scholarly journals The Arabidopsis thaliana Kinesin-5 AtKRP125b Is a Processive, Microtubule-Sliding Motor Protein with Putative Plant-Specific Functions

2021 ◽  
Vol 22 (21) ◽  
pp. 11361
Author(s):  
Tobias Strauß ◽  
Saskia Schattner ◽  
Stefan Hoth ◽  
Wilhelm J. Walter
Keyword(s):  
Arabidopsis Thaliana ◽  
Mitotic Spindle ◽  
Vascular Tissue ◽  
Lateral Roots ◽  
Protein A ◽  
Biochemical Properties ◽  
Cross Link ◽  
Low Velocity ◽  
In Vitro Motility

The formation and maintenance of the mitotic spindle during cell division requires several microtubule-interacting motor proteins. Members of the kinesin-5 family play an essential role in the bipolar organization of the spindle. These highly conserved, homotetrameric proteins cross-link anti-parallel microtubules and slide them apart to elongate the spindle during the equal separation of chromosomes. Whereas vertebrate kinesin-5 proteins are well studied, knowledge about the biochemical properties and the function of plant kinesin-5 proteins is still limited. Here, we characterized the properties of AtKRP125b, one of four kinesin-5 proteins in Arabidopsis thaliana. In in vitro motility assays, AtKRP125b displayed the archetypal characteristics of a kinesin-5 protein, a low velocity of about 20 nm·s−1 , and a plus end-directed, processive movement. Moreover, AtKRP125b was able to cross-link microtubules and to slide them apart, as required for developing and maintaining the mitotic spindle. In line with such a function, GFP-AtKRP125b fusion proteins were predominantly detected in the nucleus when expressed in Arabidopsis thaliana leaf protoplasts or Nicotiana benthamiana epidermis cells and analyzed by confocal microscopy. However, we also detected GFP signals in the cytoplasm, suggesting additional functions. By generating and analyzing AtKRP125b promoter-reporter lines, we showed that the AtKRP125b promoter was active in the vascular tissue of roots, lateral roots, cotyledons, and true leaves. Remarkably, we could not detect promoter activity in meristematic tissues. Taken together, our biochemical data support a role of AtKRP125b in mitosis, but it may also have additional functions outside the nucleus and during interphase.

scholarly journals Kinesin-14 motors drive a right-handed helical motion of antiparallel microtubules around each other

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aniruddha Mitra ◽  
Laura Meißner ◽  
Rojapriyadharshini Gandhimathi ◽  
Roman Renger ◽  
Felix Ruhnow ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Three Dimensional ◽  
Helical Motion ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Torque Generation ◽  
Free Microtubules ◽  
20 Nm

Abstract Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

scholarly journals Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

2001 ◽  
Vol 21 (23) ◽  
pp. 8035-8044 ◽  
Author(s):  
Daphné Seigneurin-Berny ◽  
André Verdel ◽  
Sandrine Curtet ◽  
Claudie Lemercier ◽  
Jérôme Garin ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Specific Binding ◽  
Protein A ◽  
Biochemical Properties ◽  
Mouse Testis ◽  
Histone Deacetylase 6 ◽  
Protein Ubiquitination ◽  
Ubiquitin Binding ◽  
Associated Proteins

ABSTRACT The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Maleimidobenzoyl Actin: Its Biochemical Properties and In Vitro Motility

1996 ◽  
Vol 119 (1) ◽  
pp. 151-156 ◽  
Author(s):  
T. Hozumi ◽  
M. Miki ◽  
S. Higashi-Fujime
Keyword(s):  
Biochemical Properties ◽  
In Vitro Motility

scholarly journals Emerin Is Required for Proper Nucleus Reassembly after Mitosis: Implications for New Pathogenetic Mechanisms for Laminopathies Detected in EDMD1 Patients

