scholarly journals The human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments

2018 ◽  
Author(s):  
Luke A. Helgeson ◽  
Alex Zelter ◽  
Michael Riffle ◽  
Michael J. MacCoss ◽  
Charles L. Asbury ◽  
...  
Keyword(s):  
Cell Division ◽  
Optical Tweezers ◽  
Coiled Coil ◽  
Microtubule Binding ◽  
C Terminus ◽  
Switching Dynamics ◽  
Microtubule Binding Domain ◽  
Ndc80 Complex ◽  
Bearing Assembly

ABSTRACTAccurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Crosslinking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C-terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together our results indicate that the Ska complex can directly strengthen the kinetochore microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.SIGNIFICANCE STATEMENTMicrotubules are dynamic, tube-like structures that drive the segregation of duplicated chromosomes during cell division. The Ska complex is part of a molecular machine that forms force-bearing connections between chromosomes and microtubule ends. Depletion of the Ska complex destabilizes these connections and disrupts cell division. The Ska complex binds microtubules but it is unknown if it directly holds force at microtubules or indirectly stabilizes the connections. Here, we show that the Ska complex makes a direct force-bearing linkage with microtubule ends and assembles with another microtubule binding component, the Ndc80 complex, to strengthen its ability to withstand force. Our results suggest that the Ska and Ndc80 complexes work together to maintain the connections between chromosomes and microtubule ends.

scholarly journals Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore–microtubule attachments

2018 ◽  
Vol 115 (11) ◽  
pp. 2740-2745 ◽  
Author(s):  
Luke A. Helgeson ◽  
Alex Zelter ◽  
Michael Riffle ◽  
Michael J. MacCoss ◽  
Charles L. Asbury ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Coiled Coil ◽  
C Terminus ◽  
Switching Dynamics ◽  
Microtubule Binding Domain ◽  
Ndc80 Complex ◽  
Bearing Assembly ◽  
Segregation Of Chromosomes ◽  
Dynamic Microtubule

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore–microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex–microtubule interaction is sufficiently strong to make a significant contribution to kinetochore–microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore–microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.

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 Divisome under Construction: Distinct Domains of the Small Membrane Protein FtsB Are Necessary for Interaction with Multiple Cell Division Proteins

2009 ◽  
Vol 191 (8) ◽  
pp. 2815-2825 ◽  
Author(s):  
Mark D. Gonzalez ◽  
Jon Beckwith
Keyword(s):  
Cell Division ◽  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Envelope ◽  
Coiled Coil ◽  
Main Function ◽  
Protein Protein Interactions ◽  
Molecular Scaffold ◽  
C Terminus ◽  
Under Construction

ABSTRACT Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

scholarly journals Modulation of tau phosphorylation and intracellular localization by cellular stress

2000 ◽  
Vol 345 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Scott M. JENKINS ◽  
Marcus ZINNERMAN ◽  
Craig GARNER ◽  
Gail V. W. JOHNSON
Keyword(s):  
Osmotic Stress ◽  
Protein Kinases ◽  
Intracellular Localization ◽  
Tau Phosphorylation ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

Tau is a microtubule-associated protein that is functionally modulated by phosphorylation and hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of great interest to identify the signalling pathways and enzymes capable of modulating tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and localization in response to osmotic stress, which activates the stress-activated protein kinases (SAPKs), a family of proline-directed protein kinases shown to phosphorylate tau in vitro and hypothesized to phosphorylate tau in Alzheimer's disease. Immunoblot analysis with phosphorylation-dependent antibodies revealed that osmotic stress increased tau phosphorylation at the non-Ser/Thr-Pro sites Ser-262/356, within the microtubule-binding domain, as well as Ser/Thr-Pro sites outside of tau's microtubule-binding domain. Although all SAPKs examined were activated by osmotic stress, none of the endogenous SAPKs mediated the increase in tau phosphorylation. However, when transfected into SH-SY5Y cells, SAPK3, but not the other SAPKs examined, phosphorylated tau in situ in response to activation by osmotic stress. Osmotic-stress-induced tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and an increase in the amount of soluble tau. This stress-induced alteration in tau localization was only partially due to phosphorylation at Ser-262/356 by a staurosporine-sensitive, non-proline-directed, protein kinase. Taken together, these results suggest that osmotic stress activates at least two tau-directed protein kinases, one proline-directed and one non-proline-directed, that SAPK3 can phosphorylate tau on Ser/Thr-Pro residues in situ, and that Ser-262/356 phosphorylation only partially regulates tau localization in the cell.

scholarly journals The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

2015 ◽  
Vol 211 (2) ◽  
pp. 309-322 ◽  
Author(s):  
Lindsay G. Lammers ◽  
Steven M. Markus
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Coiled Coil ◽  
Binding Domain ◽  
Cell Cortex ◽  
Wild Type ◽  
Microtubule Binding ◽  
Coiled Coil Domain ◽  
Microtubule Binding Domain

