The spindle protein CKAP2 regulates microtubule dynamics and ensures faithful chromosome segregation.

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore-microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.

An Aurora kinase A-BOD1L1-PP2A B56 Axis promotes chromosome segregation fidelity.

Cancer cells are often aneuploid and frequently display elevated rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). CIN is commonly caused by hyperstable kinetochore-microtubule (K-MT) attachments that reduces the efficiency of correction of erroneous K-MT attachments. We recently showed that UMK57, a chemical agonist of MCAK (alias KIF2C) improves chromosome segregation fidelity in CIN cancer cells although cells rapidly develop adaptive resistance. To determine the mechanism of resistance we performed unbiased proteomic screens which revealed increased phosphorylation in cells adapted to UMK57 at two Aurora kinase A phosphoacceptor sites on BOD1L1 (alias FAM44A). BOD1L1 depletion or Aurora kinase A inhibition eliminated resistance to UMK57 in CIN cancer cells. BOD1L1 localizes to spindles/kinetochores during mitosis, interacts with the PP2A phosphatase, and regulates phosphorylation levels of kinetochore proteins, chromosome alignment, mitotic progression and fidelity. Moreover, the BOD1L1 gene is mutated in a subset of human cancers, and BOD1L1 depletion reduces cell growth in combination with clinically relevant doses of taxol or Aurora kinase A inhibitor. Thus, an Aurora kinase A -BOD1L1-PP2A axis promotes faithful chromosome segregation during mitosis.

EML2-S constitutes a new class of proteins that recognizes and regulates the dynamics of tyrosinated microtubules.

Tubulin post-translational modifications (PTMs) alter microtubule properties by affecting the binding of microtubule-associated proteins (MAPs). Microtubule detyrosination, which occurs by proteolytic removal of the C-terminal tyrosine from ɑ-tubulin, generates the oldest known tubulin PTM, but we lack comprehensive knowledge of MAPs that are regulated by this PTM. We developed a screening pipeline to identify proteins that discriminate between Y- and ΔY-microtubules and found that echinoderm microtubule-associated protein-like 2 (EML2) preferentially interacts with Y-microtubules. This activity depends on a Y-microtubule interaction motif built from WD40 repeats. We show that EML2 tracks the tips of shortening microtubules, a behavior not previously seen among human MAPs in vivo, and influences dynamics to increase microtubule stability. Our screening pipeline is readily adapted to identify proteins that specifically recognize a wide range of microtubule PTMs.

The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules.

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding.

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.

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications.

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and ß-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for Myelination.

Oligodendrocytes extend elaborate microtubule arbors that contact up to 50 axon segments per cell, then spiral around myelin sheaths, penetrating from outer to inner layers. However, how they establish this complex cytoarchitecture is unclear. Here, we show that oligodendrocytes contain Golgi outposts, an organelle that can function as an acentrosomal microtubule-organizing center (MTOC). We identify a specific marker for Golgi outposts-TPPP (tubulin polymerization promoting protein)-that we use to purify this organelle and characterize its proteome. In in vitro cell-free assays, recombinant TPPP nucleates microtubules. Primary oligodendrocytes from Tppp knockout (KO) mice have aberrant microtubule branching, mixed microtubule polarity, and shorter myelin sheaths when cultured on 3-dimensional (3D) microfibers. Tppp KO mice exhibit hypomyelination with shorter, thinner myelin sheaths and motor coordination deficits. Together, our data demonstrate that microtubule nucleation outside the cell body at Golgi outposts by TPPP is critical for elongation of the myelin sheath.

Microtubule-associated proteins control the kinetics of microtubule nucleation.

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.

Doublecortin recognizes the longitudinal curvature of the microtubule end and lattice.

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.

Collapsin response mediator protein 4 regulates growth cone dynamics through the actin and microtubule cytoskeleton.

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.

Microtubules accelerate the kinase activity of Aurora-B by a reduction in dimensionality.

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.

Measuring ligand-receptor binding kinetics and dynamics using k-space image correlation spectroscopy.

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.