Cells ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 240 ◽  
Author(s):  
Magda Dubińska-Magiera ◽  
Katarzyna Kozioł ◽  
Magdalena Machowska ◽  
Katarzyna Piekarowicz ◽  
Daria Filipczak ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Genetic Disorders ◽  
Genetic Disorder ◽  
Protein A ◽  
Cardiac Conduction ◽  
Deletion Mutants ◽  
Chromatin Binding ◽  
Microtubule Network ◽  
Spindle Microtubules

Emerin is an essential LEM (LAP2, Emerin, MAN1) domain protein in metazoans and an integral membrane protein associated with inner and outer nuclear membranes. Mutations in the human EMD gene coding for emerin result in the rare genetic disorder: Emery–Dreifuss muscular dystrophy type 1 (EDMD1). This disease belongs to a broader group called laminopathies—a heterogeneous group of rare genetic disorders affecting tissues of mesodermal origin. EDMD1 phenotype is characterized by progressive muscle wasting, contractures of the elbow and Achilles tendons, and cardiac conduction defects. Emerin is involved in many cellular and intranuclear processes through interactions with several partners: lamins; barrier-to-autointegration factor (BAF), β-catenin, actin, and tubulin. Our study demonstrates the presence of the emerin fraction which associates with mitotic spindle microtubules and centrosomes during mitosis and colocalizes during early mitosis with lamin A/C, BAF, and membranes at the mitotic spindle. Transfection studies with cells expressing EGFP-emerin protein demonstrate that the emerin fusion protein fraction also localizes to centrosomes and mitotic spindle microtubules during mitosis. Transient expression of emerin deletion mutants revealed that the resulting phenotypes vary and are mutant dependent. The most frequent phenotypes include aberrant nuclear shape, tubulin network mislocalization, aberrant mitosis, and mislocalization of centrosomes. Emerin deletion mutants demonstrated different chromatin binding capacities in an in vitro nuclear assembly assay and chromatin-binding properties correlated with the strength of phenotypic alteration in transfected cells. Aberrant tubulin staining and microtubule network phenotype appearance depended on the presence of the tubulin binding region in the expressed deletion mutants. We believe that the association with tubulin might help to “deliver” emerin and associated membranes to decondensing chromatin. Preliminary analyses of cells from Polish patients with EDMD1 revealed that for several mutations thought to be null for emerin protein, a truncated emerin protein was present. We infer that the EDMD1 phenotype may be strengthened by the toxicity of truncated emerin expressed in patients with certain nonsense mutations in EMD.

scholarly journals Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

2007 ◽  
Vol 18 (9) ◽  
pp. 3601-3606 ◽  
Author(s):  
Abiola Oladipo ◽  
Ann Cowan ◽  
Vladimir Rodionov
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
In Vitro Motility ◽  
Microtubule Motor ◽  
Microscopy Analysis ◽  
Green Fluorescent ◽  
First Time ◽  
Segregation Of Chromosomes

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)–Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic “looping” behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 μm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis.

Utilization of a hydrate cellulose film for investigation of Arabidopsis thaliana L. root system growth and development

2020 ◽  
Vol 36 (1) ◽  
pp. 36-43
Author(s):  
I.O. Konovalova ◽  
T.N. Kudelina ◽  
S.O. Smolyanina ◽  
A.I. Lilienberg ◽  
T.N. Bibikova
Keyword(s):  
Arabidopsis Thaliana ◽  
Root System ◽  
Life Support ◽  
Higher Plants ◽  
Plant Root ◽  
Lateral Roots ◽  
Basic Research ◽  
Cellulose Film ◽  
A New Technique ◽  
Basic Research Project

