scholarly journals Antagonism between the dynein and Ndc80 complexes at kinetochores controls the stability of kinetochore-microtubule attachments during mitosis

2018 ◽  
Author(s):  
Mohammed A. Amin ◽  
Richard J. McKenney ◽  
Dileep Varma
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Protein Complexes ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
C Elegans ◽  
Dynein Motor ◽  
Chromosome Alignment ◽  
Directed Motility ◽  
The Stability ◽  
Early Mitosis

AbstractChromosome alignment and segregation during mitosis depends critically on kinetochoremicrotubule (kMT) attachments that are mediated by the function of the molecular motor cytoplasmic dynein, and the kinetochore microtubule (MT) binding complex, Ndc80. The RZZ (Rod-ZW10-Zwilch) complex is central to this coordination as it has an important role in dynein recruitment and has recently been reported to have a key function in the regulation of stable kMT attachment formation in C. elegans. However, the mechanism by which kMT attachments are controlled by the coordinated function of these protein complexes to drive chromosome motility during early mitosis is still unclear. In this manuscript, we provide evidence to show that Ndc80 and dynein directly antagonize each other’s MT-binding. We also find that severe chromosome alignment defects induced by depletion of dynein, or the dynein adapter spindly, are rescued by codepletion of the RZZ component, Rod, in human cells. Interestingly, the rescue of chromosome alignments defects was independent of Rod function in activation of the spindle assembly checkpoint and was accompanied by a remarkable restoration of stable kMT attachments. Furthermore, rescue of chromosome alignment was critically dependent on the plus-end-directed motility of CENP-E, as cells codepleted of CENP-E along with Rod and dynein were unable to establish stable kMT attachments or align their chromosomes properly. Taken together, our findings support the idea that the dynein motor may control the function of the Ndc80 complex in stabilizing kMT attachments either directly by interfering with Ndc80-MT binding, and/or indirectly by modulating the Rod-mediated inhibition of Ndc80.

scholarly journals Reconstitution of dynein transport to the microtubule plus end by kinesin

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Anthony J Roberts ◽  
Brian S Goodman ◽  
Samara L Reck-Peterson
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Directed Motion ◽  
Microtubule Associated Proteins ◽  
Spatial Regulation ◽  
Intracellular Movement ◽  
Associated Proteins ◽  
Directed Motility ◽  
Dynamic Microtubule

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.

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 Multiple Sex-Specific Differences in the Regulation of Meiotic Progression in C. elegans

2020 ◽  
Author(s):  
Sara M. Fielder ◽  
Rieke Kempfer ◽  
William G. Kelly
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Elevated Temperatures ◽  
Male Meiosis ◽  
Checkpoint Proteins ◽  
C Elegans ◽  
Meiotic Progression ◽  
Dynein Motor ◽  
Sexual Process ◽  
Speed Up ◽  
Female Specific

ABSTRACTMeiosis is a highly conserved sexual process, yet significant differences exist between males and females in meiotic regulation in many species. Meiotic progression in C. elegans males proceeds more rapidly than female meiosis, suggesting that female meiotic regulation may be more stringent than in males. We have identified multiple differences in the regulation of synapsis, including a difference that suggests the presence of a female-specific meiotic checkpoint that senses the proper initiation of synapsis. This checkpoint is detected by sex differences in the targeting of histone H3 lysine 9 dimethylation (H3K9me2) to unsynapsed chromatin. During oogenic meiosis in hermaphrodites, the failure to initiate synapsis leads to failure to target H3K9me2 enrichment on unsynapsed chromosomes. Loss of the pachytene checkpoint does not reintroduce H3K9me2 enrichment in hermaphrodites, indicating these checkpoints are separable. In contrast, widespread H3K9me2 enrichment occurs as a result of loss of synapsis initiation in both male meiosis and during spermatogenic meiosis in larval XX hermaphrodites. Additionally, male synapsis is insensitive to loss of the dynein motor light chain DLC-1 and to elevated temperatures, whereas female synapsis is prevented by both conditions. We also show that loss of spindle assembly checkpoint proteins, which provide a kinetic barrier to meiotic progression and are required for DLC-1-dependent synapsis phenotypes in hermaphrodites, does not speed up the rate of synapsis in spermatogenic meiosis. These results indicate that meiosis proceeds more rapidly in males because males lack barriers to meiotic progression that are activated by defective synapsis initiation in females.