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal.

scholarly journals Dissociating the Centrosomal Matrix Protein AKAP450 from Centrioles Impairs Centriole Duplication and Cell Cycle Progression

2003 ◽  
Vol 14 (6) ◽  
pp. 2436-2446 ◽  
Author(s):  
Guy Keryer ◽  
Oliwia Witczak ◽  
Annie Delouvée ◽  
Wolfram A. Kemmner ◽  
Danielle Rouillard ◽  
...  
Keyword(s):  
Cell Division ◽  
Hela Cells ◽  
Cellular Localization ◽  
Cell Cycle Progression ◽  
Matrix Protein ◽  
Coiled Coil ◽  
Cell Division Cycle ◽  
Scaffolding Protein ◽  
Centriole Duplication ◽  
C Terminus

Centrosomes provide docking sites for regulatory molecules involved in the control of the cell division cycle. The centrosomal matrix contains several proteins, which anchor kinases and phosphatases. The large A-Kinase Anchoring Protein AKAP450 is acting as a scaffolding protein for other components of the cell signaling machinery. We selectively perturbed the centrosome by modifying the cellular localization of AKAP450. We report that the expression in HeLa cells of the C terminus of AKAP450, which contains the centrosome-targeting domain of AKAP450 but not its coiled-coil domains or binding sites for signaling molecules, leads to the displacement of the endogenous centrosomal AKAP450 without removing centriolar or pericentrosomal components such as centrin, γ-tubulin, or pericentrin. The centrosomal protein kinase A type II α was delocalized. We further show that this expression impairs cytokinesis and increases ploidy in HeLa cells, whereas it arrests diploid RPE1 fibroblasts in G1, thus further establishing a role of the centrosome in the regulation of the cell division cycle. Moreover, centriole duplication is interrupted. Our data show that the association between centrioles and the centrosomal matrix protein AKAP450 is critical for the integrity of the centrosome and for its reproduction.

scholarly journals Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system

2015 ◽  
Vol 26 (8) ◽  
pp. 1491-1508 ◽  
Author(s):  
Robin Beaven ◽  
Nikola S. Dzhindzhev ◽  
Yue Qu ◽  
Ines Hahn ◽  
Federico Dajas-Bailador ◽  
...  
Keyword(s):  
Nervous System ◽  
Current Knowledge ◽  
Coiled Coil ◽  
Growth Dynamics ◽  
Loss Of Function ◽  
Axon Extension ◽  
Coiled Coil Domain ◽  
C Terminus

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end–tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain–containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin–dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.

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.

C-terminal inhibition of tau assembly in vitro and in Alzheimer's disease

2000 ◽  
Vol 113 (21) ◽  
pp. 3737-3745 ◽  
Author(s):  
A. Abraha ◽  
N. Ghoshal ◽  
T.C. Gamblin ◽  
V. Cryns ◽  
R.W. Berry ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Amino Acids ◽  
Disease Process ◽  
Brain Regions ◽  
Microtubule Binding ◽  
C Terminus ◽  
Microtubule Binding Domain ◽  
Tau Polymerization

Alzheimer's disease (AD) is, in part, defined by the polymerization of tau into paired helical and straight filaments (PHF/SFs) which together comprise the fibrillar pathology in degenerating brain regions. Much of the tau in these filaments is modified by phosphorylation. Additionally, a subset also appears to be proteolytically truncated, resulting in the removal of its C terminus. Antibodies that recognize tau phosphorylated at S(396/404)or truncated at E(391) do not stain control brains but do stain brain sections very early in the disease process. We modeled these phosphorylation and truncation events by creating pseudo-phosphorylation and deletion mutants derived from a full-length recombinant human tau protein isoform (ht40) that contains N-terminal exons 2 and 3 and all four microtubule-binding repeats. In vitro assembly experiments demonstrate that both modifications greatly enhance the rates of tau filament formation and that truncation increases the mass of polymer formed, as well. Removal of as few as 12 or as many as 121 amino acids from the C terminus of tau greatly increases the rate and extent of tau polymerization. However, deletion of an additional 7 amino acids, (314)DLSKVTS(320), from the third microtubule-binding repeat results in the loss of tau's ability to form filaments in vitro. These results suggest that only part of the microtubule-binding domain (repeats 1, 2 and a small portion of 3) is crucial for tau polymerization. Moreover, the C terminus of tau clearly inhibits the assembly process; this inhibition can be partially reversed by site-specific phosphorylation and completely removed by truncation events at various sites from S(320) to the end of the molecule.

scholarly journals The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Kacper B Rogala ◽  
Nicola J Dynes ◽  
Georgios N Hatzopoulos ◽  
Jun Yan ◽  
Sheng Kai Pong ◽  
...  
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Structural Analysis ◽  
Coiled Coil ◽  
Higher Order ◽  
Molecular Architecture ◽  
Eukaryotic Evolution ◽  
Centriole Duplication ◽  
Functional Implications

Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

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