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1.
Mol Biol Cell ; 34(11): ar111, 2023 10 01.
Article in English | MEDLINE | ID: mdl-37610838

ABSTRACT

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer). We determined the x-ray structure of the extended, 34-nm BASS domain. Guided by these structural studies, we generated active bipolar kinesin-5 mini-tetramer motors from Drosophila melanogastor and human orthologues which are half the length of native kinesin-5. We then used these kinesin-5 mini-tetramers to examine the role of two unique structural adaptations of kinesin-5: 1) the length and flexibility of the tetramer, and 2) the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, we find that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.


Subject(s)
Kinesins , Microtubules , Humans , Animals , Spindle Apparatus , Cluster Analysis , Drosophila
2.
Elife ; 112022 04 29.
Article in English | MEDLINE | ID: mdl-35485925

ABSTRACT

Doublecortin (DCX) is a microtubule (MT)-associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX that explain these disorders are incompletely determined. Here, we describe the molecular architecture of the DCX-MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-electron microscopy, and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving the C-terminal structured domain and its disordered tail, in a conformation that favors an open, domain-swapped state. The networked state can accommodate multiple different attachment points on the MT lattice, all of which orient the C-terminal tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in its tail, our study shows that lattice-driven self-assembly is an important property of DCX.


Subject(s)
Neuropeptides , Cryoelectron Microscopy , Doublecortin Domain Proteins , Doublecortin Protein , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Neuropeptides/metabolism
3.
Dev Cell ; 57(4): 466-479.e6, 2022 02 28.
Article in English | MEDLINE | ID: mdl-35231427

ABSTRACT

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.


Subject(s)
Cytoplasm/metabolism , Microtubules/metabolism , Polymerization , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Cell Nucleus/metabolism , Interphase/physiology , Spindle Apparatus/metabolism
4.
Structure ; 29(6): 572-586.e6, 2021 06 03.
Article in English | MEDLINE | ID: mdl-33529594

ABSTRACT

The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly.


Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chlamydomonas reinhardtii/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Tubulin/metabolism , Axoneme/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Microfilament Proteins/genetics , Microscopy, Fluorescence , Molecular Chaperones/genetics , Multiprotein Complexes/chemistry , Mutation , Protein Binding , Protein Conformation , Protein Domains , Single Molecule Imaging
5.
Biophys J ; 118(6): 1455-1465, 2020 03 24.
Article in English | MEDLINE | ID: mdl-32070477

ABSTRACT

Physical models of biological systems can become difficult to interpret when they have a large number of parameters. But the models themselves actually depend on (i.e., are sensitive to) only a subset of those parameters. This phenomenon is due to parameter space compression (PSC), in which a subset of parameters emerges as "stiff" as a function of time or space. PSC has only been used to explain analytically solvable physics models. We have generalized this result by developing a numerical approach to PSC that can be applied to any computational model. We validated our method against analytically solvable models of a random walk with drift and protein production and degradation. We then applied our method to a simple computational model of microtubule dynamic instability. We propose that numerical PSC has the potential to identify the low-dimensional structure of many computational models in biophysics. The low-dimensional structure of a model is easier to interpret and identifies the mechanisms and experiments that best characterize the system.


Subject(s)
Models, Theoretical , Proteins , Biophysics , Models, Biological , Physical Phenomena
6.
Dev Cell ; 47(2): 191-204.e8, 2018 10 22.
Article in English | MEDLINE | ID: mdl-30245157

ABSTRACT

The dynamic instability of microtubules is a conserved and fundamental mechanism in eukaryotes. Yet microtubules from different species diverge in their growth rates, lattice structures, and responses to GTP hydrolysis. Therefore, we do not know what limits microtubule growth, what determines microtubule structure, or whether the mechanisms of dynamic instability are universal. Here, we studied microtubules from the nematode C. elegans, which have strikingly fast growth rates and non-canonical lattices in vivo. Using a reconstitution approach, we discovered that C. elegans microtubules combine intrinsically fast growth with very frequent catastrophes. We solved the structure of C. elegans microtubules to 4.8 Å and discovered sequence divergence in the lateral contact loops, one of which is ordered in C. elegans but unresolved in other species. We provide direct evidence that C. elegans tubulin has a higher free energy in solution and propose a model wherein the ordering of lateral contact loops activates tubulin for growth.


