data indicating these activities enhance microtubule dynamics in vivo via repair or removal of αβ-tubulins from the soluble pools. |
2015 |
Nithianantham, Stanley; Le, Sinh; E, Elbert Seto; Jia, Weitao; Leary, Julie; Corbett, Kevin D; Moore, Jeffrey K; Al-Bassam, Jawdat Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble aß-tubulin pool for microtubule dynamics Journal Article eLife, 2015. @article{S2015, title = {Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble aß-tubulin pool for microtubule dynamics}, author = {Stanley Nithianantham and Sinh Le and Elbert Seto E and Weitao Jia and Julie Leary and Kevin D Corbett and Jeffrey K Moore and Jawdat Al-Bassam}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/e08811.full_.pdf, PDF}, doi = {10.7554/eLife.08811}, year = {2015}, date = {2015-07-24}, journal = {eLife}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
2014 |
Scholey*, Jessica; Nithianantham*, Stanley; Scholey, Jonathan M; Al-Bassam, Jawdat Structural basis for the Assembly of the Mitotic Motor Kinesin-5 into Bipolar Tetramers Journal Article eLife, 2014. @article{JE2014, title = {Structural basis for the Assembly of the Mitotic Motor Kinesin-5 into Bipolar Tetramers}, author = {Jessica Scholey* and Stanley Nithianantham* and Jonathan M Scholey and Jawdat Al-Bassam}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/elife02217.pdf, PDF}, doi = {10.7554/eLife.02217}, year = {2014}, date = {2014-04-08}, journal = {eLife}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Al-Bassam, Jawdat Reconstituting Dynamic Microtubule Polymerization Regulation by TOG Domain Proteins Book Chapter 540 , Chapter 8, pp. 131-148, Elsevier, 2014. @inbook{J2014, title = {Reconstituting Dynamic Microtubule Polymerization Regulation by TOG Domain Proteins}, author = {Jawdat Al-Bassam}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/methods_2014.pdf, PDF}, doi = {10.1016/B978-0-12-397924-7.00008-X}, year = {2014}, date = {2014-03-12}, volume = {540}, pages = {131-148}, publisher = {Elsevier}, chapter = {8}, abstract = {Microtubules (MTs) polymerize from soluble αβ-tubulin and undergo rapid dynamic transitions to depolymerization at their ends. Microtubule-associated regulator proteins modulate polymerization dynamics in vivo by altering microtubule plus end conformations or influencing αβ-tubulin incorporation rates. Biochemical reconstitution of dynamic MT polymerization can be visualized with total internal reflection fluorescence (TIRF) microscopy using purified MT regulators. This approach has provided extensive details on the regulation of microtubule dynamics. Here, I describe a general approach to reconstitute MT dynamic polymerization with TOG domain microtubule regulators from the XMAP215/Dis1 and CLASP families using TIRF microscopy. TIRF imaging strategies require nucleation of microtubule polymerization from surface-attached, stabilized MTs. The approaches described here can be used to study the mechanism of a wide variety of microtubule regulatory proteins.}, keywords = {}, pubstate = {published}, tppubtype = {inbook} } Microtubules (MTs) polymerize from soluble αβ-tubulin and undergo rapid dynamic transitions to depolymerization at their ends. Microtubule-associated regulator proteins modulate polymerization dynamics in vivo by altering microtubule plus end conformations or influencing αβ-tubulin incorporation rates. Biochemical reconstitution of dynamic MT polymerization can be visualized with total internal reflection fluorescence (TIRF) microscopy using purified MT regulators. This approach has provided extensive details on the regulation of microtubule dynamics. Here, I describe a general approach to reconstitute MT dynamic polymerization with TOG domain microtubule regulators from the XMAP215/Dis1 and CLASP families using TIRF microscopy. TIRF imaging strategies require nucleation of microtubule polymerization from surface-attached, stabilized MTs. The approaches described here can be used to study the mechanism of a wide variety of microtubule regulatory proteins. |
2012 |
