Short Stop provides an essential link between F-actin and microtubules during axon extension

Development ◽  
2002 ◽  
Vol 129 (5) ◽  
pp. 1195-1204 ◽  
Author(s):  
Seungbok Lee ◽  
Peter A. Kolodziej
Keyword(s):  
Cultured Cells ◽  
Binding Motif ◽  
Cellular Motility ◽  
Connecting Rod ◽  
Neuronal Morphogenesis ◽  
Microtubule Binding ◽  
Axon Extension ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Underlying Mechanisms

Coordination of F-actin and microtubule dynamics is important for cellular motility and morphogenesis, but little is known about underlying mechanisms. short stop (shot) encodes an evolutionarily conserved, neuronally expressed family of rod-like proteins required for sensory and motor axon extension in Drosophila melanogaster. We identify Shot isoforms that contain N-terminal F-actin and C-terminal microtubule-binding domains, and that crosslink F-actin and microtubules in cultured cells. The F-actin- and microtubule-binding domains of Shot are required in the same molecule for axon extension, though the length of the connecting rod domain can be dramatically reduced without affecting activity. Shot therefore functions as a cytoskeletal crosslinker in axon extension, rather than mediating independent interactions with F-actin and microtubules. A Ca2+-binding motif located adjacent to the microtubule-binding domain is also required for axon extension, suggesting that intracellular Ca2+ release may regulate Shot activity. These results suggest that Shot coordinates regulated interactions between F-actin and microtubules that are crucial for neuronal morphogenesis.

scholarly journals The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

2018 ◽  
Vol 217 (11) ◽  
pp. 3886-3900 ◽  
Author(s):  
Aida Llauró ◽  
Hanako Hayashi ◽  
Megan E. Bailey ◽  
Alex Wilson ◽  
Patryk Ludzia ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Binding Activity ◽  
Significant Similarity ◽  
Load Bearing ◽  
Kinetochore Protein ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Coupling Protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.

scholarly journals MTCL2 is a new Golgi-resident microtubule-regulating protein, essential for organizing asymmetric microtubule network

2020 ◽  
Author(s):  
Risa Matsuoka ◽  
Masateru Miki ◽  
Sonoko Mizuno ◽  
Yurina Ito ◽  
Atsushi Suzuki
Keyword(s):  
Coiled Coil ◽  
Golgi Membrane ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Ribbon Structure ◽  
Golgi Ribbon

AbstractThe Golgi apparatus plays important roles in organizing the asymmetric microtubule network essential for polarized vesicle transport. The Golgi-associated coiled-coil protein MTCL1 is crucially involved in Golgi functioning by interconnecting and stabilizing microtubules on the Golgi membrane through its N- and C-terminal microtubule-binding domains. Here, we report the presence of a mammalian paralog of MTCL1, named MTCL2, lacking the N-terminal microtubule-binding domain. MTCL2 localizes to the Golgi membrane through the N-terminal region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown experiments demonstrated essential roles of MTCL2 in accumulating MTs around the Golgi and regulating the Golgi ribbon structure. In vitro wound healing assays further suggested a possible intriguing activity of MTCL2 in integrating the centrosomal and Golgi-associated microtubules around the Golgi ribbon, thus supporting directional migration. Altogether, the present results demonstrate that cells utilize two members of the MTCL protein family to differentially regulate the Golgi-associated microtubules for controlling cell polarity.

The Proline-Rich Domain and the Microtubule Binding Domain of Protein Tau Acting as RNA Binding Domains

2006 ◽  
Vol 13 (7) ◽  
pp. 679-685 ◽  
Author(s):  
Xing Sheng Wang ◽  
Dong Liang Wang ◽  
Jing Zhao ◽  
Mei Hua Qu ◽  
Xiao Hong Zhou ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Protein Tau ◽  
Binding Domains ◽  
Rich Domain ◽  
Microtubule Binding Domain ◽  
Rna Binding Domains

scholarly journals The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation

2020 ◽  
Vol 295 (9) ◽  
pp. 2614-2628 ◽  
Author(s):  
Kseniya Ustinova ◽  
Zora Novakova ◽  
Makoto Saito ◽  
Marat Meleshin ◽  
Jana Mikesova ◽  
...  
Keyword(s):  
Single Molecule ◽  
Recombinant Protein Expression ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Histone Deacetylase 6 ◽  
Catalytic Core ◽  
Microtubule Binding ◽  
Catalytic Domains ◽  
Microtubule Binding Domain

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo. Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.

scholarly journals Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Samuel E Lacey ◽  
Shaoda He ◽  
Sjors HW Scheres ◽  
Andrew P Carter
Keyword(s):  
Conformational Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Cross Sectional ◽  
Microtubule Binding ◽  
High Affinity ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein ◽  
Cilia And Flagella

Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

scholarly journals Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

2019 ◽  
Author(s):  
Samuel E. Lacey ◽  
Shaoda He ◽  
Sjors H. W. Scheres ◽  
Andrew P. Carter
Keyword(s):  
Conformational Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Cross Sectional ◽  
Microtubule Binding ◽  
High Affinity ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein ◽  
Cilia And Flagella

AbstractDyneins are motor proteins responsible for transport in the cytoplasm and the beating of the axoneme in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a long coiled-coil stalk. Here we address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using the stand-alone Relion package. We show the majority of the MTBD is remarkably rigid upon binding, with the transition to the high affinity state controlled by the movement of a single helix at the MTBD interface. In addition DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that reaches over and contacts the adjacent protofilament. Unexpectedly we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

scholarly journals The unconventional kinetoplastid kinetochore protein KKT4 tracks with dynamic microtubule tips

