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 The Affinity of the Dynein Microtubule-binding Domain Is Modulated by the Conformation of Its Coiled-coil Stalk

2005 ◽  
Vol 280 (25) ◽  
pp. 23960-23965 ◽  
Author(s):  
I. R. Gibbons ◽  
Joan E. Garbarino ◽  
Carol E. Tan ◽  
Samara L. Reck-Peterson ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

scholarly journals Communication between the AAA+ ring and microtubule-binding domain of dyneinThis paper is one of a selection of papers published in this special issue entitled 8th International Conference on AAA Proteins and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (1) ◽  
pp. 15-21 ◽  
Author(s):  
Andrew P. Carter ◽  
Ronald D. Vale
Keyword(s):  
Conformational Changes ◽  
Peer Review Process ◽  
Coiled Coil ◽  
Binding Domain ◽  
Power Stroke ◽  
Microtubule Binding ◽  
Microtubule Motors ◽  
Microtubule Binding Domain ◽  
Mechanical Element ◽  
Linker Domain

Dyneins are microtubule motors, the core of which consists of a ring of AAA+ domains. ATP-driven conformational changes of the AAA+ ring are used to drive the movement of a mechanical element (termed the linker domain) that provides the motor’s powerstroke and to change the affinity of the motor for microtubules (strong binding during the power stroke and weak binding to allow stepping and recocking of the linker domain). Dynein’s microtubule-binding domain (MTBD) is located at the end of a 10 nm long anti-parallel coiled coil (the stalk) and conformational changes that alter the affinity for microtubules must propagate through this coiled coil. A recent crystal structure of dynein’s MTBD sheds new light on how this long-range communication along a coiled coil might occur.

scholarly journals Structural characterisation of KKT4, an unconventional microtubule-binding kinetochore protein

2020 ◽  
Author(s):  
Patryk Ludzia ◽  
Edward D. Lowe ◽  
Gabriele Marcianò ◽  
Shabaz Mohammed ◽  
Christina Redfield ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Brct Domain ◽  
Interaction Domain ◽  
Kinetochore Protein ◽  
Microtubule Binding ◽  
Structural Characterisation ◽  
X Ray Crystallography ◽  
Protein Protein Interaction ◽  
Microtubule Binding Domain

AbstractThe kinetochore is the macromolecular protein machinery that drives chromosome segregation by interacting with spindle microtubules. Unlike most other eukaryotes that have canonical kinetochore proteins, a group of evolutionarily divergent eukaryotes called kinetoplastids (such as Trypanosoma brucei) have a unique set of kinetochore proteins. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterise the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The crystal structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Finally, we show that KKT4 binds DNA with high affinity. Taken together, these results provide the first structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

scholarly journals Structure and Functional Role of Dynein's Microtubule-Binding Domain

Science ◽  
2008 ◽  
Vol 322 (5908) ◽  
pp. 1691-1695 ◽  
Author(s):  
A. P. Carter ◽  
J. E. Garbarino ◽  
E. M. Wilson-Kubalek ◽  
W. E. Shipley ◽  
C. Cho ◽  
...  
Keyword(s):  
Functional Role ◽  
Binding Domain ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

scholarly journals Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics

2003 ◽  
Vol 14 (10) ◽  
pp. 4181-4195 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Ted R. Zarzar ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Regulatory Mechanism ◽  
Metaphase Plate ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Kinetochore Function

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

scholarly journals Autoregulatory control of microtubule binding in doublecortin-like kinase 1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Regina L Agulto ◽  
Melissa M Rogers ◽  
Tracy C Tan ◽  
Amrita Ramkumar ◽  
Ashlyn M Downing ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Therapeutic Target ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Binding Domain ◽  
Cancer Development ◽  
Microtubule Binding ◽  
Microtubule Binding Domain

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

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