Subject(s)
Microtubules/physiology , Tubulin/metabolism , Tubulin/physiology , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/physiology , Guanosine Triphosphate , Microtubules/metabolism , Microtubules/ultrastructure , Models, Molecular , Structure-Activity Relationship
7.
Nat Rev Mol Cell Biol ; 19(7): 451-463, 2018 07.
Article in English | MEDLINE | ID: mdl-29674711

ABSTRACT

Microtubules are dynamic polymers of αß-tubulin that are essential for intracellular organization, organelle trafficking and chromosome segregation. Microtubule growth and shrinkage occur via addition and loss of αß-tubulin subunits, which are biochemical processes. Dynamic microtubules can also engage in mechanical processes, such as exerting forces by pushing or pulling against a load. Recent advances at the intersection of biochemistry and mechanics have revealed the existence of multiple conformations of αß-tubulin subunits and their central role in dictating the mechanisms of microtubule dynamics and force generation. It has become apparent that microtubule-associated proteins (MAPs) selectively target specific tubulin conformations to regulate microtubule dynamics, and mechanical forces can also influence microtubule dynamics by altering the balance of tubulin conformations. Importantly, the conformational states of tubulin dimers are likely to be coupled throughout the lattice: the conformation of one dimer can influence the conformation of its nearest neighbours, and this effect can propagate over longer distances. This coupling provides a long-range mechanism by which MAPs and forces can modulate microtubule growth and shrinkage. These findings provide evidence that the interplay between biochemistry and mechanics is essential for the cellular functions of microtubules.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Tubulin/metabolism , Biological Transport/physiology , Humans , Organelles/metabolism
8.
Mol Biol Cell ; 28(22): 2924-2931, 2017 Nov 01.
Article in English | MEDLINE | ID: mdl-29084910

ABSTRACT

Microtubules are long, slender polymers of αß-tubulin found in all eukaryotic cells. Tubulins associate longitudinally to form protofilaments, and adjacent protofilaments associate laterally to form the microtubule. In the textbook view, microtubules are 1) composed of 13 protofilaments, 2) arranged in a radial array by the centrosome, and 3) built into the 9+2 axoneme. Although these canonical structures predominate in eukaryotes, microtubules with divergent protofilament numbers and higher-order microtubule assemblies have been discovered throughout the last century. Here we survey these noncanonical structures, from the 4-protofilament microtubules of Prosthecobacter to the 40-protofilament accessory microtubules of mantidfly sperm. We review the variety of protofilament numbers observed in different species, in different cells within the same species, and in different stages within the same cell. We describe the determinants of protofilament number, namely nucleation factors, tubulin isoforms, and posttranslational modifications. Finally, we speculate on the functional significance of these diverse polymers. Equipped with novel tubulin-purification tools, the field is now prepared to tackle the long-standing question of the evolutionary basis of microtubule structure.


Subject(s)
Microtubules/metabolism , Microtubules/physiology , Animals , Centrosome/metabolism , Cytoskeleton/metabolism , Humans , Models, Molecular , Polymers/analysis , Protein Isoforms/metabolism , Protein Processing, Post-Translational , Tubulin/metabolism , Tubulin/ultrastructure
9.
J Cell Biol ; 215(5): 631-647, 2016 Dec 05.
Article in English | MEDLINE | ID: mdl-27881713

ABSTRACT

The dynamic regulation of microtubules (MTs) during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore MTs have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The MT depolymerase MCAK (mitotic centromere-associated kinesin) can influence CIN through its impact on MT stability, but how its potent activity is controlled in cells remains unclear. In this study, we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumors, regulates MT stability during mitosis by inhibiting MCAK MT depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning as a result of MT instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, whereas artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus, GTSE1 inhibition of MCAK activity regulates the balance of MT stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability.


Subject(s)
Chromosome Segregation , Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Anaphase , Cell Line, Tumor , Chromosomal Instability , Chromosomes, Human/metabolism , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/metabolism , Humans , Kinetochores/metabolism , Mitosis , Protein Binding , Spindle Apparatus/metabolism
10.
Sci Transl Med ; 8(365): 365ra159, 2016 11 16.
Article in English | MEDLINE | ID: mdl-27856798

ABSTRACT

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.