2017 ◽  
Author(s):  
Aida Llauró ◽  
Hanako Hayashi ◽  
Megan E. Bailey ◽  
Alex Wilson ◽  
Patryk Ludzia ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Binding Activity ◽  
Load Bearing ◽  
Kinetochore Protein ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Primary Element ◽  
Microtubule Binding Domain ◽  
Spindle Microtubules

AbstractKinetochores are multiprotein machines that drive chromosome segregation in all eukaryotes by maintaining persistent, load-bearing linkages between the chromosomes and the tips of dynamic spindle microtubules. Kinetochores in commonly studied eukaryotes are assembled from widely conserved components like the Ndc80 complex that directly binds microtubules. However, in evolutionarily-divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here we show that a kinetochore protein from T. brucei called KKT4 binds directly to microtubules, diffuses along the microtubule lattice, and tracks with disassembling microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a minimal microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Laser trapping experiments show that KKT4 can maintain load-bearing attachments to both growing and shortening microtubule tips. Thus, despite its lack of similarity to other known microtubule-binding proteins, KKT4 has key functions required for harnessing microtubule dynamics to drive chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.

scholarly journals Processivity vs. Beating: Comparing Cytoplasmic and Axonemal Dynein Microtubule Binding Domain Association with Microtubule

2019 ◽  
Vol 20 (5) ◽  
pp. 1090 ◽  
Author(s):  
Nayere Tajielyato ◽  
Emil Alexov
Keyword(s):  
Conformational Dynamics ◽  
Cytoplasmic Dynein ◽  
Weakly Bound ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Binding Interface ◽  
Common Effect ◽  
Microtubule Binding Domain ◽  
Axonemal Dynein

This study compares the role of electrostatics in the binding process between microtubules and two dynein microtubule-binding domains (MTBDs): cytoplasmic and axonemal. These two dyneins are distinctively different in terms of their functionalities: cytoplasmic dynein is processive, while axonemal dynein is involved in beating. In both cases, the binding requires frequent association/disassociation between the microtubule and MTBD, and involves highly negatively charged microtubules, including non-structured C-terminal domains (E-hooks), and an MTBD interface that is positively charged. This indicates that electrostatics play an important role in the association process. Here, we show that the cytoplasmic MTBD binds electrostatically tighter to microtubules than to the axonemal MTBD, but the axonemal MTBD experiences interactions with microtubule E-hooks at longer distances compared with the cytoplasmic MTBD. This allows the axonemal MTBD to be weakly bound to the microtubule, while at the same time acting onto the microtubule via the flexible E-hooks, even at MTBD–microtubule distances of 45 Å. In part, this is due to the charge distribution of MTBDs: in the cytoplasmic MTBD, the positive charges are concentrated at the binding interface with the microtubule, while in the axonemal MTBD, they are more distributed over the entire structure, allowing E-hooks to interact at longer distances. The dissimilarities of electrostatics in the cases of axonemal and cytoplasmic MTBDs were found not to result in a difference in conformational dynamics on MTBDs, while causing differences in the conformational states of E-hooks. The E-hooks’ conformations in the presence of the axonemal MTBD were less restricted than in the presence of the cytoplasmic MTBD. In parallel with the differences, the common effect was found that the structural fluctuations of MTBDs decrease as either the number of contacts with E-hooks increases or the distance to the microtubule decreases.

Identification of a novel microtubule-binding domain in microtubule-associated protein 1A (MAP1A)

1994 ◽  
Vol 107 (3) ◽  
pp. 661-672 ◽  
Author(s):  
A. Cravchik ◽  
D. Reddy ◽  
A. Matus
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Binding Domain ◽  
Adult Brain ◽  
Microtubule Binding ◽  
Cultured Cell ◽  
Microtubule Associated Proteins ◽  
Binding Domains ◽  
Microtubule Binding Domain ◽  
Associated Proteins

Several microtubule-associated proteins (MAPs) have been shown to bind to microtubules via short sequences with repeated amino acids motifs. A microtubule-binding domain has hitherto not been defined for the adult brain microtubule-associated protein 1A (MAP1A). We have searched for a microtubule-binding domain by expressing different protein regions of MAP1A in cultured cell lines using cDNA constructs. One construct included an area with homology to the microtubule-binding domain of MAP1B (Noble et al. (1989) J. Cell Biol. 109, 437–448), but this did not bind to microtubules in transfected cells. Further investigation of other areas of MAP1A revealed a protein domain, capable of autonomously binding to microtubules, which bears no homology to any previously described microtubule-binding sequence. This MAP1A domain is rich in charged amino acids, as are other mammalian microtubule-binding domains, but unlike them has no identifiable sequence repeats. Whereas all previously described mammalian microtubule-binding domains are basic, this novel microtubule-binding domain of MAP1A is acidic. The expression of this polypeptide in cultured cell lines led to a rearrangement of the microtubules in a pattern distinct from that produced by MAP2 or tau, and increased their resistance to treatment with the microtubule depolymerising agent nocodazole. When the MAP1A microtubule-binding domain was co-expressed in cultured cell lines together with MAP2c, the MAP1A microtubule-binding domain was able to bind to the MAP2c-induced microtubule bundles. These results suggest that different microtubule-binding sequences have a common ability to stabilise microtubules but differ in their influence on microtubule arrangement in the cell. This may be significant in neurons, where microtubule-associated proteins with different microtubule-binding sequences are expressed in different cell compartments and at different times during development.

Sign in / Sign up

Export Citation Format

Share Document