Cold-induced Depolymerization Research Articles

Dysfunction of the ciliary ARMC9/TOGARAM1 protein module causes Joubert syndrome.

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.

Small molecules targeted to the microtubule\u2013Hec1 interaction inhibit cancer cell growth through microtubule stabilization

Highly expressed in cancer protein 1 (Hec1) is a subunit of the kinetochore (KT)-associated Ndc80 complex, which ensures proper segregation of sister chromatids at mitosis by mediating the interaction between KTs and microtubules (MTs). HEC1 mRNA and protein are highly expressed in many malignancies as part of a signature of chromosome instability. These properties render Hec1 a promising molecular target for developing therapeutic drugs that exert their anticancer activities by producing massive chromosome aneuploidy. A virtual screening study aimed at identifying small molecules able to bind at the Hec1–MT interaction domain identified one positive hit compound and two analogs of the hit with high cytotoxic, pro-apoptotic and anti-mitotic activities. The most cytotoxic analog (SM15) was shown to produce chromosome segregation defects in cancer cells by inhibiting the correction of erroneous KT–MT interactions. Live cell imaging of treated cells demonstrated that mitotic arrest and segregation abnormalities lead to cell death through mitotic catastrophe and that cell death occurred also from interphase. Importantly, SM15 was shown to be more effective in inducing apoptotic cell death in cancer cells as compared to normal ones and effectively reduced tumor growth in a mouse xenograft model. Mechanistically, cold-induced MT depolymerization experiments demonstrated a hyper-stabilization of both mitotic and interphase MTs. Molecular dynamics simulations corroborate this finding by showing that SM15 can bind the MT surface independently from Hec1 and acts as a stabilizer of both MTs and KT–MT interactions. Overall, our studies represent a clear proof of principle that MT-Hec1-interacting compounds may represent novel powerful anticancer agents.

PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division.

Phospholipase D2 (PLD2) is involved in cytoskeletal reorganization, cell migration, cell cycle progression, transcriptional control and vesicle trafficking. There is no evidence about PLD2 function in oocytes during meiosis. Herein, we analyzed PLD2 expression and its relationship with spindle formation and positioning in mouse oocyte meiosis. High protein level of PLD2 was revealed in oocytes by Western blot, which remained consistently stable from prophase I with intact germinal vesicle (GV) up to metaphase II (MII) stage. Immunofluorescence showed that PLD2 appeared and gathered around the condensed chromosomesafter germinal vesicle breakdown (GVBD), and co-localized with spindle from pro-metaphase I (pro-MI) to metaphase I (MI) and at MII stage. During anaphase I (Ana I) to telophase I (Tel I) transition, PLD2 was concentrated in the spindle polar area but absent from the midbody. In oocytes incubated with NFOT, an allosteric and catalytic inhibitor to PLD2, the spindle was enlarged and center-positioned, microtubules were resistant to cold-induced depolymerization and, additionally, the meiotic progression was arrested at MI stage. However, spindle migration could not be totally prevented by PLD2 catalytic specific inhibitors, FIPI and 1-butanol, implying at least partially, that PLD2 effect on spindle migration needs non-catalytic domain participation. NFOT-induced defects also resulted in actin-related molecules’ distribution alteration, such as RhoA, phosphatidylinosital 4, 5- biphosphate (PIP2), phosphorylated Colifin and, consequently, unordered F-actin dynamics. Taken together, these data indicate PLD2 is required for the regulation of microtubule dynamics and spindle migration toward the cortex in mammalian oocytes during meiotic progression.

Induction of acetylation and bundling of cellular microtubules by 9-(4-vinylphenyl) noscapine elicits S-phase arrest in MDA-MB-231 cells

Noscapine is an alkaloid present in the latex of Papaver somniferum. It has been known for its anticancer efficacy and lack of severe toxicities to normal tissues. Structural alterations in noscapine core architecture have produced a number of potent analogues of noscapine. Here, we report an unusual activity of a novel noscapine analogue, 9-(4-vinylphenyl)noscapine (VinPhe-Nos) on cancer cells. As we reported earlier, VinPhe-Nos inhibited MDA-MB-231 cell proliferation with an IC50 of 6μM. The present study elucidated a possible antiproliferative mechanism of action of VinPhe-Nos. The noscapinoid significantly inhibited clonogenic propagation of MDA-MB-231 cells. However, unlike the majority of tubulin-binding agents, it did not induce mitotic arrest; instead, it prolonged S-phase. Although prolonged presence of the drug show some disruption of cellular microtubule architecture, it did not affect microtubule recovery after cold-induced depolymerization. VinPhe-Nos, nevertheless, induced acetylation and bundling of microtubules. Our data suggest that rational modification of parent compound can alter its mechanism of action on cell cycle and that VinPhe-Nos can be investigated further as a less-toxic, S-phase-preferred, cytostatic anticancer agent.

Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity

Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity.

Randomized, open-label, phase II study comparing the efficacy and the safety of cabazitaxel versus weekly paclitaxel given as neoadjuvant treatment in patients with operable triple-negative or luminal b/HER2 normal breast cancer (GENEVIEVE).

TPS1138 Background: Cabazitaxel is a new taxoid promoting the tubulin assembly in vitro and stabilizing microtubules against cold-induced depolymerization as efficiently as docetaxel. It has shown superior survival against mitoxantrone plus prednisone in docetaxel pre-treated hormone refractory metastatic prostate cancer pts leading to its registration. It showed a favorable toxicity profile with a low rate of alopecia. In the GENEVIEVE study it will be compared to weekly paclitaxel, which is currently most widely used in breast cancer (BC) pts. Methods: This is a prospective multicenter, randomized, open label study investigating efficacy and safety of cabazitaxel. Pts with uni- or bilateral primary BC (stage cT3/T2/T1c and cN+/T1c and pNSLN+), tumor lesion ≥ 2cm (palpation) or ≥ 1cm (sonography) and centrally confirmed TNBC or luminal B/HER2- can be included. Pts will be randomized to four q3w cabazitaxel (25mg/m² i.v.) vs. 12 q1w paclitaxel (80mg/m² i.v.). Randomization will be stratified by nodal status and subtype. Treatment will be given until surgery, disease progression, unacceptable toxicity or withdrawal of consent. The primary objective is pathologic complete response (pCR) (ypT0/is ypN0/+). Secondary objectives are pCR in stratified subgroups and by other definitions, objective response rate, pCR and local recurrence free survival in pts with clinical complete response and neg. core biopsy before surgery, breast conservation rate, toxicity, compliance, survival rates, biomarkers predicting response. Assuming 15% pCR in controls and targeting a smallest clinical improvement of 10% (i.e. pCR = 25% in experimental arm), a total of 326 pts (163/arm) are required for the one-sided proportion comparison test (α=0.1) with 80% power. The trial is registered under NCT01779479. Results: Recruitment is planned for 12 mths in 45 (+10 back-up) sites in Germany. 1st pt in is planned for Feb 2013. Conclusion: GENEVIEVE will rapidly and precisely compare efficacy and tolerability of cabacitaxel vs. paclitaxel to decide if further development in BC is reasonable. Clinical trial information: NCT01779479.

Abstract 4495: Fisetin, a dietary flavonoid, binds to and disrupts microtubule dynamic instability in prostate cancer cells.

Abstract Taxanes are the first class of chemotherapy drugs shown to improve survival in advanced prostate cancer (PCa). Taxanes bind β-tubulin and stabilize microtubules, resulting in mitotic arrest and cell death. Therefore, disruption of microtubule function may provide an effective therapy for human PCa, which has been observed in clinical settings using paclitaxel. However, these drugs are associated with severe side effects and are sensitive to resistance mechanisms. Thus, search for novel and non-toxic tubulin targeting agents is urgently needed. We hypothesized that fisetin may offer PCa chemotherapeutic and/or chemopreventive effects by interfering with microtubule assembly, either by inhibiting or enhancing tubulin polymerization. Using an in vitro microtubule polymerization assay, we observed that fisetin enhanced tubulin polymerization whose kinetics paralleled that of paclitaxel. Because acetylation of α-tubulin is considered as a marker of stable microtubule structure, we next tested whether increased acetylated α-tubulin correlates with increased microtubule stability in PC-3 and DU-145 cells. We found that fisetin (20-80 μM) treatment increased α-tubulin acetylation in a dose dependent manner in PC-3 and DU-145 cells. To further investigate the stabilization of the microtubule network in fisetin-treated cells, we evaluated microtubule resistance to cold-induced depolymerization. Immunofluorescence labeling revealed that fisetin-treated PC-3 and DU-145 cells were resistant to cold-induced microtubule depolymerization. To identify microtubule-dependent tumor-specific pathways modulated by fisetin, we determined its effect on MAP2 expression in PC-3 and DU-145 cells. We found increased expression of MAP-2 with fisetin treatment. Stabilization of microtubules resulted in arrest of the cells in the G2/M phase of cell cycle and subsequent cell growth inhibition. Activation of microtubule-stabilizing proteins in PCa cells may inhibit their proliferation and correlate with a delay or inhibition of cell migration. Therefore, we investigated the efficacy of fisetin on the invasion and metastasis of PCa using scratch wound assay. We found that fisetin treatment (20-80 μM) for 24 hours inhibited PC-3 cells migration and invasion, which suggested fisetin as an anti-metastatic agent. As a mechanism for these effects we observed that fisetin increased the expression of nuclear migration protein Nud C in PC-3 and DU-145 cells in a dose dependent manner. Because Nud C protein regulates microtubule dynamics in the cell, the result reveal anti-microtubule effects of fisetin. The data reported here provide the first evidence of the dietary flavonoid fisetin as a microtubule stabilizing agent in PCa. Our observation suggests that fisetin could be used for the treatment of advanced PCa alone or as an adjuvant with other chemotherapy drugs. Citation Format: Eiman Mukhtar, Vaqar M. Adhami, Deeba N. Syed, Hasan Mukhtar. Fisetin, a dietary flavonoid, binds to and disrupts microtubule dynamic instability in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2013-4495