scholarly journals Spindle assembly checkpoint proteins regulate and monitor meiotic synapsis in C. elegans

2015 ◽  
Vol 211 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Tisha Bohr ◽  
Christian R. Nelson ◽  
Erin Klee ◽  
Needhi Bhalla
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Meiotic Chromosome ◽  
Spindle Assembly ◽  
Anaphase Promoting Complex ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
Cis Acting ◽  
C Elegans ◽  
Meiotic Synapsis ◽  
The Stability

Homologue synapsis is required for meiotic chromosome segregation, but how synapsis is initiated between chromosomes is poorly understood. In Caenorhabditis elegans, synapsis and a checkpoint that monitors synapsis depend on pairing centers (PCs), cis-acting loci that interact with nuclear envelope proteins, such as SUN-1, to access cytoplasmic microtubules. Here, we report that spindle assembly checkpoint (SAC) components MAD-1, MAD-2, and BUB-3 are required to negatively regulate synapsis and promote the synapsis checkpoint response. Both of these roles are independent of a conserved component of the anaphase-promoting complex, indicating a unique role for these proteins in meiotic prophase. MAD-1 and MAD-2 localize to the periphery of meiotic nuclei and interact with SUN-1, suggesting a role at PCs. Consistent with this idea, MAD-1 and BUB-3 require full PC function to inhibit synapsis. We propose that SAC proteins monitor the stability of pairing, or tension, between homologues to regulate synapsis and elicit a checkpoint response.

scholarly journals Nudel functions in membrane traffic mainly through association with Lis1 and cytoplasmic dynein

2004 ◽  
Vol 164 (4) ◽  
pp. 557-566 ◽  
Author(s):  
Yun Liang ◽  
Wei Yu ◽  
Yan Li ◽  
Zhenye Yang ◽  
Xiumin Yan ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Neuronal Migration ◽  
Time Lapse ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Time Lapse Microscopy ◽  
Dynein Motor ◽  
Defective Mutant ◽  
Directed Motility

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains elusive. Herein, overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end–directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end–directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain.

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.

scholarly journals Rational thermostabilisation of four-helix bundle dimeric de novo proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shin Irumagawa ◽  
Kaito Kobayashi ◽  
Yutaka Saito ◽  
Takeshi Miyata ◽  
Mitsuo Umetsu ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Protein Complexes ◽  
Salt Bridge ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Helix Bundle ◽  
Stable Mutant ◽  
Four Helix Bundle ◽  
The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

scholarly journals The influence of sugar–protein complexes on the thermostability of C-reactive protein (CRP)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andreea Lorena Mateescu ◽  
Nicolae-Bogdan Mincu ◽  
Silvana Vasilca ◽  
Roxana Apetrei ◽  
Diana Stan ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Protein Complexes ◽  
C Reactive Protein ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Reactive Protein ◽  
In Vitro Diagnostic ◽  
The Stability ◽  
Positive Controls

AbstractThe purpose of the present study was to evaluate de influence of protein–sugar complexation on the stability and functionality of C-reactive protein, after exposure to constant high temperatures, in order to develop highly stable positive controls for in-vitro diagnostic tests. C-reactive protein is a plasmatic protein used as a biomarker for the diagnosis of a series of health problems such as ulcerative colitis, cardiovascular diseases, metabolic syndrome, due to its essential role in the evolution of chronic inflammation. The sugar–protein interaction was investigated using steady state and time resolved fluorescence. The results revealed that there are more than two classes of tryptophan, with different degree of accessibility for the quencher molecule. Our study also revealed that sugar–protein complexes have superior thermostability, especially after gamma irradiation at 2 kGy, the protein being stable and functional even after 22 days exposure to 40 °C.

scholarly journals Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

2011 ◽  
Vol 22 (15) ◽  
pp. 2690-2701 ◽  
Author(s):  
Melissa D. Stuchell-Brereton ◽  
Amanda Siglin ◽  
Jun Li ◽  
Jeffrey K. Moore ◽  
Shubbir Ahmed ◽  
...  
Keyword(s):  
Light Chain ◽  
Direct Evidence ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Spindle Positioning ◽  
Dynein Motor ◽  
Intermediate Chains ◽  
Activator Complex

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

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