Subject(s)
Antineoplastic Agents/pharmacology , Lactams, Macrocyclic/pharmacology , Microtubules/drug effects , Neurons/drug effects , Oxazoles/pharmacology , Vinca Alkaloids/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Genomics , Humans , Lactams, Macrocyclic/chemistry , Mice , Microscopy, Electron , Mitosis , Neoplasm Transplantation , Oxazoles/chemistry , Tubulin/chemistry , Vinblastine/analogs & derivatives , Vinblastine/chemistry , Vinblastine/pharmacology , Vinca Alkaloids/chemistry , Vinorelbine
11.
Biol Open ; 5(8): 1040-51, 2016 Aug 15.
Article in English | MEDLINE | ID: mdl-27422905

ABSTRACT

The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3'UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila.

12.
Nat Cell Biol ; 17(7): 907-16, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26098575

ABSTRACT

Microtubules are born and reborn continuously, even during quiescence. These polymers are nucleated from templates, namely γ-tubulin ring complexes (γ-TuRCs) and severed microtubule ends. Using single-molecule biophysics, we show that nucleation from γ-TuRCs, axonemes and seed microtubules requires tubulin concentrations that lie well above the critical concentration. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule-associated proteins (MAPs) alter these time lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, whereas a polymerase (XMAP215) and an anti-catastrophe factor (TPX2) promoted nucleation. We observed similar phenomena in cells. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Our results explain how MAPs establish the spatial and temporal profile of microtubule nucleation.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Tubulin/metabolism , Animals , Axoneme/metabolism , CHO Cells , Cell Line, Tumor , Centrosome/metabolism , Cricetinae , Cricetulus , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/metabolism , Humans , Hydrolysis , Immunoblotting , Kinetics , LLC-PK1 Cells , Microscopy, Electron , Microscopy, Fluorescence/methods , Microtubule-Associated Proteins/genetics , Microtubules/drug effects , Microtubules/ultrastructure , Nocodazole/pharmacology , Polymerization/drug effects , Swine , Tubulin Modulators/pharmacology
13.
Mol Biol Cell ; 26(7): 1207-10, 2015 Apr 01.
Article in English | MEDLINE | ID: mdl-25823928

ABSTRACT

Microtubules are not like other polymers. Whereas polymers such as F-actin will grow continuously as long as the subunit concentration is high enough, a steadily growing microtubule can suddenly shrink even when there is ample αß-tubulin around. This remarkable behavior was discovered in 1984 when Tim Mitchison and Marc Kirschner deduced that microtubules switch from growth to shrinkage when they lose their GTP caps. Here, I review the canonical explanation of dynamic instability that was fleshed out in the years after its discovery. Many aspects of this explanation have been recently subverted, particularly those related to how GTP-tubulin forms polymers and why GTP hydrolysis disrupts them. I describe these developments and speculate on how our explanation of dynamic instability can be changed to accommodate them.


Subject(s)
Microtubules/metabolism , Tubulin/metabolism , Animals , Guanosine Triphosphate/metabolism
14.
J Cell Biol ; 207(3): 323-34, 2014 Nov 10.
Article in English | MEDLINE | ID: mdl-25385183

ABSTRACT

Microtubules are dynamic polymers of αß-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αß-tubulin. The ability of αß-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αß-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.


Subject(s)
Microtubules/ultrastructure , Tubulin/chemistry , Animals , Humans , Microtubule-Associated Proteins/physiology , Microtubules/physiology , Protein Multimerization , Protein Stability , Protein Structure, Quaternary , Tubulin/ultrastructure
15.
Curr Biol ; 24(20): 2366-75, 2014 Oct 20.
Article in English | MEDLINE | ID: mdl-25283777

ABSTRACT

BACKGROUND: Microtubule ends have distinct biochemical and structural features from those of the lattice. Several proteins that control microtubule behavior can distinguish the end of a microtubule from the lattice. The end-binding protein EB1, for example, recognizes the nucleotide state of microtubule ends, which are enriched in GTP-tubulin. EB1 shares its binding site with Doublecortin (DCX), a protein expressed in developing neurons. We showed recently that DCX binds with highest affinity to microtubule ends. RESULTS: Here we show that DCX recognizes microtubule ends by a novel mechanism based on lattice curvature. Using single-molecule microscopy, we show that DCX "comets" do not elongate at faster microtubule growth rates and DCX does not recognize two out of three GTP analogs. We demonstrate that DCX binds with higher affinity to curved microtubule lattices than to straight ones. We find that curvature recognition is a property of single DCX molecules. Straightening of protofilaments (pfs) at microtubule ends with paclitaxel significantly attenuates end-recognition by DCX, but not EB1. Mutations in DCX found in patients with double cortex syndrome disrupted curvature recognition. CONCLUSIONS: We propose a model in which DCX recognizes microtubule ends through specific interactions with their structure. We conclude that microtubule ends have two distinct features that proteins can recognize independently, namely a structural feature related to curvature and nucleotide state.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Neuropeptides/metabolism , Animals , Cattle , Doublecortin Domain Proteins , Doublecortin Protein , Gene Expression Regulation/physiology , Guanosine Triphosphate/analogs & derivatives , Humans , Microtubule-Associated Proteins/genetics , Neurons/metabolism , Neuropeptides/genetics , Protein Binding , Protein Conformation
16.
J Biol Chem ; 289(43): 30133-43, 2014 Oct 24.
Article in English | MEDLINE | ID: mdl-25225289

