scholarly journals Dynactin's pointed-end complex is a cargo-targeting module

2012 ◽  
Vol 23 (19) ◽  
pp. 3827-3837 ◽  
Author(s):  
Ting-Yu Yeh ◽  
Nicholas J. Quintyne ◽  
Brett R. Scipioni ◽  
D. Mark Eckley ◽  
Trina A. Schroer
Keyword(s):  
Cytoplasmic Dynein ◽  
Cross Linking ◽  
Structural Domain ◽  
Mitotic Spindles ◽  
Protein Overexpression ◽  
Recycling Endosome ◽  
Binding Partners ◽  
Dynein Motor ◽  
Pointed End ◽  
Chemical Cross Linking

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin's interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the “pointed-end complex.” We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin's interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin's Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.

scholarly journals Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

2009 ◽  
Vol 20 (12) ◽  
pp. 2885-2899 ◽  
Author(s):  
Krysten J. Palmer ◽  
Helen Hughes ◽  
David J. Stephens
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Dominant Negative ◽  
Automated Image Analysis ◽  
Recycling Endosome ◽  
Microtubule Network ◽  
Dynein Motor ◽  
Specific Subset ◽  
Biochemical Analyses

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.

scholarly journals Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment

2008 ◽  
Vol 182 (6) ◽  
pp. 1045-1054 ◽  
Author(s):  
Dileep Varma ◽  
Pascale Monzo ◽  
Stephanie A. Stehman ◽  
Richard B. Vallee
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Cytoplasmic Dynein ◽  
Oscillatory Behavior ◽  
Supporting Evidence ◽  
Mitotic Spindles ◽  
Direct Role ◽  
Regulatory Factors ◽  
Microtubule Attachment ◽  
Dynein Motor ◽  
Show Evidence

Cytoplasmic dynein has been implicated in diverse mitotic functions, several involving its association with kinetochores. Much of the supporting evidence comes from inhibition of dynein regulatory factors. To obtain direct insight into kinetochore dynein function, we expressed a series of dynein tail fragments, which we find displace motor-containing dynein heavy chain (HC) from kinetochores without affecting other subunits, regulatory factors, or microtubule binding proteins. Cells with bipolar mitotic spindles progress to late prometaphase-metaphase at normal rates. However, the dynein tail, dynactin, Mad1, and BubR1 persist at the aligned kinetochores, which is consistent with a role for dynein in self-removal and spindle assembly checkpoint inactivation. Kinetochore pairs also show evidence of misorientation relative to the spindle equator and abnormal oscillatory behavior. Further, kinetochore microtubule bundles are severely destabilized at reduced temperatures. Dynein HC RNAi and injection of anti-dynein antibody in MG132-arrested metaphase cells produced similar effects. These results identify a novel function for the dynein motor in stable microtubule attachment and maintenance of kinetochore orientation during metaphase chromosome alignment.

scholarly journals Activation of cytoplasmic dynein through microtubule crossbridging

2020 ◽  
Author(s):  
Manas Chakraborty ◽  
Algirdas Toleikis ◽  
Nida Siddiqui ◽  
Robert A. Cross ◽  
Anne Straube
Keyword(s):  
Single Molecules ◽  
Cytoplasmic Dynein ◽  
Mitotic Spindles ◽  
Force Output ◽  
Animal Cells ◽  
Physical Separation ◽  
Cargo Transport ◽  
Dynein Motor

SummaryCytoplasmic dynein is the main microtubule-minus-end-directed transporter of cellular cargo in animal cells [1, 2]. Cytoplasmic dynein also functions in the organisation and positioning of mitotic spindles [3, 4] and the formation of ordered microtubule arrays in neurons and muscle [5, 6]. Activation of the motor for cargo transport is thought to require formation of a complex with dynactin and a cargo adapter [7-10]. Here we show that recombinant human dynein can crossbridge neighbouring microtubules and can be activated by this crossbridging to slide and polarity-sort microtubule bundles. While single molecules of human dynein are predominantly static or diffusive on single microtubules, they walk processively for 1.5 μm on average along the microtubule bundles they form. Speed and force output of dynein are doubled on bundles compared to single microtubules, indicating that the crossbridging dynein steps equivalently on two microtubules. Our data are consistent with a model of autoactivation through the physical separation of dynein motor domains when crossbridging two microtubules. This enables cytoplasmic dynein to function effectively as a microtubule organiser and transporter without needing to first form a complex with dynactin and a cargo adapter.

scholarly journals Analysis of Dynactin Subcomplexes Reveals a Novel Actin-Related Protein Associated with the Arp1 Minifilament Pointed End

1999 ◽  
Vol 147 (2) ◽  
pp. 307-320 ◽  
Author(s):  
D. Mark Eckley ◽  
Steven R. Gill ◽  
Karin A. Melkonian ◽  
James B. Bingham ◽  
Holly V. Goodson ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Cytoplasmic Dynein ◽  
Related Protein ◽  
Flexible Assembly ◽  
Dynein Motor ◽  
Structure Treatment ◽  
Comprehensive Picture ◽  
End Capping ◽  
And Function ◽  
Pointed End

The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150Glued sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150Glued, dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.

Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

2018 ◽  
Author(s):  
Allan J. R. Ferrari ◽  
Fabio C. Gozzo ◽  
Leandro Martinez
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Force Field ◽  
Protein Structures ◽  
Protein Structure Determination ◽  
Structural Modeling ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Distance Constraints ◽  
Chemical Cross Linking

<div><p>Chemical cross-linking/Mass Spectrometry (XLMS) is an experimental method to obtain distance constraints between amino acid residues, which can be applied to structural modeling of tertiary and quaternary biomolecular structures. These constraints provide, in principle, only upper limits to the distance between amino acid residues along the surface of the biomolecule. In practice, attempts to use of XLMS constraints for tertiary protein structure determination have not been widely successful. This indicates the need of specifically designed strategies for the representation of these constraints within modeling algorithms. Here, a force-field designed to represent XLMS-derived constraints is proposed. The potential energy functions are obtained by computing, in the database of known protein structures, the probability of satisfaction of a topological cross-linking distance as a function of the Euclidean distance between amino acid residues. The force-field can be easily incorporated into current modeling methods and software. In this work, the force-field was implemented within the Rosetta ab initio relax protocol. We show a significant improvement in the quality of the models obtained relative to current strategies for constraint representation. This force-field contributes to the long-desired goal of obtaining the tertiary structures of proteins using XLMS data. Force-field parameters and usage instructions are freely available at http://m3g.iqm.unicamp.br/topolink/xlff <br></p></div><p></p><p></p>

Sign in / Sign up

Export Citation Format

Share Document