Non-catalytic motor domains enable processive movement and functional diversification of the kinesin-14 Kar3

Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a ‘foothold’ that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus.

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Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3576 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3576-3586 ◽

Cited By ~ 77

Author(s):

C H Yang ◽

J Tomkiel ◽

H Saitoh ◽

D H Johnson ◽

W C Earnshaw

Keyword(s):

Dna Binding ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Mammalian Cells ◽

In Vitro Assays ◽

Centromeric Heterochromatin ◽

Structural Protein ◽

Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

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SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A

Journal of Cell Science ◽

10.1242/jcs.249771 ◽

2021 ◽

Author(s):

Bin Yu ◽

Qiaoyu Lin ◽

Chao Huang ◽

Boyan Zhang ◽

Ying Wang ◽

...

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Kinase Activity ◽

In Vitro Assays ◽

Aurora A ◽

Sumo Proteases ◽

Spatiotemporal Control ◽

Early Mitosis

Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator, Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupted the deSUMOylation of AurA, leading to an increased kinase activity and abnormalities in spindle assembly and chromosomes segregation which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render a spatiotemporal control on its kinase activity in mitosis.

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Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)–related protein Sli15 during chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.200105029 ◽

2001 ◽

Vol 155 (5) ◽

pp. 763-774 ◽

Cited By ~ 121

Author(s):

Jung-seog Kang ◽

Iain M. Cheeseman ◽

George Kallstrom ◽

Soundarapandian Velmurugan ◽

Georjana Barnes ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Kinase Activity ◽

Related Protein ◽

Centromeric Dna ◽

Centromere Protein ◽

Kinetochore Function

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1–Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome–spindle interactions or in the movement of kinetochores along microtubules.

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Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

eLife ◽

10.7554/elife.00943 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 50

Author(s):

Marvin E Tanenbaum ◽

Ronald D Vale ◽

Richard J McKenney

Keyword(s):

Single Molecule ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Motor Proteins ◽

Cytoplasmic Dynein ◽

Eukaryotic Cells ◽

Reverse Direction ◽

Tail Domain ◽

Mitotic Spindles

Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins.

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Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis

Journal of Biological Chemistry ◽

10.1074/jbc.m900257200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16501-16512 ◽

Cited By ~ 13

Author(s):

Kyung Uk Hong ◽

Hyun-Jun Kim ◽

Hyo-Sil Kim ◽

Yeon-Sun Seong ◽

Kyeong-Man Hong ◽

...

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Cyclin B1 ◽

Mutant Form ◽

Metaphase Chromosomes ◽

C Terminus ◽

Mitotic Spindle Assembly ◽

Bipolar Spindles

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.

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Mitotic kinesins in action: diffusive searching, directional switching, and ensemble coordination

Molecular Biology of the Cell ◽

10.1091/mbc.e17-10-0612 ◽

2018 ◽

Vol 29 (10) ◽

pp. 1153-1156 ◽

Cited By ~ 1

Author(s):

Allison M. Gicking ◽

Weihong Qiu ◽

William O. Hancock

Keyword(s):

Single Molecule ◽

Mitotic Spindle ◽

Cell Biology ◽

Motor Systems ◽

Microtubule Motors ◽

Biophysical Studies ◽

Cellular Behaviors ◽

Mitotic Spindle Assembly

Mitotic spindle assembly requires the collective action of multiple microtubule motors that coordinate their activities in ensembles. However, despite significant advances in our understanding of mitotic kinesins at the single-motor level, multi-motor systems are challenging to reconstitute in vitro and thus less well understood. Recent findings highlighted in this perspective demonstrate how various properties of kinesin-5 and -14 motors—diffusive searching, directional switching, and multivalent interactions—allow them to achieve their physiological roles of cross-linking parallel microtubules and sliding antiparallel ones during cell division. Additionally, we highlight new experimental techniques that will help bridge the gap between in vitro biophysical studies and in vivo cell biology investigations and provide new insights into how specific single-molecule mechanisms generate complex cellular behaviors.

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The SUMO-Specific Protease SENP5 Is Required for Cell Division

Molecular and Cellular Biology ◽

10.1128/mcb.02301-05 ◽

2006 ◽

Vol 26 (12) ◽

pp. 4489-4498 ◽

Cited By ~ 111

Author(s):

Alessandra Di Bacco ◽

Jian Ouyang ◽

Hsiang-Ying Lee ◽

Andre Catic ◽

Hidde Ploegh ◽

...