ABSTRACT

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.


Subject(s)
Actin Cytoskeleton/metabolism , Growth Cones/metabolism , Microtubules/metabolism , Nerve Tissue Proteins/metabolism , Animals , Axons/metabolism , Cell Size , Female , Hippocampus/cytology , Mice, Inbred BALB C , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/deficiency , Protein Structure, Tertiary , Tubulin/metabolism
17.
PLoS One ; 9(2): e86786, 2014.
Article in English | MEDLINE | ID: mdl-24498282

ABSTRACT

Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a "reduction in dimensionality." We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.


Subject(s)
Aurora Kinase B/metabolism , Centromere/metabolism , Microtubules/metabolism , Xenopus Proteins/metabolism , Animals , Aurora Kinase B/chemistry , Aurora Kinase B/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Kinetics , Microtubules/chemistry , Models, Biological , Models, Molecular , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Substrate Specificity , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus laevis/genetics , Xenopus laevis/metabolism
18.
Methods ; 66(2): 273-82, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-23938869

ABSTRACT

Accurate measurements of kinetic rate constants for interacting biomolecules are crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the relevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.


Subject(s)
Molecular Docking Simulation , Animals , COS Cells , Chlorocebus aethiops , Cholera Toxin/chemistry , Doublecortin Domain Proteins , Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Humans , Kinetics , Ligands , Microscopy, Fluorescence , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Microtubules/chemistry , Microtubules/metabolism , Neuropeptides/chemistry , Neuropeptides/metabolism , Spectrometry, Fluorescence
19.
Methods Cell Biol ; 115: 343-54, 2013.
Article in English | MEDLINE | ID: mdl-23973082

ABSTRACT

In vitro fluorescence-based assays have enabled the direct observation of single microtubule-associated proteins (MAPs) alongside the measurement of microtubule growth and shrinkage. Fluorescence-based assays have not, however, been able to address questions of "microtubule architecture." Tubulin can form diverse polymer structures in vitro. Importantly, microtubules nucleated spontaneously have different numbers of protofilaments (pfs), ranging from 11-pf to 16-pf, as well as sheet-like structures, indicating flexibility in tubulin-tubulin bonds. This structural diversity influences microtubule dynamics and the binding of MAPs to microtubules. Observation of microtubule architecture has required the imaging of microtubules by electron microscopy (EM). Because EM requires chemical fixation or freezing, it has not been possible to observe, in real time, how microtubule dynamics might influence structure and vice versa; it also remains technically challenging to directly observe some MAPs, especially small ones, by EM. It is therefore imperative to develop fluorescence-based assays that enable the direct, real-time observation of microtubule architecture alongside growth, shrinkage, and MAP binding. In this chapter, we describe our efforts to control microtubule architecture for fluorescence-based assays. We also describe how microtubule structure can be probed with the help of GFP-tagged doublecortin, a MAP that binds preferentially to 13-pf microtubules.


Subject(s)
Microscopy, Fluorescence/methods , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Tubulin/metabolism , Animals , Brain/cytology , Brain/metabolism , Cattle , Microtubule-Associated Proteins/analysis , Tubulin/analysis
20.
Dev Cell ; 26(2): 118-20, 2013 Jul 29.
Article in English | MEDLINE | ID: mdl-23906062

ABSTRACT

Midzone microtubules keep chromosomes apart after segregation and provide a platform for cytokinesis factors. Reporting recently in Cell, Subramanian et al. (2013) describe how the motor protein kinesin-4 and the microtubule-associated protein PRC1 work together to mark microtubule ends for incorporation into the midzone in a length-dependent manner.


Subject(s)
Cell Cycle Proteins/metabolism , Microtubules/metabolism , Humans
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