The biology and medicinal chemistry of epothilones.

Epothilones are naturally occurring 16-membered macrolides with the ability to promote tubulin polymerization in vitro and to stabilize preformed microtubules against Ca(2+)- or cold-induced depolymerization. At the cellular level, interference with microtubule functionality results in potent inhibition of cancer cell proliferation at nM to even sub-nM concentrations. Most significantly, epothilones, unlike paclitaxel (Taxol), are equally active against drug-sensitive and multidrug-resistant cell lines in vitro and epothilone B has also shown potent in vivo antitumor activity in Taxol-resistant human tumor models. Epothilone B is currently undergoing Novartis-sponsored Phase II clinical trials. In addition to naturally occurring epothilones, numerous synthetic and semi-synthetic analogs have been prepared since the absolute stereochemistry of epothilone B was first disclosed in mid-1996 and their in vitro biological activity has been determined. These studies have generated a wealth of SAR data in a remarkably short period of time, given the complexity of the synthetic targets pursued. One of these analogs, BMS-247550, is presently in Phase II clinical trials by Bristol-Myers Squibb. In a first part this review is intended to provide a summary of the basic features of the in vitro biological profile of epothilones A and B, including emerging data on potential cellular epothilone resistance mechanisms. The second and third part will feature a comprehensive discussion of the epothilone SAR as it has emerged from the work of various (industrial and academic) laboratories across the world, including our own, with regard to effects on tubulin polymerization, in vitro antiproliferative activity, and in vivo antitumor activity.

MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.

Hsp90 binds microtubules and is involved in the reorganization of the microtubular network in angiosperms

Microtubules (MTs) are essential for many processes in plant cells. MT-associated proteins (MAPs) influence MT polymerization dynamics and enable them to perform their functions. The molecular chaperone Hsp90 has been shown to associate with MTs in animal and plant cells. However, the role of Hsp90–MT binding in plants has not yet been investigated. Here, we show that Hsp90 associates with cortical MTs in tobacco cells and decorates MTs in the phragmoplast. Further, we show that tobacco Hsp90_MT binds directly to polymerized MTs in vitro. The inhibition of Hsp90 by geldanamycin (GDA) severely impairs MT re-assembly after cold-induced de-polymerization. Our results indicate that the plant Hsp90 interaction with MTs plays a key role in cellular events, where MT re-organization is needed.

C-Src but Not Fyn Promotes Proper Spindle Orientation in Early Prometaphase

Src family tyrosine kinases (SFKs) participate in mitotic signal transduction events, including mitotic entry, cleavage furrow ingression, and cytokinesis abscission. Although SFKs have been shown to associate with the mitotic spindle, the role of SFKs in mitotic spindle formation remains unclear. Here, we show that c-Src promotes proper spindle orientation in early prometaphase. Src localizes close to spindle poles in a manner independent of Src kinase activity. Three-dimensional analyses showed that Src inhibition induced spindle misorientation, exhibiting a tilting spindle in early prometaphase. Spindle misorientation is frequently seen in SYF cells, which harbor triple knock-out mutations of c-Src, c-Yes, and Fyn, and reintroduction of c-Src but not Fyn into SYF cells rescued spindle misorientation. Spindle misorientation was also observed upon Src inhibition under conditions in which Aurora B was inhibited. Inducible expression of c-Src promoted a properly oriented bipolar spindle, which was suppressed by Src inhibition. Aster formation was severely inhibited in SYF cells upon Aurora B inhibition, which was rescued by reintroduction of c-Src into SYF cells. Furthermore, reintroduction of c-Src facilitated microtubule regrowth from cold-induced depolymerization and accelerated M phase progression. These results suggest that c-Src is involved in spindle orientation through centrosome-mediated aster formation.

Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions.

Ubiquitination is a reversible modification that influences a broad range of physiological processes. There are approximately 90 deubiquitinases (DUBs) encoded in the human genome, of which 79 are predicted to have catalytic activity. We tagged 66 DUBs with green fluorescent protein and systematically surveyed their subcellular distribution, identifying enzymes specific to the nucleus, plasma membrane, and secretory and endocytic pathways. USP21 is unique in showing clear association with both centrosomes and microtubules. Using an in vitro assay, we show that microtubule binding is direct and identify a novel microtubule-binding motif encompassed within amino acids 59-75 of the N-terminus of USP21. Our functional studies indicate a key role for USP21 in the governance of microtubule- and centrosome-associated physiological processes: Depletion of USP21 in A549 cells compromises the reestablishment of a radial array of microtubules during recovery from cold-induced depolymerization and also reduces the probability of primary cilium formation, whereas USP21 knockdown in PC12 cells inhibits nerve growth factor-induced neurite outgrowth.

Dysferlin Interacts with Histone Deacetylase 6 and Increases alpha-Tubulin Acetylation

Dysferlin is a multi-C2 domain transmembrane protein involved in a plethora of cellular functions, most notably in skeletal muscle membrane repair, but also in myogenesis, cellular adhesion and intercellular calcium signaling. We previously showed that dysferlin interacts with alpha-tubulin and microtubules in muscle cells. Microtubules are heavily reorganized during myogenesis to sustain growth and elongation of the nascent muscle fiber. Microtubule function is regulated by post-translational modifications, such as acetylation of its alpha-tubulin subunit, which is modulated by the histone deacetylase 6 (HDAC6) enzyme. In this study, we identified HDAC6 as a novel dysferlin-binding partner. Dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme, via its C2D domain, and to the substrate, alpha-tubulin, via its C2A and C2B domains. We further show that dysferlin expression promotes alpha-tubulin acetylation, as well as increased microtubule resistance to, and recovery from, Nocodazole- and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrate that myotube formation was impaired when alpha-tubulin was hyperacetylated early in the myogenic process; however, myotube elongation occurred when alpha-tubulin was hyperacetylated in myotubes. This study suggests a novel role for dysferlin in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein.

Abstract 2522: Therapeutic synergism of cabazitaxel in combination with cisplatin in tumor-bearing mice

Abstract Cabazitaxel (Jevtana), a semi-synthetic taxane derived from 10-deacetyl baccatin III, is as efficient as docetaxel in promoting tubulin assembly in vitro and stabilizing microtubules against cold-induced depolymerization. Cabazitaxel was selected for clinical development based on better antiproliferative activity than docetaxel against chemotherapy resistant tumor-cell lines (Bissery M.C. et al, AACR 2000) and a broad spectrum of in vivo antitumor activity (Vrignaud P. et al, AACR 2000), not only in docetaxel-sensitive tumor models, such as the human gastric N87 xenograft, but also in tumors models in which docetaxel was either poorly active or inactive. As an example, cabazitaxel was highly active in mice bearing UISO-BCA1 tumors, a human mammary xenograft expressing Her-2, which is poorly sensitive to ixabepilone and not sensitive to docetaxel. In addition, cabazitaxel penetrates the blood brain barrier (Cisternino S. et al, British J. Pharm. 2003) and has marked antitumor activity in nude mice bearing intracranial glioblastomas (Dykes D.J. et al, AACR 2000). Cabazitaxel was recently approved by the FDA for patients with metastatic hormone-refractory prostate cancer previously treated with docetaxel-based therapy. In order to explore other therapeutic indications, the cabazitaxel-cisplatin combination was evaluated in C51 tumor-bearing mice. A 5-arm dose-response study was performed with cabazitaxel and cisplatin administered IV on days 5 and 12 after tumor implantation as single agents and in combination using 3 different sequences of administration: simultaneous in group A and sequentially separated by a 24h interval in groups B (cabazitaxel first) and C (cisplatin first). At their highest nontoxic doses (HNTD), both single agents were found active with tumor growth delays (T-C) of 15.2 days for cabazitaxel at 20 mg/kg/inj and 22.1 days for cisplatin at 4.3 mg/kg/inj. The 3 combination arms showed greater antitumor activity at their respective HNTD with T-C of 38.4 days in group A, 37.8 days in B, and 36.2 days in C. Of interest, 2 (A) and 3 (B & C) dose levels were found more active than the single agents in the combination arms. In conclusion, a therapeutic synergism was achieved when cabazitaxel was combined with cisplatin in C51 tumor-bearing mice. These data are supporting the ongoing Phase I clinical study of cabazitaxel-cisplatin combination in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2522. doi:10.1158/1538-7445.AM2011-2522

