Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes

1999 ◽  
Vol 112 (9) ◽  
pp. 1291-1302 ◽  
Author(s):  
R.E. Palazzo ◽  
E.A. Vaisberg ◽  
D.G. Weiss ◽  
S.A. Kuznetsov ◽  
W. Steffen
Keyword(s):  
Monoclonal Antibody ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Inhibitory Effect ◽  
Spisula Solidissima ◽  
Germinal Vesicles ◽  
N Terminus ◽  
Similar Inhibitory Effect ◽  
Meiosis I

Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.

scholarly journals Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment

2013 ◽  
Vol 451 (2) ◽  
pp. 195-204 ◽  
Author(s):  
Yuko Iwakiri ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Mitotic Apparatus ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Correct Alignment ◽  
Spindle Microtubules

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5-depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5.

scholarly journals Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro.

1995 ◽  
Vol 128 (1) ◽  
pp. 107-115 ◽  
Author(s):  
V A Lombillo ◽  
C Nislow ◽  
T J Yen ◽  
V I Gelfand ◽  
J R McIntosh
Keyword(s):  
Polyclonal Antibodies ◽  
Neck Region ◽  
Cytoplasmic Dynein ◽  
Inhibitory Effect ◽  
Microtubule Depolymerization ◽  
Cell Biol ◽  
Immunological Investigation ◽  
Chromosome Motion ◽  
Electrophoretic Component

Chromosomes can move with the ends of depolymerizing microtubules (MTs) in vitro, even in the absence of nucleotide triphosphates (Coue, M., V. A. Lombillo, and J. R. McIntosh. 1991. J. Cell Biol. 112:1165-1175.) Here, we describe an immunological investigation of the proteins important for this form of motility. Affinity-purified polyclonal antibodies to kinesin exert a severe inhibitory effect on depolymerization-dependent chromosome motion. These antibodies predominantly recognize a polypeptide of M(r) approximately 250 kD on immunoblots of CHO chromosomes and stain kinetochores as well as some vesicles that are in the chromosome preparation. Antibodies to CENP-E, a kinetochore-associated kinesin-like protein, also recognize a 250-kD electrophoretic component, but they stain only the kinetochroe region of isolated chromosomes. Polyclonal antibodies that recognize specific domains of the CENP-E polypeptide affect MT disassembly-dependent chromosome motion in different ways; antibodies to the head or tail portions slow motility threefold, while those raised against the neck region stop motion completely. Analogous antibodies that block conventional, ATP-dependent motility of cytoplasmic dynein (Vaisberg, G., M. P. Koonce, and J. R. McIntosh. 1993. J. Cell Biol. 123:849-858) have no effect on disassembly-dependent chromosome motion, even though they bind to kinetochores. These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. A similar coupling activity may allow spindle MTs to remain kinetochore-bound while their lengths change during both prometaphase and anaphase A.

scholarly journals Dynein-dynactin segregate meiotic chromosomes in C. elegans spermatocytes

Development ◽  
2021 ◽  
Vol 148 (3) ◽  
pp. dev197780
Author(s):  
Daniel J. Barbosa ◽  
Vanessa Teixeira ◽  
Joana Duro ◽  
Ana X. Carvalho ◽  
Reto Gassmann
Keyword(s):  
Protein Misfolding ◽  
Meiotic Chromosome ◽  
Spindle Assembly ◽  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Early Embryo ◽  
Loss Of Function ◽  
C Elegans ◽  
Spindle Elongation

ABSTRACTThe microtubule motor cytoplasmic dynein 1 (dynein) and its essential activator dynactin have conserved roles in spindle assembly and positioning during female meiosis and mitosis, but their contribution to male meiosis remains poorly understood. Here, we characterize the G33S mutation in the C. elegans dynactin subunit DNC-1, which corresponds to G59S in human p150Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents the segregation of homologous chromosomes. By contrast, chromosomes segregate without errors in the early dnc-1(G33S) embryo. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to the inability of DNC-1 to interact with microtubules. Instead, our results suggest that the DNC-1(G33S) protein, which is aggregation prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins

2012 ◽  
Vol 442 (2) ◽  
pp. 403-412 ◽  
Author(s):  
Yi-Ling Chen ◽  
Kai-Fa Huang ◽  
Wen-Chih Kuo ◽  
Yan-Chung Lo ◽  
Yu-May Lee ◽  
...  
Keyword(s):  
Cell Migration ◽  
Inhibitory Effect ◽  
Glutaminyl Cyclase ◽  
Monocyte Chemoattractant ◽  
Monocyte Migration ◽  
Similar Inhibitory Effect ◽  
And Migration ◽  
Interfering Rna

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.

scholarly journals Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Joseph M. Varberg ◽  
Kaice A. LaFavers ◽  
Gustavo Arrizabalaga ◽  
William J. Sullivan
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Inhibitory Effect ◽  
Protozoan Parasites ◽  
Conjugation System ◽  
Content Type ◽  
Similar Inhibitory Effect ◽  
High Concentrations

ABSTRACT Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma. While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.