Al-Bassam, Jawdat; Corbett, Kevin D a-Tubulin acetylation from the inside out Journal Article Proc Natl Acad Sci U S A., 48 , pp. 19515-19516, 2012. @article{J2012, title = {a-Tubulin acetylation from the inside out}, author = {Jawdat Al-Bassam and Kevin D Corbett }, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/02/Albassam_corbett_2012.pdf, PUB}, doi = {10.1073/pnas.1217594109}, year = {2012}, date = {2012-11-27}, journal = {Proc Natl Acad Sci U S A.}, volume = {48}, pages = {19515-19516}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Al-Bassam, Jawdat; Hwajin Kim, Ignacio Flor-Parra ; Lal, Neeraj; Velji, Hamida; Chang, Fred C Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase. Journal Article Mol Biol Cell, 23 (15), pp. 2878-90, 2012. @article{Al-Bassam2012, title = {Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase.}, author = {Jawdat Al-Bassam and Hwajin Kim, Ignacio Flor-Parra and Neeraj Lal and Hamida Velji and Fred C Chang }, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/mbc.e12-03-0205.pdf, PDF}, doi = {10.1091/mbc.E12-03-0205}, year = {2012}, date = {2012-08-12}, journal = {Mol Biol Cell}, volume = {23}, number = {15}, pages = {2878-90}, abstract = {XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer-bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly.}, keywords = {}, pubstate = {published}, tppubtype = {article} } XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer-bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly. |
2011 |
Al-Bassam, Jawdat; Chang, Fred Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP. Journal Article Trends Cell Biol., 21 (10), pp. 604-14, 2011. @article{Al-Bassam2011, title = {Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP.}, author = {Jawdat Al-Bassam and Fred Chang}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1-s2.0-S0962892411001280-main.pdf, PDF}, doi = {10.1016/j.tcb.2011.06.007}, year = {2011}, date = {2011-10-21}, journal = {Trends Cell Biol.}, volume = {21}, number = {10}, pages = {604-14}, abstract = {The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The molecular mechanisms by which microtubule-associated proteins (MAPs) regulate the dynamic properties of microtubules (MTs) are still poorly understood. We review recent advances in our understanding of two conserved families of MAPs, the XMAP215/Dis1 and CLASP family of proteins. In vivo and in vitro studies show that XMAP215 proteins act as microtubule polymerases at MT plus ends to accelerate MT assembly, and CLASP proteins promote MT rescue and suppress MT catastrophe events. These are structurally related proteins that use conserved TOG domains to recruit tubulin dimers to MTs. We discuss models for how these proteins might use these individual tubulin dimers to regulate dynamic behavior of MT plus ends. |
2010 |
Al-Bassam, Jawdat; Kim, Hwajin; Brouhard, Gary; van Oijen, Antoine; Harrison, Stephen C; Chang, Fred C CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule. Journal Article Development Cell, 19 (2), pp. 245-58, 2010. @article{Al-Bassam2010, title = {CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule.}, author = {Jawdat Al-Bassam and Hwajin Kim and Gary Brouhard and Antoine van Oijen and Stephen C Harrison and Fred C Chang}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1-s2.0-S1534580710003448-main.pdf, PDF}, doi = {10.1016/j.devcel.2010.07.016}, year = {2010}, date = {2010-08-17}, journal = {Development Cell}, volume = {19}, number = {2}, pages = {245-58}, abstract = {Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an alphabeta-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an alphabeta-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer. |
2008 |
Brouhard, Gary J; Stear, Jeff H; Noetzel, Timothy L; Al-Bassam, Jawdat; Kazuhisa Kinoshita Stephen C Harrison, Jonathon Howard ; Hyman, Anthony A XMAP215 is a processive microtubule polymerase. Journal Article Cell, 132 (1), pp. 79-88, 2008. @article{Brouhard2008, title = {XMAP215 is a processive microtubule polymerase.}, author = {Gary J Brouhard and Jeff H Stear and Timothy L Noetzel and Jawdat Al-Bassam and Kazuhisa Kinoshita, Stephen C Harrison, Jonathon Howard and Anthony A Hyman}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1-s2.0-S0092867407015474-main.pdf, PDF}, doi = {10.1016/j.cell.2007.11.043}, year = {2008}, date = {2008-01-08}, journal = {Cell}, volume = {132}, number = {1}, pages = {79-88}, abstract = {Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers. |