Motors and MAPs collaborate to size up microtubules.

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.

Fluorescence-based assays for microtubule architecture.

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.

Drosophila auditory organ genes and genetic hearing defects.

The Drosophila auditory organ shares equivalent transduction mechanisms with vertebrate hair cells, and both are specified by atonal family genes. Using a whole-organ knockout strategy based on atonal, we have identified 274 Drosophila auditory organ genes. Only four of these genes had previously been associated with fly hearing, yet one in five of the genes that we identified has a human cognate that is implicated in hearing disorders. Mutant analysis of 42 genes shows that more than half of them contribute to auditory organ function, with phenotypes including hearing loss, auditory hypersusceptibility, and ringing ears. We not only discover ion channels and motors important for hearing, but also show that auditory stimulus processing involves chemoreceptor proteins as well as phototransducer components. Our findings demonstrate mechanosensory roles for ionotropic receptors and visual rhodopsins and indicate that different sensory modalities utilize common signaling cascades.

Doublecortin recognizes the 13-protofilament microtubule cooperatively and tracks microtubule ends.

Neurons, like all cells, face the problem that tubulin forms microtubules with too many or too few protofilaments (pfs). Cells overcome this heterogeneity with the γ-tubulin ring complex, which provides a nucleation template for 13-pf microtubules. Doublecortin (DCX), a protein that stabilizes microtubules in developing neurons, also nucleates 13-pf microtubules in vitro. Using fluorescence microscopy assays, we show that the binding of DCX to microtubules is optimized for the lateral curvature of the 13-pf lattice. This sensitivity depends on a cooperative interaction wherein DCX molecules decrease the dissociation rate of their neighbors. Mutations in DCX found in patients with subcortical band heterotopia weaken these cooperative interactions. Using assays with dynamic microtubules, we discovered that DCX binds to polymerization intermediates at growing microtubule ends. These results support a mechanism for stabilizing 13-pf microtubules that allows DCX to template new 13-pf microtubules through associations with the sides of the microtubule lattice.

Variations on the single-molecule assay for microtubule-associated proteins and kinesins.

The direct observation of single kinesins and microtubule-associated proteins (MAPs) has become a core tool for cytoskeleton research. We outline several variations to the core experiment that allow the researcher to explore structural and biophysical mechanisms underlying kinesin motility and MAP function.

Hearing mechanics: a fly in your ear.

Recent work suggests that the auditory organ of Drosophila may serve as an excellent model system for understanding the complex mechanical signal processing that takes place in sensory hair cells of the vertebrate inner ear.

Models of hair cell mechanotransduction.

Hair cell mechanotransduction is based on a finely tuned machinery residing in the hair bundle, the hair cell's receptive organelle. The machinery consists of a transduction channel, an adaptation motor, the tip link, and many other components that reside in the stereocilia. The transduction channel is connected to and opened by a gating spring, for which there are several molecular candidates. The interplay among the motor, the spring, the channel, and the tip link assures that the channel is always working at its most sensitive point of this machine, allowing very fast responses to a force stimulus. This chapter addresses the mechanisms and molecular components underlying mechanotransduction, adaptation, and motility in the hair bundle. Bundle deflection in the excitatory direction is thought to increase tension in the tip link, which leads to an opening of the mechanoelectrical transduction channel, located close to the insertion site of the tip link.

Molecular profiling reveals synaptic release machinery in Merkel cells.

Merkel cell-neurite complexes are somatosensory receptors that initiate the perception of gentle touch. The role of epidermal Merkel cells within these complexes is disputed. To ask whether Merkel cells are genetically programmed to be excitable cells that may participate in touch reception, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts, including neuronal transcription factors, presynaptic molecules, and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, vesicular glutamate transporter 2, and cholecystokinin 26-33. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, by using Ca(2+) imaging, we discovered that Merkel cells have L- and P/Q-type voltage-gated Ca(2+) channels, which have been shown to trigger vesicle release at synapses. These results demonstrate that Merkel cells are excitable cells and suggest that they release neurotransmitters to shape touch sensitivity.