The posttranslational modification of tubulin undergoes a switch from detyrosination to acetylation as epithelial cells become polarized

Tubulin posttranslational modifications (PTMs) have been suggested to provide navigational cues for molecular motors to deliver cargo to spatially segregated subcellular domains, but the molecular details of this process remain unclear. Here we show that in Madin-Darby Canine Kidney (MDCK) epithelial cells, microtubules express several tubulin PTMs. These modifications, however, are not coordinated, and cells have multiple subpopulations of microtubules that are marked by different combinations of PTMs. Furthermore these subpopulations show differential sensitivity to both drug- and cold-induced depolymerization, suggesting that they are functionally different as well. The composition and distribution of modified microtubules change as cells undergo the morphogenesis associated with polarization. Two-dimensionally polarized spreading cells have more detyrosinated microtubules that are oriented toward the leading edge, but three-dimensionally polarized cells have more acetylated microtubules that are oriented toward the apical domain. These data suggest that the transition from 2D polarity to 3D polarity involves both a reorganization of the microtubule cytoskeleton and a change in tubulin PTMs. However, in both 2D polarized and 3D polarized cells, the modified microtubules are oriented to support vectorial cargo transport to areas of high need.

Dystrophin is a microtubule-associated protein.

Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a Kd of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.

Doublecortin Kinase-2, a Novel Doublecortin-related Protein Kinase Associated with Terminal Segments of Axons and Dendrites

The microtubule (MT)-associated DCX protein plays an essential role in the development of the mammalian cerebral cortex. We report on the identification of a protein kinase, doublecortin kinase-2 (DCK2), with a domain (DC) highly homologous to DCX. DCK2 has MT binding activity associated with its DC domain and protein kinase activity mediated by a kinase domain, organized in a structure in which the two domains are functionally independent. Overexpression of DCK2 stabilizes the MT cytoskeleton against cold-induced depolymerization. Autophosphorylation of DCK2 strongly reduces its affinity for MTs. DCK2 and DCX mRNAs are nervous system-specific and are expressed during the period of cerebrocortical lamination. DCX is down-regulated postnatally, whereas DCK2 persists in abundance into adulthood, suggesting that the DC sequence has previously unrecognized functions in the mature nervous system. In sympathetic neurons, DCK2 is localized to the cell body and to the terminal segments of axons and dendrites. DCK2 may represent a phosphorylation-dependent switch for the reversible control of MT dynamics in the vicinity of neuronal growth cones.

NuMA expression and function in mouse oocytes and early embryos.

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage oocytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the re-formed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.

NuMA Expression and Function in Mouse Oocytes and Early Embryos

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage oocytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the re-formed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.

Direct Regulation of Microtubule Dynamics by Protein Kinase CK2

Microtubule dynamics is essential for many vital cellular processes such as morphogenesis and motility. Protein kinase CK2 is a ubiquitous protein kinase that is involved in diverse cellular functions. CK2 holoenzyme is composed of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show that the alpha subunit of CK2 binds directly to both microtubules and tubulin heterodimers. CK2 holoenzyme but neither of its individual subunits exhibited a potent effect of inducing microtubule assembly and bundling. Moreover, the polymerized microtubules were strongly stabilized by CK2 against cold-induced depolymerization. Interestingly, the kinase activity of CK2 is not required for its microtubule-assembling and stabilizing function because a kinase-inactive mutant of CK2 displayed the same microtubule-assembling activity as the wild-type protein. Knockdown of CK2alpha/alpha' in cultured cells by RNA interference dramatically destabilized their microtubule networks, and the destabilized microtubules were readily destructed by colchicine at a very low concentration. Further, over-expression of chicken CK2alpha or its kinaseinactive mutant in the endogenous CK2alpha/alpha'-depleted cells fully restored the microtubule resistance to the low dose of colchicine. Taken together, CK2 is a microtubule-associated protein that confers microtubule stability in a phosphorylation-independent manner.