MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule dynamics and intracellular transport

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1‐mediated ubiquitination of α‐tubulin in interphase cells. Here, we show that MGRN1‐mediated ubiquitination regulates dynamics of EB1‐labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule‐dependent organellar transport are evident in cells where noncanonical K6‐mediated ubiquitination of α‐tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late‐onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (^CtmPrP) interacts with MGRN1 leading to its loss of function. Expression of ^CtmPrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1‐mediated ubiquitination of α‐tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non‐transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.      

Effects of abscisic acid,low temperature,and plant age on cytoskeleton and phosphorylated proteins

The effects of exogenous abscisic acid (ABA), low temperature, and seedling age on the content of tubulin, actin, and phosphorylated proteins and the structural organization of microtubules (MTs) in cells of different tissues and organs of winter wheat cultivars contrasting in cold hardiness were studied by immunocytochemical methods using monoclonal (against - and -tubulin and actin) and polyclonal (phosphothreonine) antibodies. The leaves and roots of five- and nine- day-old seedlings of three cultivars were characterized by unequal proportion of actin/tubulin proteins. ABA decreased the content of the cytoskeleton and the 60-kD phosphorylated proteins, thus promoting a decrease in the number of MTs and occurrence of a less branched network of weakly fluorescent tubulin components in the cells of the root differentiating zone (which is most responsible for the development of cold hardiness in wheat). Although the cold acclimation of plants (3°C, 7 days) did not change the level of tubulin and actin proteins, it evoked the spatial aggregation of MT, leading to formation of a dense network of tubulin cytoskeleton comprised of thick bundles of intensively fluorescent MTs. In the case of a combined action of the studied factors, low temperatures abolished the hormone effect described above, evoking an increase in the content of the cytoskeletal and 60-kD phosphorylated proteins and MT structures. We suggest that the ABA-induced decrease in the levels of proteins and MTs occurs at the initial stages of plant cold acclimation (3°C, 2-3 days). It may be the signal that triggers the processes of low-temperature adaptation. As the duration of cold acclimation increased (3°C, 7 days), the role of ABA in the formation of plant tolerance decreased. Apparently, in this case other hormone-independent mechanisms of frost hardiness development are triggered, in which the role of the cytoskeleton components and cytoskeleton-associated proteins increases.     

H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo

Summary We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells, H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.     

Microtubular Systems of Paramecium in Division: Pattern of Cytospindle Assembly

The pattern of cytospindle assembly and the modifications of the microtubular cytoplasmic network during division of Paramecium are studied by means of indirect immunofluorescence. The assembly of cytospindle starts at two independent areas placed respectively around the proter's and opisthe's buccal overture. The moment of the microtubule bundles’appearance depends on their distance from the buccal opening, with those closest appearing 1st. The existence of microtubule organizing centers that act transiently during division of Paramecium is discussed.     

Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β-tubulin

To obtain information on plant microtubule stability to low temperature and Ca²⁺, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβ_s-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβ_s-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβ_s-microtubules. Ca²⁺ enhanced the cold-induced depolymerization of both αβ- and αβ_s-microtubules. However, αβ_s-microtubules were significantly more stable to depolymerization by cold and Ca²⁺ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca²⁺. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.     

Effect of melatonin on β-tubulin and MAP2 expression in NIE-115 cells

Physiological concentrations of the pineal hormone melatonin induce an increase of microtubules in neuroblastoma NIE-115 cells. This effect is due to an increase in the polymerization state of tubulin. Concomitantly, higher levels of soluble β-tubulin are present in the treated cells. Unexpectedly, no significant changes in the levels of β-tubulin or its mRNA occur in the presence of melatonin reflecting perhaps a strict control of its steady state in a physiological context. In contrast, higher amounts of microtubule-associated-protein 2 are found when the cells are exposed to melatonin. These findings support the idea that tubulin polymerization process is one of the targets of melatonin action. Furthermore, our results might explain the increase in the length and number of neurites present in these cells when they are treated with this hormone.     

An essential quality control mechanism at the eukaryotic basal body prior to intraflagellar transport

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.     

Centrosome function in normal and tumor cells

Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process.     

Versatile substrate protein recognition mechanism of the eukaryotic chaperonin CCT

Group II chaperonins, found in eukaryotic and archaeal organisms, recognize substrate proteins through diverse mechanisms that involve either hydrophobic‐ or electrostatic‐dominated interactions. This action is distinct from the universal substrate recognition mechanism of group I chaperonins, which bind a wide spectrum of non‐native proteins primarily through hydrophobic interactions. We use computational approaches to pinpoint the substrate protein binding sites of the γ‐subunit of the eukaryotic chaperonin CCT and to identify its interactions with the stringent substrate β‐tubulin. Protein–protein docking methods reveal intrinsic binding sites of CCT comprising a helical (HL) region, homologous to the GroEL‐binding site, and the helical protrusion (HP) region. We performed molecular dynamics simulations of the solvated CCTγ apical domain, β‐tubulin peptide‐CCTγ complexes, and isolated β‐tubulin peptides. We find that tubulin binds to CCTγ through an extensive interface that spans both the HL region and the HP region. HL interactions involve both hydrophobic and electrostatic contacts, while binding to the HP region is stabilized almost exclusively by a salt bridge network. On the basis of additional simulations of a β‐tubulin‐CCTγ complex that involves a reduced interface, centered onto the HP region, we conclude that this salt bridge network is the minimal stabilizing interaction required. Strong conservation of the charged amino acids that participate in the salt bridge network, Arg306 and Glu271, indicates a general mechanism across the nonidentical CCT subunits and group II chaperonins. Proteins 2010. © 2009 Wiley‐Liss, Inc.