scholarly journals MAST/Orbit has a role in microtubule–kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

2002 ◽  
Vol 157 (5) ◽  
pp. 749-760 ◽  
Author(s):  
Helder Maiato ◽  
Paula Sampaio ◽  
Catarina L. Lemos ◽  
John Findlay ◽  
Mar Carmena ◽  
...  
Keyword(s):  
Metaphase Plate ◽  
Microtubule Associated Proteins ◽  
New Family ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Associated Proteins ◽  
In Vivo Analysis ◽  
Bipolar Spindles ◽  
Functional Kinetochore

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals Marliolide Derivative Induces Melanosome Degradation via Nrf2/p62-Mediated Autophagy

2021 ◽  
Vol 22 (8) ◽  
pp. 3995
Author(s):  
Cheong-Yong Yun ◽  
Nahyun Choi ◽  
Jae Un Lee ◽  
Eun Jung Lee ◽  
Ji Young Kim ◽  
...  
Keyword(s):  
Related Factor ◽  
Melanin Pigmentation ◽  
Microtubule Associated Proteins ◽  
Melanocyte Stimulating Hormone ◽  
Associated Proteins ◽  
Autophagy Pathway ◽  
Nrf2 Activator ◽  
Promising Therapeutic Agent

Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.

scholarly journals KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle

2019 ◽  
Vol 2 (1) ◽  
pp. e201800169 ◽  
Author(s):  
Heidi LH Malaby ◽  
Dominique V Lessard ◽  
Christopher L Berger ◽  
Jason Stumpff
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Binding Proteins ◽  
Mitotic Chromosome ◽  
Chromosome Movement ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Chromosome Alignment ◽  
Associated Proteins ◽  
Fiber Dynamics

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber–binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

scholarly journals Prodigiosin-Emerged PI3K/Beclin-1-Independent Pathway Elicits Autophagic Cell Death in Doxorubicin-Sensitive and -Resistant Lung Cancer

2018 ◽  
Vol 7 (10) ◽  
pp. 321 ◽  
Author(s):  
Wei-Jun Chiu ◽  
Shian-Ren Lin ◽  
Yu-Hsin Chen ◽  
May-Jwan Tsai ◽  
Max Leong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Death ◽  
Mammalian Target Of Rapamycin ◽  
Underlying Mechanism ◽  
Beclin 1 ◽  
Class Iii ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Anti Tumor Activity

Prodigiosin (PG) belongs to a family of prodiginines isolated from gram-negative bacteria. It is a water insoluble red pigment and a potent proapoptotic compound. This study elucidates the anti-tumor activity and underlying mechanism of PG in doxorubicin-sensitive (Dox-S) and doxorubicin-resistant (Dox-R) lung cancer cells. The cytotoxicity and cell death characteristics of PG in two cells were measured by MTT assay, cell cycle analysis, and apoptosis/autophagic marker analysis. Then, the potential mechanism of PG-induced cell death was evaluated through the phosphatidylinositol-4,5-bisphosphate 3-kinase-p85/Protein kinase B /mammalian target of rapamycin (PI3K-p85/Akt/mTOR) and Beclin-1/phosphatidylinositol-4,5-bisphosphate 3-kinase-Class III (Beclin-1/PI3K-Class III) signaling. Finally, in vivo efficacy was examined by intratracheal inoculation and treatment. There was similar cytotoxicity with PG in both Dox-S and Dox-R cells, where the half maximal inhibitory concentrations (IC50) were all in 10 μM. Based on a non-significant increase in the sub-G1 phase with an increase of microtubule-associated proteins 1A/1B light chain 3B-phosphatidylethanolamine conjugate (LC3-II), the cell death of both cells was categorized to achieve autophagy. Interestingly, an increase in cleaved-poly ADP ribose polymerase (cleaved-PARP) also showed the existence of an apoptosis-sensitive subpopulation. In both Dox-S and Dox-R cells, PI3K-p85/Akt/mTOR signaling pathways were reduced, which inhibited autophagy initiation. However, Beclin-1/PI3K-Class III downregulation implicated non-canonical autophagy pathways were involved in PG-induced autophagy. At completion of the PG regimen, tumors accumulated in the mice trachea and were attenuated by PG treatment, which indicated the efficacy of PG for both Dox-S and Dox-R lung cancer. All the above results concluded that PG is a potential chemotherapeutic agent for lung cancer regimens regardless of doxorubicin resistance.