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Posttranslational Modification ◽

Catalytic Domain ◽

Biological Processes ◽

Nucleocytoplasmic Trafficking ◽

Binucleate Cells ◽

Specific Protease

ABSTRACT Posttranslational modification of substrates by the small ubiquitin-like modifier, SUMO, regulates diverse biological processes, including transcription, DNA repair, nucleocytoplasmic trafficking, and chromosome segregation. SUMOylation is reversible, and several mammalian homologs of the yeast SUMO-specific protease Ulp1, termed SENPs, have been identified. We demonstrate here that SENP5, a previously uncharacterized Ulp1 homolog, has SUMO C-terminal hydrolase and SUMO isopeptidase activities. In contrast to other SENPs, the C-terminal catalytic domain of SENP5 preferentially processed SUMO-3 compared to SUMO-1 precursors and preferentially removed SUMO-2 and SUMO-3 from SUMO-modified RanGAP1 in vitro. In cotransfection assays, SENP5 preferentially reduced high-molecular-weight conjugates of SUMO-2 compared to SUMO-1 in vivo. Full-length SENP5 localized to the nucleolus. Deletion of the noncatalytic N-terminal domain led to loss of nucleolar localization and increased de-SUMOylation activity in vivo. Knockdown of SENP5 by RNA interference resulted in increased levels of SUMO-1 and SUMO-2/3 conjugates, inhibition of cell proliferation, defects in nuclear morphology, and appearance of binucleate cells, revealing an essential role for SENP5 in mitosis and/or cytokinesis. These findings establish SENP5 as a SUMO-specific protease required for cell division and suggest that mechanisms involving both the catalytic and noncatalytic domains determine the distinct substrate specificities of the mammalian SUMO-specific proteases.

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The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽

10.3390/biom11030360 ◽

2021 ◽

Vol 11 (3) ◽

pp. 360

Author(s):

Pieterjan Debie ◽

Noemi B. Declerck ◽

Danny van Willigen ◽

Celine M. Huygen ◽

Bieke De Sloovere ◽

...

Keyword(s):

Single Molecule ◽

Gamma Ray ◽

Nuclear Imaging ◽

Primary Amines ◽

Resection Margins ◽

Fluorescent Light ◽

Single Structure ◽

Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Microbiology ◽

10.1099/mic.0.079111-0 ◽

2014 ◽

Vol 160 (10) ◽

pp. 2157-2169 ◽

Cited By ~ 13

Author(s):

Sudarson Sundarrajan ◽

Junjappa Raghupatil ◽

Aradhana Vipra ◽

Nagalakshmi Narasimhaswamy ◽

Sanjeev Saravanan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mechanism Of Action ◽

Catalytic Domain ◽

Antibiotic Sensitivity ◽

High Sensitivity ◽

Common Mechanism ◽

Cross Bridge ◽

Sensitivity Pattern

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

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Overexpressed Pituitary Tumor-Transforming Gene Causes Aneuploidy in Live Human Cells

Endocrinology ◽

10.1210/en.2003-0305 ◽

2003 ◽

Vol 144 (11) ◽

pp. 4991-4998 ◽

Cited By ~ 76

Author(s):

Run Yu ◽

Wenge Lu ◽

Jiandong Chen ◽

Chris J. McCabe ◽

Shlomo Melmed

Keyword(s):

Cancer Cells ◽

Chromosome Segregation ◽

Pituitary Tumor ◽

Fluorescent Protein ◽

Human Cancer ◽

Live Cells ◽

Pituitary Tumor Transforming Gene ◽

Transforming Gene

Abstract The mammalian securin, pituitary tumor-transforming gene (PTTG), is overexpressed in several tumors and transforms cells in vitro and in vivo. To test the hypothesis that PTTG overexpression causes aneuploidy, enhanced green fluorescent protein (EGFP)-tagged PTTG (PTTG-EGFP) was expressed in human H1299 cancer cells (with undetectable endogenous PTTG expression) and mitosis of individual live cells observed. Untransfected cells and cells expressing EGFP alone exhibited appropriate mitosis. PTTG-EGFP markedly prolonged prophase and metaphase, indicating that PTTG blocks progression of mitosis to anaphase. In cells that underwent apparently normal mitosis (35 of 65 cells), PTTG-EGFP was degraded about 1 min before anaphase onset. Cells that failed to degrade PTTG-EGFP exhibited asymmetrical cytokinesis without chromosome segregation (18 of 65 cells) or chromosome decondensation without cytokinesis (9 of 65 cells), resulting in appearance of a macronucleus. Fifty-one of 55 cells expressing a nondegradable mutant PTTG exhibited asymmetrical cytokinesis without chromosome segregation, and some (4 of 55) decondensed chromosomes, both resulting in macronuclear formation. During this abnormal cytokinesis, all chromosomes and spindles and both centrosomes moved to one daughter cell, suggesting potential chaos in the subsequent mitosis. In conclusion, failure of PTTG degradation or enhanced PTTG accumulation, as a consequence of overexpression, inhibits mitosis progression and chromosome segregation but does not directly affect cytokinesis, resulting in aneuploidy. These results demonstrate that PTTG induces aneuploidy in single, live, human cancer cells.

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