2007 |
Al-Bassam, Jawdat; N, Nicholas Larsen A; Hyman, Anthony A; Harrison, Stephen C Crystal structure of a TOG domain: conserved features of XMAP215/Dis1-family TOG domains and implications for tubulin binding. Journal Article Structure, 15 (3), pp. 355-62, 2007. @article{Al-Bassam2007, title = {Crystal structure of a TOG domain: conserved features of XMAP215/Dis1-family TOG domains and implications for tubulin binding.}, author = {Jawdat Al-Bassam and Nicholas A Larsen N and Anthony A Hyman and Stephen C Harrison}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1-s2.0-S0969212607000676-main.pdf, PDF}, doi = {10.1016/j.str.2007.01.012}, year = {2007}, date = {2007-05-01}, journal = {Structure}, volume = {15}, number = {3}, pages = {355-62}, abstract = {Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 A resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Members of the XMAP215/Dis1 family of microtubule-associated proteins (MAPs) are essential for microtubule growth. MAPs in this family contain several 250 residue repeats, called TOG domains, which are thought to bind tubulin dimers and promote microtubule polymerization. We have determined the crystal structure of a single TOG domain from the Caenorhabditis elegans homolog, Zyg9, to 1.9 A resolution, and from it we describe a structural blueprint for TOG domains. These domains are flat, paddle-like structures, composed of six HEAT-repeat elements stacked side by side. The two wide faces of the paddle contain the HEAT-repeat helices, and the two narrow faces, the intra- and inter-HEAT repeat turns. Solvent-exposed residues in the intrarepeat turns are conserved, both within a particular protein and across the XMAP215/Dis1 family. Mutation of some of these residues in the TOG1 domain from the budding yeast homolog, Stu2p, shows that this face indeed participates in the tubulin contact. |
Al-Bassam, Jawdat; Roger, Benoit; Halpain, Shelley; Milligan., Ronald A Analysis of the weak interactions of ADP-Unc104 and ADP-kinesin with microtubules and their inhibition by MAP2c. Journal Article Cell Motil Cytoskeleton, 64 (5), pp. 377-89, 2007. @article{Al-Bassam2007b, title = {Analysis of the weak interactions of ADP-Unc104 and ADP-kinesin with microtubules and their inhibition by MAP2c.}, author = {Jawdat Al-Bassam and Benoit Roger and Shelley Halpain and Ronald A Milligan.}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/Al-Bassam_et_al-2007-Cell_Motility_and_the_Cytoskeleton.pdf, PDF}, doi = {10.1002/cm.20190}, year = {2007}, date = {2007-05-01}, journal = {Cell Motil Cytoskeleton}, volume = {64}, number = {5}, pages = {377-89}, abstract = {Microtubule based motors like conventional kinesin (Kinesin-1) and Unc104 (Kinesin-3), and classical microtubule associated proteins (MAPs), including MAP2, are intimately involved in neurite formation and organelle transport. The processive motility of both these kinesins involves weak microtubule interactions in the ADP-bound states. Using cosedimentation assays, we have investigated these weak interactions and characterized their inhibition by MAP2c. We show that Unc104 binds microtubules with five-fold weaker affinity and two-fold higher stoichiometry compared with conventional kinesin. Unc104 and conventional kinesin binding affinities are primarily dependent on positively charged residues in the Unc104 K-loop and conventional kinesin neck coiled-coil and removal of these residues affects Unc104 and conventional kinesin differently. We observed that MAP2c acts primarily as a competitive inhibitor of Unc104 but a mixed inhibitor of conventional kinesin. Our data suggest a specific model in which MAP2c differentially interferes with each kinesin motor by inhibiting its weak attachment to the tubulin C-termini. This is reminiscent of the defects we have observed in Unc104 and kinesin mutants in which the positively charged residues in K-loop and neck coiled-coil domains were removed.