Modulation of microtubule dynamic instability in vivo by brain microtubule associated proteins

1995 ◽  
Vol 108 (4) ◽  
pp. 1679-1689 ◽  
Author(s):  
R. Dhamodharan ◽  
P. Wadsworth
Keyword(s):  
Dynamic Behavior ◽  
Dynamic Instability ◽  
Average Rate ◽  
Stable Maps ◽  
Microtubule Dynamic ◽  
Unit Distance ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins

Heat-stable brain microtubule associated proteins (MAPs) and purified microtubule associated protein 2 (MAP-2) were microinjected into cultured BSC-1 cells which had been previously injected with rhodamine-labeled tubulin. The dynamic instability behavior of individual microtubules was then examined using low-light-level fluorescence microscopy and quantitative microtubule tracking methods. Both MAP preparations suppressed microtubule dynamics in vivo, by reducing the average rate and extent of both growing and shortening events. The average duration of growing events was not affected. When measured as events/unit time, heat-stable MAPs and MAP-2 did not significantly alter the frequency of rescue; the frequency of catastrophe was decreased approximately two-fold by heat-stable MAPs and MAP-2. When transition frequencies were calculated as events/unit distance, both MAP preparations increased the frequency of rescue, without altering the frequency of catastrophe. The percentage of total time spent in the phases of growth, shrink and pause was determined. Both MAP-2 and heat-stable MAPs decreased the percentage of time spent shortening, increased the percentage of time spent paused, and had no effect on percentage of time spent growing. Heat-stable MAPs increased the average pause duration, decreased the average number of events per minute per microtubule and increased the probability that a paused microtubule would switch to growing rather than shortening. The results demonstrate that addition of MAPs to living cells reduces the dynamic behavior of individual microtubules primarily by suppressing the magnitude of dynamic events and increasing the time spent in pause, where no change in the microtubule length can be detected. The results further suggest that the expression of MAPs directly contributes to cell type-specific microtubule dynamic behavior.

scholarly journals The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Vol 134 (1) ◽  
pp. jcs251025
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

scholarly journals Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.

1994 ◽  
Vol 126 (4) ◽  
pp. 1017-1029 ◽  
Author(s):  
S Barlow ◽  
M L Gonzalez-Garay ◽  
R R West ◽  
J B Olmsted ◽  
F Cabral
Keyword(s):  
Sodium Butyrate ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Constitutive Expression ◽  
Microtubule Assembly ◽  
Western Blots ◽  
Transfected Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.

scholarly journals Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.

1989 ◽  
Vol 109 (6) ◽  
pp. 2977-2991 ◽  
Author(s):  
D R Kellogg ◽  
C M Field ◽  
B M Alberts
Keyword(s):  
Affinity Chromatography ◽  
Mitotic Spindle ◽  
Polyclonal Antibodies ◽  
Drosophila Embryo ◽  
Microtubule Cytoskeleton ◽  
Microtubule Associated Proteins ◽  
Early Drosophila Embryo ◽  
Individual Mouse ◽  
Associated Proteins

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

scholarly journals The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics

2011 ◽  
Vol 22 (22) ◽  
pp. 4343-4361 ◽  
Author(s):  
Joshua D. Currie ◽  
Shannon Stewman ◽  
Gregory Schimizzi ◽  
Kevin C. Slep ◽  
Ao Ma ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
Function Analysis ◽  
Microtubule Dynamics ◽  
Cell Periphery ◽  
Cell Interior ◽  
Microtubule Associated Proteins ◽  
Contact Sites ◽  
Associated Proteins ◽  
Fine Tune

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure–function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH2-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability.

scholarly journals Yeast proteins associated with microtubules in vitro and in vivo.

1992 ◽  
Vol 3 (1) ◽  
pp. 29-47 ◽  
Author(s):  
G Barnes ◽  
K A Louie ◽  
D Botstein
Keyword(s):  
Self Assembly ◽  
Synthetic Peptides ◽  
Immunofluorescence Microscopy ◽  
The Self ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Associated Proteins

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

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