A new technique for Arabidopsis thaliana cultivation has been proposed that combines the use of a phytogel-based nutrient medium and a hydrophilic membrane of hydrate cellulose film, separating the root system of the plant from the medium thickness. Growth rates of both main and lateral roots were faster in the plants cultivated on the surface of hydrate cellulose film than in the plants grown in the phytogel volume. The location of the root system on the surface of the transparent hydrate film simplifies its observation and analysis and facilitates plant transplantation with preservation of the root system configuration. The proposed technique allowed us to first assess the effect of exogenous auxin on the growth of lateral roots at the 5-6 developmental stage. methods to study plant root systems, hydrate cellulose film, A. thaliana, lateral roots, differential root growth rate, auxin The work was financially supported by the Russian Foundation for Basic Research (Project Bel_mol_a 19-54-04015) and the basic topic of the Russian Academy of Sciences - IBMP RAS «Regularities of the Influence of Extreme Environmental Factors on the Processes of Cultivation of Higher Plants and the Development of Japanese Quail Tissues at Different Stages of its Ontogenesis under the Conditions of Regenerative Life Support Systems».

Surface Modification by Nanobiomaterials for Vascular Tissue Engineering Applications

2020 ◽  
Vol 27 (10) ◽  
pp. 1634-1646 ◽  
Author(s):  
Huey-Shan Hung ◽  
Shan-hui Hsu
Keyword(s):  
Tissue Engineering ◽  
Vascular Tissue ◽  
Thrombus Formation ◽  
Vascular Grafts ◽  
Vascular Tissue Engineering ◽  
Great Success ◽  
Natural Polymers ◽  
Vascular Biomaterials

Treatment of cardiovascular disease has achieved great success using artificial implants, particularly synthetic-polymer made grafts. However, thrombus formation and restenosis are the current clinical problems need to be conquered. New biomaterials, modifying the surface of synthetic vascular grafts, have been created to improve long-term patency for the better hemocompatibility. The vascular biomaterials can be fabricated from synthetic or natural polymers for vascular tissue engineering. Stem cells can be seeded by different techniques into tissue-engineered vascular grafts in vitro and implanted in vivo to repair the vascular tissues. To overcome the thrombogenesis and promote the endothelialization effect, vascular biomaterials employing nanotopography are more bio-mimic to the native tissue made and have been engineered by various approaches such as prepared as a simple surface coating on the vascular biomaterials. It has now become an important and interesting field to find novel approaches to better endothelization of vascular biomaterials. In this article, we focus to review the techniques with better potential improving endothelization and summarize for vascular biomaterial application. This review article will enable the development of biomaterials with a high degree of originality, innovative research on novel techniques for surface fabrication for vascular biomaterials application.

scholarly journals Effective decellularisation of human saphenous veins for biocompatible arterial tissue engineering applications: Bench optimisation and feasibility in vivo testing

2021 ◽  
Vol 12 ◽  
pp. 204173142098752
Author(s):  
Nadiah S Sulaiman ◽  
Andrew R Bond ◽  
Vito D Bruno ◽  
John Joseph ◽  
Jason L Johnson ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Mechanical Strength ◽  
Vascular Tissue ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Replacement Model ◽  
In Vivo Testing ◽  
Vascular Scaffolds

Human saphenous vein (hSV) and synthetic grafts are commonly used conduits in vascular grafting, despite high failure rates. Decellularising hSVs (D-hSVs) to produce vascular scaffolds might be an effective alternative. We assessed the effectiveness of a detergent-based method using 0% to 1% sodium dodecyl sulphate (SDS) to decellularise hSV. Decellularisation effectiveness was measured in vitro by nuclear counting, DNA content, residual cell viability, extracellular matrix integrity and mechanical strength. Cytotoxicity was assessed on human and porcine cells. The most effective SDS concentration was used to prepare D-hSV grafts that underwent preliminary in vivo testing using a porcine carotid artery replacement model. Effective decellularisation was achieved with 0.01% SDS, and D-hSVs were biocompatible after seeding. In vivo xeno-transplantation confirmed excellent mechanical strength and biocompatibility with recruitment of host cells without mechanical failure, and a 50% patency rate at 4-weeks. We have developed a simple biocompatible methodology to effectively decellularise hSVs. This could enhance vascular tissue engineering toward future clinical applications.

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