Identification of an anti-sperm auto-monoclonal antibody (Ts4)-recognized molecule in the mouse sperm acrosomal region and its inhibitory effect on fertilization in vitro

2016 ◽  
Vol 115 ◽  
pp. 6-13
Author(s):  
Hiroshi Yoshitake ◽  
Risako Oda ◽  
Mitsuaki Yanagida ◽  
Yu Kawasaki ◽  
Mayumi Sakuraba ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Inhibitory Effect ◽  
Fertilization In Vitro ◽  
Mouse Sperm

DN 9693: A Phosphodiesterase Inhibitor with a Platelet Membrane Effect

1989 ◽  
Vol 61 (02) ◽  
pp. 266-269 ◽  
Author(s):  
C Jackson ◽  
J Ball ◽  
J Peel ◽  
J Lawry ◽  
M Greaves ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Reactivity ◽  
Surface Membrane ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Platelet Membrane ◽  
Dependent Manner ◽  
Platelet Surface

SummaryWe have examined the in vitro effects of DN 9693 (piperidinylimidazo-quinazolinone) on various aspects of platelet reactivity. Our results are consistent with its known function as a phosphodiesterase inhibitor in that it increased platelet cyclic AMP, particularly in conjunction with an adenylate cyclase stimulator, and exerted a profound inhibitory effect on platelet aggregation responses to a variety of agonists. DN 9693 also inhibited ristocetin-induced platelet agglutination (RIPA). We therefore examined its effect on ristocetin co-factor assays and on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein lb (GPIb). The drug inhibited the binding of the monoclonal antibody in a dose-dependent manner. This suggests an effect of the drug on the platelet surface membrane with reduced expression of GPIb. Our results indicate that in addition to its anticipated inhibitory effect on platelet aggregation, DN 9693 may also inhibit platelet adhesion.

scholarly journals Competition between microtubule-associated proteins directs motor transport

2017 ◽  
Author(s):  
Brigette Y. Monroy ◽  
Danielle L. Sawyer ◽  
Bryce E. Ackermann ◽  
Melissa M. Borden ◽  
Tracy C. Tan ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Inhibitory Effect ◽  
Microtubule Associated Proteins ◽  
Motor Transport ◽  
Branch Formation ◽  
Microtubule Motor ◽  
Associated Proteins

Within cells, numerous motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule lattice. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and distribution of microtubule motor transport is unknown. Here, we examine the relationship between MAP7 and tau due to their antagonistic effects on neuronal branch formation and kinesin motility in vivo1–8. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. In striking contrast to the inhibitory effect of tau, MAP7 promotes kinesin-based transport in vivo and strongly enhances kinesin-1 binding to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. In contrast, both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs exhibit differential control over distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the correct distribution and balance of molecular motor activity.

Meiotic spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes

1993 ◽  
Vol 51 ◽  
pp. 180-181
Author(s):  
Robert E. Palazzo ◽  
Eugeni Vaisberg ◽  
Richard W. Cole ◽  
Conly L. Rieder
Keyword(s):  
Polarized Light ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Spisula Solidissima ◽  
Animal Cells ◽  
Parthenogenetic Activation ◽  
Unique System ◽  
Tubulin Antibodies

Critical for karyokinesis in animal cells, is the assembly of a spindle-chromosome-aster complex which serves as the force-generation system for chromosome segregation. Spisula solidissima oocytes offer a unique system for the study of spindle assembly since they can be obtained in large quantities, are naturally synchronized in the cell cycle and can be induced to assemble meiotic spindles within 15 min after parthenogenetic activation. Oocytes contain a large nucleus [germinal vesicle (GV)] and are arrested at the G2/M border of meiosis I. No centrosomes or asters have been observed in oocytes at this stage by either electron microscopy or fluorescence microscopy using anti-tubulin antibodies. Observation of live oocytes with polarized light microscopy revealed that within 4-6 min after parthenogenetic activation, asters appeared and invaded the GV as it breaks down (GVBD). 15 min after activation a complete bipolar meiotic spindle was observed.As a first step towards the development of an in vitro biochemical reconstitution system for the study of spindle assembly, concentrated lysates were prepared at various times following parthenogenetic activation and analyzed by phase-contrast microscopy.

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