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Microtubule based motors like conventional kinesin (Kinesin-1) and Unc104 (Kinesin-3), and classical microtubule associated proteins (MAPs), including MAP2, are intimately involved in neurite formation and organelle transport. The processive motility of both these kinesins involves weak microtubule interactions in the ADP-bound states. Using cosedimentation assays, we have investigated these weak interactions and characterized their inhibition by MAP2c. We show that Unc104 binds microtubules with five-fold weaker affinity and two-fold higher stoichiometry compared with conventional kinesin. Unc104 and conventional kinesin binding affinities are primarily dependent on positively charged residues in the Unc104 K-loop and conventional kinesin neck coiled-coil and removal of these residues affects Unc104 and conventional kinesin differently. We observed that MAP2c acts primarily as a competitive inhibitor of Unc104 but a mixed inhibitor of conventional kinesin. Our data suggest a specific model in which MAP2c differentially interferes with each kinesin motor by inhibiting its weak attachment to the tubulin C-termini. This is reminiscent of the defects we have observed in Unc104 and kinesin mutants in which the positively charged residues in K-loop and neck coiled-coil domains were removed. |
Larsen, Nicholas A; Al-Bassam, Jawdat; Wei, Ronnie R; Harrison., Stephen C Structural analysis of Bub3 interactions in the mitotic spindle checkpoint. Journal Article Proc Natl Acad Sci U S A, 104 (4), pp. 201-6, 2007. @article{Larsen2007, title = {Structural analysis of Bub3 interactions in the mitotic spindle checkpoint.}, author = {Nicholas A Larsen and Jawdat Al-Bassam and Ronnie R Wei and Stephen C Harrison.}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1201.full_.pdf, PDF}, doi = {10.1073/pnas.0610358104}, year = {2007}, date = {2007-01-06}, journal = {Proc Natl Acad Sci U S A}, volume = {104}, number = {4}, pages = {201-6}, abstract = {The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (approximately 200 A long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are approximately 5 microM. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the beta-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (approximately 200 A long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are approximately 5 microM. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the beta-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores. |
Wei, Ronnie R; Al-Bassam, Jawdat; Harrison, Stephen C The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment. Journal Article Nat Struct Mol Biol, 4 (1), pp. 54-9, 2007. @article{Wei2007, title = {The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment.}, author = {Ronnie R Wei and Jawdat Al-Bassam and Stephen C Harrison}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/nsmb1186.pdf, PDF}, doi = {10.1038/nsmb1186}, year = {2007}, date = {2007-01-01}, journal = {Nat Struct Mol Biol}, volume = {4}, number = {1}, pages = {54-9}, abstract = {Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 complex forms a direct link between kinetochore core components and spindle microtubules.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 complex forms a direct link between kinetochore core components and spindle microtubules. |
2006 |
Al-Bassam, Jawdat; van Breugel, Mark; Harrison, Stephen C; Hyman, Anthony A Stu2p binds tubulin and undergoes an open-to-closed conformational change Journal Article J Cell Biol, 172 (7), pp. 1009-22, 2006. @article{Al-Bassam2006, title = {Stu2p binds tubulin and undergoes an open-to-closed conformational change}, author = {Jawdat Al-Bassam and Mark van Breugel and Stephen C Harrison and Anthony A Hyman}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1009.full_.pdf, PDF}, doi = {10.1083/jcb.200511010}, year = {2006}, date = {2006-03-27}, journal = {J Cell Biol}, volume = {172}, number = {7}, pages = {1009-22}, abstract = {Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat-containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat-containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs. |
2004 |
Roger*, Benoit; Al-Bassam*, Jawdat; Dehmelt, Leif; Milligan, Ronald A; Halpain, Shelley MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain. Journal Article Curr Biol, 14 (5), pp. 363-71, 2004. @article{Roger*2004, title = {MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain.}, author = {Benoit Roger* and Jawdat Al-Bassam* and Leif Dehmelt and Ronald A Milligan and Shelley Halpain}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1-s2.0-S0960982204000491-main.pdf, PDF}, doi = {10.1016/j.cub.2004.01.058}, year = {2004}, date = {2004-03-09}, journal = {Curr Biol}, volume = {14}, number = {5}, pages = {363-71}, abstract = {BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.}, keywords = {}, pubstate = {published}, tppubtype = {article} } BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons. |
2003 |
Al-Bassam, Jawdat; Cui, Yujia; Klopfenstein, Dieter; Carragher, Bridget O; Vale, Ronald D; Milligan., Ronald A Distinct conformations of the kinesin Unc104 neck regulate a monomer to dimer motor transition. Journal Article J Cell Biol, 163 (4), pp. 743-53, 2003. @article{Al-Bassam2003, title = {Distinct conformations of the kinesin Unc104 neck regulate a monomer to dimer motor transition.}, author = {Jawdat Al-Bassam and Yujia Cui and Dieter Klopfenstein and Bridget O Carragher and Ronald D Vale and Ronald A Milligan.}, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/743.full_.pdf, PDF}, doi = {10.1083/jcb.200308020}, year = {2003}, date = {2003-11-24}, journal = {J Cell Biol}, volume = {163}, number = {4}, pages = {743-53}, abstract = {Caenhorhabditis elegans Unc104 kinesin transports synaptic vesicles at rapid velocities. Unc104 is primarily monomeric in solution, but recent motility studies suggest that it may dimerize when concentrated on membranes. Using cryo-electron microscopy, we observe two conformations of microtubule-bound Unc104: a monomeric state in which the two neck helices form an intramolecular, parallel coiled coil; and a dimeric state in which the neck helices form an intermolecular coiled coil. The intramolecular folded conformation is abolished by deletion of a flexible hinge separating the neck helices, indicating that it acts as a spacer to accommodate the parallel coiled-coil configuration. The neck hinge deletion mutation does not alter motor velocity in vitro but produces a severe uncoordinated phenotype in transgenic C. elegans, suggesting that the folded conformation plays an important role in motor regulation. We suggest that the Unc104 neck regulates motility by switching from a self-folded, repressed state to a dimerized conformation that can support fast processive movement.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Caenhorhabditis elegans Unc104 kinesin transports synaptic vesicles at rapid velocities. Unc104 is primarily monomeric in solution, but recent motility studies suggest that it may dimerize when concentrated on membranes. Using cryo-electron microscopy, we observe two conformations of microtubule-bound Unc104: a monomeric state in which the two neck helices form an intramolecular, parallel coiled coil; and a dimeric state in which the neck helices form an intermolecular coiled coil. The intramolecular folded conformation is abolished by deletion of a flexible hinge separating the neck helices, indicating that it acts as a spacer to accommodate the parallel coiled-coil configuration. The neck hinge deletion mutation does not alter motor velocity in vitro but produces a severe uncoordinated phenotype in transgenic C. elegans, suggesting that the folded conformation plays an important role in motor regulation. We suggest that the Unc104 neck regulates motility by switching from a self-folded, repressed state to a dimerized conformation that can support fast processive movement. |
2002 |
Al-Bassam, Jawdat; Ozer, Rachel S; D, Daniel Safer; Halpain, Shelley; Milligan, Ronald A MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments. Journal Article J Cell Biol, 157 (7), pp. 1187-96, 2002. @article{Al-Bassam2002, title = {MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments.}, author = {Jawdat Al-Bassam and Rachel S Ozer and Daniel Safer D and Shelley Halpain and Ronald A Milligan }, url = {https://microtubule.faculty.ucdavis.edu/wp-content/uploads/sites/228/2019/03/1187.full_.pdf, PDF}, doi = {10.1083/jcb.200201048}, year = {2002}, date = {2002-06-24}, journal = {J Cell Biol}, volume = {157}, number = {7}, pages = {1187-96}, abstract = {MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.}, keywords = {}, pubstate = {published}, tppubtype = {article} } MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces. |